EPA/600/R-04/075
July 2004
LITERATURE REVIEW ON THE USE OF
COMMERCIAL BIOREMEDIATION AGENTS
FOR CLEANUP OF OIL-CONTAMINATED
ESTUARINE ENVIRONMENTS
by
^ueqing Zhu, 2Albert D. Venosa, and ^akram T. Suidan,
University of Cincinnati
Cincinnati, OH 45221
U.S. Environmental Protection Agency
National Risk Management Research Laboratory
Cincinnati, OH 45268
EPA Contract No. 68-C-00-159
Task Order No. 19
Task Order Manager
Albert D. Venosa
Land Remediation and Pollution Control Division
National Risk Management Research Laboratory
Cincinnati, OH 45268
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Disclaimer
The information in this document has been funded by the United States Environmental
Protection Agency (U.S. EPA) under Task Order No. 19 of Contract No. 68-C-00-159 to the
University of Cincinnati. It has been subjected to the Agency's peer and administrative reviews
and has been approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute an endorsement or recommendation for use.
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
Nation's land, air, and water resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, EPA's research program is providing data and technical support for solving
environmental problems today and building a science knowledge base necessary to manage our
ecological resources wisely, understand how pollutants affect our health, and prevent or reduce
environmental risks in the future.
The National Risk Management Research Laboratory (NRMRL) is the Agency's center for
investigation of technological and management approaches for preventing and reducing risks
from pollution that threaten human health and the environment. The focus of the Laboratory's
research program is on methods and their cost-effectiveness for prevention and control of
pollution to air, land, water, and subsurface resources; protection of water quality in public water
systems; remediation of contaminated sites, sediments and ground water; prevention and control
of indoor air pollution; and restoration of ecosystems. NRMRL collaborates with both public
and private sector partners to foster technologies that reduce the cost of compliance and to
anticipate emerging problems. NRMRL's research provides solutions to environmental problems
by: developing and promoting technologies that protect and improve the environment; advancing
scientific and engineering information to support regulatory and policy decisions; and providing
the technical support and information transfer to ensure implementation of environmental
regulations and strategies at the national, state, and community levels.
This publication has been produced as part of the Laboratory's strategic long-term research plan.
It is published and made available by EPA's Office of Research and Development to assist the
user community and to link researchers with their clients.
Lawrence W. Reiter, Acting Director
National Risk Management Research Laboratory
in
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Executive Summary
The objective of this document is to conduct a comprehensive review of the use of commercial
bioremediation products treating oil spills in all environments. Literature assessed includes peer-
reviewed articles, company reports, government reports, and reports by cleanup contractors
engaged in responses to oil spills. The scope of this review is in the general context of estuarine
environments. However, marine shorelines, terrestrial environments, freshwaters, and wetlands
are frequent candidates for bioremediation of spilled oil, and these ecosystems are also included
in the review for completeness. The review will be useful for oil spill responders (e.g., on-scene
coordinators and response contractors) to better understand the feasibility of bioremediation
technology and as an aid in selecting bioremediation products.
This state-of-science review on the efficacy of bioremediation products is conducted using
different approaches and presented accordingly as follows. Section 1 provides an overall
introduction of the background and the scope of this review. Section 2 presents an in-depth
review of field tests of bioremediation products based on the scientific literature, which includes
peer-reviewed journal articles, books, and major conference proceedings. Section 3 evaluates oil
bioremediation products based on the non-peer reviewed literature articles gathered, such as
government agency reports and vendor/service provider reports. Finally, Section 4 gives the
conclusions and recommendations based on the reviewed information.
The overall conclusions reached by this review are as follows. First, according to the peer-
reviewed literature, bioaugmentation appears to have little benefit for the treatment of spilled oil
in an open environment. Microbial addition has not been shown to work better than nutrient
addition alone in many field trials. However, case studies provided by vendors seem to suggest
that application of bioaugmentation products could still have some potential in the treatment of
specific oil components, isolated spills in confined areas, or certain environments where oil-
degrading microorganisms are deficient. Unfortunately, the evidence for such a conclusion is not
strong and in most, if not all, cases is scientifically deficient.
Biostimulation has been proven to be a promising tool to treat certain aerobic oil-contaminated
shorelines. One of the key factors for the success of oil biostimulation is to maintain an optimal
nutrient level in the interstitial pore water. In general, commercial oleophilic nutrient products
have not shown clear advantages over common agricultural fertilizers in stimulating oil
biodegradation. Effects of nutrients are also highly site-specific. For example, the availability of
oxygen rather than nutrients is often the limiting factor in wetland environments, where addition
of nutrient products has not been successful in enhancing oil biodegradation.
The extreme uncertainty associated with the efficacy of bioremediation agents is due in large part
to the poorly designed field tests that have been conducted to demonstrate efficacy. Much of the
reported literature either lacked proper controls and quality assurance, or the data were
incorrectly analyzed. If there is any hope for advancement of commercial bioremediation,
experiments based on sound scientific principles are needed. Unfortunately, due to the extreme
resource intensiveness of field studies, the benefit accruing to testing one bioremediation agent is
only applicable to the one product being tested. Testing products in the field is not within the
purview of the federal government unless such a test has the potential of advancing science in
terms of general microbiological and engineering principles.
iv
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TABLE OF CONTENTS
Executive Summary iv
1 Introduction and Background 1
1.1 Objectives and Scope 2
1.2 Organization of the Document 3
2 Assessment of Bioremediation Products in the Field: Peer-Reviewed Literature 4
2.1 Bioremediation Products and Evaluation 4
2.1.1 Bioremediation agents 4
2.1.2 Assessing oil bioremediation in the field 7
2.2 Application of Bioaugmentation Products 8
2.3 Application of Biostimulation Products 11
2.3.1 Common agricultural fertilizers 12
2.3.1.1 Water-soluble fertilizers 12
2.3.1.2 Slow-release fertilizers 14
2.3.2 Commercial biostimulation agents 17
2.3.2.1 ImpolEAP22 17
2.3.2.2 BIOREN 18
2.3.2.3 Oil Spill Eater IIฎ (OSEII) 19
2.4 Summary 20
3 Assessment of Oil Bioremediation Products: Non-Peer Reviewed Literature 22
3.1 Government Agency Reports 22
3.1.1 Application of bioaugmentation products 22
3.1.2 Application of biostimulation products 24
3.2 Vendor's Reports 25
3.2.1 Information collection 26
3.2.2 Summary of case studies submitted by vendors 28
3.2.2.1 Enviro-Zyme, Inc 28
3.2.2.2 Forrester Environmental Technologies Corp. (FET Group) 29
3.2.2.3 Garner Environmental Services, Inc 29
3.2.2.4 Industrial Wastewater Solutions Corp 30
3.2.2.5 Medina Agriculture Products Co., Inc 31
3.2.2.6 Petrol Rem, Inc 33
3.2.2.7 Verde Environmental, Inc 34
3.2.2.8 WMI International, Inc 35
3.2.3 Review of vendor reports 40
3.3 Bioremedial Approaches for Controlling Petroleum Hydrocarbons in Stormwater and
Bilge Water 42
3.3.1 Bioremediation of hydrocarbon contamination from storm water discharges 42
3.3.2 Bioremedial approaches for treating bilge oil 43
4 Summary and Findings 44
5 References 46
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1 Introduction and Background
It is estimated that between 1.7 and 8.8 million metric tons of oil are released into the
world's water every year (NAS, 1985), of which more than 90% is directly related to human
activities including deliberate waste disposal. Marine oil spills, particularly large-scale spill
accidents, have received great attention due to their catastrophic damage to the environment. For
example, the spill of 37,000 metric tons (11 million gallons) of North Slope crude oil into Prince
William Sound, Alaska, from the Exxon Valdez in 1989 led to the mortality of thousands of
seabirds and marine mammals, a significant reduction in population of many intertidal and
subtidal organisms, and many long term environmental impacts (Spies et a/., 1996). Minor oil
spills and oil contamination from non-point source discharges (e.g., urban runoff and boat bilge)
are no less threats to public health and the environment, although they have received much less
attention in the past. According to recent National Water Quality Inventory reports, non-point
source pollution remains the Nation's largest source of water quality problems (U.S. EPA,
1996&2000). It is the main reason that approximately 40 % of surveyed rivers, lakes, and
estuaries are not clean enough to meet basic uses such as fishing or swimming.
Conventional oil spill countermeasures include various physical, chemical, and biological
methods. Commonly used physical methods include booming and skimming, manual removal
(wiping), mechanical removal, water flushing, sediment relocation, and tilling. Physical
containment and recovery of bulk or free oil is the primary response option of choice in the
United States for the cleanup of oil spills in marine and freshwater shoreline environments.
Chemical methods, particularly dispersants, have been routinely used in many countries as a
response option. However, chemical methods have not been extensively used in the United
States due to the disagreement about their effectiveness and the concerns of their toxicity and
long-term environmental effects (U.S. EPA, 1999). With the recent development of less toxic
chemical dispersants, the potential for their applications may increase.
Although conventional methods, such as physical removal, often are the first response
option, they rarely achieve complete cleanup of oil spills. According to the Office of
Technology Assessment (OTA, 1990), current mechanical methods typically recover no more
than 10-15 percent of the oil after a major spill, although significantly higher recoveries have
been achieved, depending on the environment. Bioremediation is beginning to emerge as a
promising technology, particularly as a secondary treatment option for oil cleanup.
Bioremediation has been defined as "the act of adding materials to contaminated environments to
cause an acceleration of the natural biodegradation processes" (OTA, 1991). This technology is
based on the premise that a large percentage of oil components are readily biodegradable in
nature (Atlas, 1984, 1981; Prince, 1993). Bioremediation has several potential advantages over
conventional technologies, such as being less costly, less intrusive to the contaminated site, and
more environmentally benign in terms of its end products.
The success of oil spill bioremediation depends on one's ability to establish and maintain
conditions that favor enhanced oil biodegradation rates in the contaminated environment.
Numerous scientific review articles have covered various factors that influence the rate of oil
biodegradation (Zobell 1946; Atlas, 1981 & 1984; Atlas and Bartha, 1992; NAS, 1985; Focht
and Westlake, 1987; Leahy and Colwell, 1990). One important requirement is the presence of
microorganisms with the appropriate metabolic capabilities. If these microorganisms are
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present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by
ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is
between 6 and 9. The physical and chemical characteristics of the oil and oil surface area are
also important determinants of bioremediation success. There are two main approaches to oil
spill bioremediation:
Bioaugmentation, in which known oil-degrading bacteria are added to supplement the
existing microbial population, and
Biostimulation, in which the growth of indigenous oil degraders is stimulated by the
addition of nutrients or other growth-limiting co-substrates.
Although extensive research has been conducted on oil bioremediation during the last
decade, the effectiveness of this technology has only rarely been convincingly demonstrated, and
in the case of commercial bioremediation products, the literature is virtually completely lacking
in supportive evidence of success. Most existing studies have concentrated on evaluating the
factors affecting oil bioremediation or testing favored products and methods through laboratory
studies (Mearns, 1997). Only limited numbers of pilot-scale and field trials, which may provide
the most convincing demonstrations of this technology, have been reported in the peer-reviewed
literature (Prince 1993, Swannell et al, 1996, Venosa et al., 1996 and 2002). The scope of current
understanding of oil bioremediation is also limited because the emphasis of most of these field
studies and reviews has been on the evaluation of bioremediation technology for dealing with
large-scale oil spills on marine shorelines. To help oil spill responders in the selection and
application of bioremediation products, there is an immediate need to gather and evaluate
information about the field performance of commercial bioremediation products, especially for
dealing with low-level petroleum hydrocarbon contamination.
To better understand the potential effectiveness of bioremediation technology, Public
Law 105-457 entitled "Estuaries and Clean Waters Act of 2000" (the Act) was enacted, which
states specifically that "the Administrator of the Environmental Protection Agency (EPA) shall
begin a two-year study on the efficacy of bioremediation products." The Act mandated that "the
study shall evaluate and assess bioremediation technology (a) on low-level petroleum
hydrocarbon contamination from recreational boat bilges, (b) on low-level petroleum
hydrocarbon contamination from storm water discharges, (c) on non-point source petroleum
hydrocarbon discharges, and (d) as a first response tool for petroleum hydrocarbon spills." This
report is a part of EPA's efforts to address the Congressional mandate under the Act by extensive
review of literature where bioremediation products have been used for oil spill cleanup.
1.1 Objectives and Scope
The objective of this document is to conduct a thorough assessment of bioremediation
products by a comprehensive review of the actual use of bioremediation in real world cases.
Literature assessed includes peer-reviewed articles, company reports, government reports, and
actual reports by cleanup contractors engaged in responses to spills in inland, estuarine, and
marine environments. The review will be useful for oil spill responders (e.g., on-scene
coordinators and response contractors) to better understand the feasibility of bioremediation
technology and as an aid in selecting bioremediation products.
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As mentioned earlier, only a limited number of scientific, peer-reviewed journal articles
are available on the performance of bioremediation products for oil spill cleanups. However,
there are many reports pertaining to the use of bioremediation products in the non-peer reviewed
or "gray" literature. Various government agencies [e.g., U.S. EPA, U.S. Coast Guard, U.S. Navy,
U.S. Army, Department of Energy (DOE), the National Oceanic and Atmospheric
Administration (NOAA)] have involved field investigation of bioremedial approaches for
treating petroleum hydrocarbon contamination. More than 170 companies around the world are
listed in Oil Spill Intelligence Report (2000), which offer either bioremediation products or
bioremediation services. It is reasonable to assume that many field trials or applications
conducted by government agencies, vendors, and responders have been documented but are not
readily available to the public for various reasons. A thorough search for the "gray" literature is
an important part of this project. The in-depth review of these non-peer reviewed reports will fill
the "information gap" and provide a better picture in regard to the present and potential of the
use of bioremediation products as a viable option for oil spill cleanups.
It should be noted that all the reports collected are evaluated comprehensively for their
scientific merit, and only those judged appropriate and scientifically sound are earmarked for
inclusion in this document. If a report is deemed invalid due to technical deficiencies or
insufficient information, it will also be mentioned but explanations given to why it was not an
integral part of our final recommendation.
The scope of this review is in the general context of estuarine environments. However,
marine shorelines, terrestrial environments, freshwaters, and wetlands are frequent candidates for
bioremediation of spilled oil, and these ecosystems are also included in the review for
completeness.
1.2 Organization of the Document
This state-of-science review on the efficacy of bioremediation products is conducted
using three different approaches and, accordingly, presented in the following major Sections.
Section 2 presents an in-depth review of field tests of bioremediation products based on scientific
literature, which includes peer-reviewed journal articles, books, and major conference
proceedings (e.g., International Oil Spill Conference and International Bioremediation
Symposium). Section 3 evaluates oil bioremediation products based on all the non-peer reviewed
literature articles gathered, such as government agency reports and vendor/service provider
reports. A discussion is also presented in Section 3 in regard to the potential of using
bioremediation products in the areas of non-point source and stormwater runoff countermeasures
and for treating bilge oil from boats, ships, cutters, and other watercraft. Finally, Chapter 4 gives
the conclusions and recommendations based on all the information reviewed throughout this
document.
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2 Assessment of Bioremediation Products in the Field: Peer-Reviewed Literature
Field studies can provide the most convincing demonstration of the effectiveness of oil
bioremediation since laboratory studies are not always able to account for numerous real world
conditions such as spatial heterogeneity, biological interactions, and mass transfer limitations.
Swannell et al. (1996) conducted the most extensive review available on field evaluations of oil
bioremediation in marine environments. Venosa (1998) presented an in-depth critical review of
research studies emphasizing extensive inadequacies in the experimental design and control of
published field tests. Other reviews are also available (Prince, 1993; Leahy and Colwell, 1990).
However, none of existing reviews has focused on the field performance of commercial
bioremediation agents. They did not distinguish bioremediation due to addition of commercial
products and bioremediation due to application of common agricultural fertilizers/nutrient
solutions or non-commercial microbial strains. This chapter will present a comprehensive review
with the emphasis on the efficacy of commercial bioremediation products in the field by
reviewing latest peer reviewed articles, as well as summarizing major points identified in the
previous reviews. Non-commercial products or common agricultural fertilizers may also be
covered only for the purpose of comparison.
2.1 Bioremediation Products and Evaluation
2.1.1 Bioremediation agents
The U.S. EPA has defined Bioremediation agents as "microbiological cultures, enzyme
additives, or nutrient additives that significantly increase the rate of biodegradation to mitigate
the effects of the discharge" (Nichols, 2001). Bioremediation agents are also classified as
bioaugmentation agents and biostimulation agents based on the two main approaches to oil spill
bioremediation. Numerous bioremediation products have been proposed and promoted by their
vendors, especially during early 1990s, when bioremediation was popularized as "the ultimate
solution" to oil spills (Hoff, 1993). The U.S. EPA is often inundated with salespeople wanting to
have EPA endorse their products. To have bioremediation products used properly, the U.S. EPA
has compiled a list of bioremediation agents (Nichols, 2001; USEPA, 2002) as part of the
National Oil and Hazardous Substances Pollution Contingency Plan (NCP) Product Schedule,
which is required by the Clean Water Act, the Oil Pollution Act of 1990, and the National
Contingency Plan. The Schedule is intended for use by Federal On-Screen Coordinators
(FOSCs), Regional Response Teams (RRT), and other oil spill responders as an aid in
determining the most appropriate products to use in various spill scenarios.
At the time of this writing, 15 bioremediation agents were listed on the NCP Schedule as
shown in Table 2.1. This list has been modified recently, and the number has been reduced to
nine. A product can be placed on the Schedule only if all the required data have been submitted
and when its safety and effectiveness have been demonstrated under the conditions of a test
protocol developed by EPA (NETAC, 1993a; Nichols, 2001). However, the listing of a product
on the Schedule does not mean that the product is approved or certified for use on an oil spill. At
present, the only efficacy requirement for being listed is to pass the Bioremediation 28-Day
Effectiveness Test. The test protocol uses laboratory shake flasks to compare the degradation of
artificially-weathered crude oil in natural seawater with and without a bioremediation product.
This test alone cannot demonstrate that a product will be effective in the field. Studies have
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shown that bioremediation products may be effective in the laboratory but significantly less so in
the field (Lee et al, 1997, Mearns, 1997; Venosa et al, 1992 and 1996). This is because
laboratory studies cannot always simulate complicated real world conditions such as spatial
heterogeneity, biological interactions, climatic effects, and nutrient mass transport limitations.
Therefore, field studies and applications are the ultimate tests or the most convincing
demonstration of the effectiveness of bioremediation products.
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Table 2.1 Bioremediation agents in NCP product schedule (Adapted from USEPA, 2002)
Name or Trademark | Product Type Manufacture
BET BIOPETRO
BILGEPRO
INIPOL EAP 22
LAND AND SEA
RESTORATION
MICRO-BLAZE
OIL SPILL EATER II
OPPENHEIMER FORMULA
PRISTINE SEA II
STEP ONE
SYSTEM E.T. 20
VB591WATER,
VB997SOIL, AND
BINUTRIX
WMI-2000
MC
NA
NA
NA
MC
NA/EA
MC
MC
MC
MC
NA
MC
BioEnviro Tech, Tomball, TX
International Environmental Products, LLC,
Conshohocken, PA
Societe, CECA S.A., France
Land and Sea Restoration LLC, San Antonio,
TX
Verde Environmental, Inc., Houston, TX
Oil Spill Eater International, Corporation
Dallas, TX
Oppenheimer Biotechnology, Inc., Austin, TX
Marine Systems, Baton Rouge, LA
B & S Research, Inc., Embarrass, MN
Quantum Environmental Technologies, Inc.
(QET), La Jolla, CA
BioNutraTech, Inc., Houston, TX
WMI International, Inc., Houston, TX
Abbreviations of product type:
MC -- Microbial Culture
EA Enzyme Additive
NA -- Nutrient Additive
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2.1.2 Assessing oil bioremediation in the field
Compared to laboratory investigations, few tests have been carried out to evaluate the
effectiveness of bioremediation products in the field because such trials are both difficult and
expensive to conduct. One of the most difficult tasks in field studies is the proper evaluation of
oil biodegradation. Oil contaminated sites are often highly heterogeneous, where oil
concentrations can vary greatly within a small area. Physical and chemical weathering may also
significantly affect the composition and concentration of oil contamination. Consequently,
variability associated with field studies can be so high as to preclude or interfere with one's
ability to discern significant treatment differences. Nevertheless, the efficacy of bioremediation
in the field can be verified through well-designed monitoring programs and proper data
interpretation.
Evidence for the effectiveness of oil bioremediation should include: (1) faster
disappearance of oil in treated areas than in untreated areas, and (2) a demonstration that
biodegradation was the main reason for the increased rate of oil disappearance. To obtain such
evidence, one has to be careful in selecting proper oil analysis procedures as well as in
interpreting the data. Oil analysis methods can be generally classified into two categories:
nonspecific methods to measure total petroleum hydrocarbons (TPHs), and specific methods
using various chromatographic techniques to quantify target oil constituents. Total petroleum
hydrocarbons (TPH) techniques have been widely accepted methods to rapidly quantify the oil
due to their simplicity and low costs. However, these methods are severely affected by the spatial
heterogeneity and, more importantly, they are much less able to distinguish between abiotic and
biotic losses. The reason for this is that conventional TPH analysis is confounded by the presence
of plant lipids and other biogenic compounds that interfere with interpretation of the analysis.
In recent years, non-biodegradable or slowly biodegradable components in oil - often
called biomarkers - have been used successfully to distinguish between biodegradation and the
physical or chemical loss of oil and to mitigate the high variability associated with field studies
(Bragg et al, 1994; Venosa et al., 1996; Lee et al, 1997). This approach estimates the extent of
biodegradation by using GC/MS techniques and evaluating the ratios of target hydrocarbon
concentrations relative to the concentration of these recalcitrant biomarkers, such as hopanes and
steranes and to a lesser extent alkyl-substituted 4-ring PAHs such as Cs-chrysene. Studies have
shown that normalizing target oil constituents to biomarkers mitigates the spatial variability of
oil contamination when compared to other mass balance approaches and allows biodegradation
to be monitored effectively by reducing the number of samples required (Douglas et al., 1994).
To ensure that monitored results reflect reality in a highly heterogeneous environment, it
is also critical that a bioremediation sampling plan be designed according to valid statistical
principles that include the principles of randomization, replication, and the use of proper
controls. For example, to minimize bias, a random sampling plan should be used to evaluate
treatment effects and their variance within the bioremediation zone. Efforts should also be made
to ensure that an adequate number of independent samples are taken to reach a given accuracy
and confidence. A proper control or untreated set aside area is also critical to demonstrate the
true impact of a treatment. Detailed procedures to properly evaluate oil bioremediation can be
found in Guidelines for the Bioremediation of Marine Shorelines and Freshwater Wetlands (Zhu
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et a/., 2001). All these principles will be used as the basis to evaluate the technical merit of the
literature reviewed in this document.
2.2 Application of Bioaugmentation Products
Since the 1970s, bioaugmentation, or the addition of oil-degrading microorganisms to
supplement the indigenous populations, has been proposed as an alternate strategy for the
bioremediation of oil contaminated environments. The rationale for this approach is that
indigenous microbial populations may not be capable of degrading the wide range of potential
substrates present in complex mixtures such as petroleum (Leahy and Colwell, 1990) or that they
may be in a stressed state as a result of the recent exposure to the spill. Other conditions under
which bioaugmentation may be considered are when the indigenous hydrocarbon-degrading
population is low, the speed of decontamination is the primary factor, and when seeding may
reduce the lag period to start the bioremediation process (Forsyth et al, 1995). For this approach
to be successful in the field, the seed microorganisms must be able to degrade most petroleum
components, maintain genetic stability and viability during storage, survive in foreign and hostile
environments, effectively compete with indigenous microorganisms, and move through the pores
of the sediment to the contaminants (Atlas, 1977; Goldstein etal., 1985).
Methods involving the addition of selected oil-degrading microorganisms into spilled oil
have been patented and marketed since early 1970s (Azarowick, 1973; Linn, 1971; and Mohan et
al., 1975). However, before the Exxon Valdez spill in 1989, little information on the performance
of commercial bioaugmentation products was available in the peer-reviewed literature. Atlas and
Bartha (1973) conducted one of the first laboratory tests on the effectiveness of commercial
mixed bacterial cultures. Two commercial petroleum-degrading bacterial inocula, Ekolo-Gest
(also marketed as Petrobac, National Chem. Corp.) and DEC bacteria (Gerald Bauer Corp.),
were tested using shake flasks to compare the degradation of Sweden crude oil. The study found
that none of the commercial mixtures was superior to the indigenous microorganisms in coastal
marine waters.
One of first field trials on oil bioremediation using a microbial product in a marine
environment was reported by Lee and Levy (1987). The study involved seeding a mixed culture
of marine oil-degrading bacteria (strains of Pseudomonas aemginosa, Pseudomonas stutzeri, and
Bacillus subtilis grown on bran) in a Scotian Shelf Condensate (SSC) contaminated sandy beach.
The extent of biodegradation was measured by the decline in the n-Cn/pristane ratio in this
study. The results showed that the n-Cn/pristane ratio in the seeded plots did decrease slightly.
However, due to high inter-and intra-plot variability, no significant difference in the rate of oil
loss was observed among the treatments. This study also observed that the number of oil-
degrading bacteria did not increase until 10 to 15 days after the addition of oil. However, the
addition of the microbial product did not reduce this lag period, suggesting that the toxic volatile
components in the oil, which evaporated mostly during the first week, was the main cause of the
lag period.
Since the application of nutrient amendments for the cleanup of the Exxon Valdez spill in
1989, bioremediation has received increased attention, and several field tests and applications of
bioaugmentation have been reported. Venosa et al. (1992) conducted a field test in Prince
William Sound following the Exxon Valdez spill to investigate the effectiveness of two
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commercial microbial products vis-a-vis natural attenuation and nutrient addition alone. These
products were selected based on a previous laboratory study (Venosa et al., 1991). This field trial
failed to demonstrate enhanced oil biodegradation by these products. No biostimulation
occurred in the nutrient control plots either. There were no significant differences between any of
the treatment and control plots during the 27-day trial period. However, the site where the
project took place (Disk Island) was characterized as having highly weathered (degraded) oil and
very calm waters, so dissolved oxygen may have been limiting, thus precluding effective
biodegradation by any means.
One approach in overcoming the competition problem was proposed by Rosenberg et al.
(1992). They developed a product that combined a polymerized ureaformaldehyde fertilizer,
which they called F-l, with a selected oil-degrading culture capable of using this fertilizer as a
nitrogen source. Thus, the culture had a selective advantage over the indigenous population
unable to utilize F-l as nutrient source. A field trial conducted at an Israeli beach showed that
this approach seemed to be successful in enhancing oil biodegradation. However, conclusions
were confounded by the lack of adequate controls in the study (Swannell et al., 1996; Venosa,
1998).
To evaluate the effectiveness of two commercial bioaugmentation products in an
estuarine environment, a field trial was carried out in a Texas coastal wetland by a research
group from Texas A&M University (Simon et al., 1999; Townsend et al., 1999). The two
products were selected based on a previous laboratory efficacy test, in which four out of twelve
products showed an enhancement of oil biodegradation with significantly higher degradation
rates of alkanes and aromatics when compared to a nutrient control (Aldrett et al., 1997). The 21-
plot site, named San Jacinto Wetland Research Facility (SJWRF) has been used for a series of
studies on oil spills and their countermeasures. In this study, four treatment strategies were
examined: an oiled control, biostimulation with inorganic nutrient addition (diammonium
phosphate) , and commercial bioaugmentation with 2 different products. Arabian medium crude
oil was selected in this test and the 21 plots each measuring 5 x 5 m were arranged in a balanced,
incomplete block experimental design. Oil constituents were determined using gas
chromatography/mass spectrometry (GC/MS) and were normalized to 17a(H), 21(3(H)-hopane to
reduce the effects of sample heterogeneity and physical losses. The results showed that the
addition of microbial products could not significantly enhance oil biodegradation rates. No
differences were observed between treatments when comparing the first order biodegradation
rate coefficients for the total target saturates, total target aromatics, and individual hydrocarbon
target analytes. The authors also pointed out that one of the products (BP8) "did show
consistently higher biodegradation rates, though the rates were not significantly different from
the control." Because this microbial product was applied with vendor supplied inorganic
nutrients (Townsend et al., 1999), it is difficult to conclude whether the "consistently but
insignificantly" higher rates resulted from the additions of the microbial components or the
nutrient components. The fact that neither addition of bioaugmentation agents nor application of
inorganic nutrients significantly enhanced oil biodegradation suggested that other factors, such as
oxygen, could have been limiting oil degradation in that environment.
Studies comparing the performance of bioaugmentation and biostimulation have
suggested that nutrient addition alone had a greater effect on oil biodegradation than did the
addition of microbial products when oxygen supply was not limited (Jobson et al., 1974; Lee et
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a/., 1997; Venosa et al., 1996). This is probably because the hydrocarbon-degrading population
is rarely a limiting factor as compared to the nutrients since the size of the hydrocarbon-
degrading bacterial population usually increases rapidly in response to oil contamination. One of
the first comprehensive field tests evaluating various bioremediation approaches to enhance oil
biodegradation was carried out in a soil environment in northwest area of Canada in early 1970s
(Jobson et al., 1974). A randomized block design was used to examine the effects of four
treatments (control, inorganic fertilizer application, ; addition of a microbial culture alone , and
combined fertilizer and microbial culture addition) over a 308-day time period. The microbial
culture was grown in the laboratory and consisted of several genera of oil-degrading bacteria
(Flavobacterium and Cytophoga sp., Pseudomonas sp., Xanthomonas sp., Alcaligenes sp., and
Arthrobacter sp.). The study showed that the nutrient application resulted in a significant
stimulation of bacterial numbers and in the degradation rate of n-alkane components of the crude
oil. The application of the microbial agent, however, resulted in only a slightly enhanced
degradation rate of n-alkane components of chain lengths C20 to C25.
A field study conducted on a sandy beach in Delaware also showed that addition of a
microbial inoculum did not enhance oil biodegradation more than addition of inorganic nutrients
alone (Venosa et al., 1996). A randomized block design was used in this study to assess the
effects of three treatments: a no-nutrient control (natural attenuation), addition of water-soluble
nutrients, and addition of water-soluble nutrients supplemented with a natural microbial
inoculum from the site. No significant differences were observed between plots treated with
nutrients alone and plots treated with nutrients and the indigenous inoculum, suggesting that
supplementation of the natural population with indigenous cultures from the same site still did
not result in further enhancement over simple nutrient addition on marine shorelines. The authors
also indicated that this conclusion could be extended to include exogenous microbial inocula or
commercial microbial agents because "if indigenous cultures do not accelerate the degradation
rates, organisms enriched from different environments, grown in the laboratory, and not
acclimated to a particular climatic or geographic location should be even less able to compete
with the natural population."
Lee et al. (1997) conducted a 129-day field trial to compare the effect of four treatments
on biodegradation of weathered Venture Condensate on a sandy beach in Nova Scotia, Canada.
The four treatments (control, inorganic nutrient addition, a commercial bioremediation product,
and addition of inorganic nutrients along with bioremediation product) as well as an unoiled
control were replicated in a complete block design using 20 enclosures or plots. C2-chrysene
was used as the normalizing biomarker due to the low concentration of hopane in the condensate.
PRP (PetrolRem, Inc.) was selected to be the representative commercial bioremediation agent in
this study. This product is no longer listed in the current NCP Product Schedule. According to
Lee et al. (1997), PRP contains mineral nutrients and nonpathogenic bacteria within spherical
particles made from plant derived natural products (beeswax) and exhibits both bioaugmentation
and biostimulation properties. The agricultural fertilizer used in this study was a mixture of
granular forms of ammonium nitrate (N:P:K: 33-0-0) and triple super phosphate (N:P:K: 0-46-0).
The study showed that an average of 11.0% of the n-alkanes remained in the oiled control plots,
and only 0.1% of the oil remained in the enclosures treated with inorganic nutrients alone; 5.4%
of the alkanes were found in the plots treated with inorganic nutrients and PRP, and 25.3%
remained in the plots treated with PRP alone. The results indicate that periodic addition of
inorganic nutrients was the most effective strategy for enhancing oil degradation and that the full
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potential of the bioremediation product was limited by nutrient availability. This field trial
demonstrated that adding the bioremediation product did not perform better in terms of
enhancing alkane degradation than applying inorganic agricultural fertilizers alone.
Several other possible reasons for the failure of inocula in degrading contaminants in
nature were summarized by Goldstein et al. (1985), which include: (1) the concentration of the
contaminant may be too low to support the growth of the inoculated species, (2) the natural
environment may contain substances inhibiting growth or activity of the inocula, (3) the growth
rate of the inoculated species may be limited by predation such as protozoa, (4) the added species
may use other substrates in nature rather than the targeted contaminants, and (5) the seeded
microorganisms may be unable to move through the pores of the sediment to the contaminants.
A few field trials did claim success in demonstrating the effectiveness of oil
bioaugmentation, such as using Alpha BioSea (Alpha Environmental, Inc.) to treat the
Angolan Palanca crude oil spilled from Mega Borg off Texas coast (Mauro and Wynne, 1990;
Swannell et al., 1996) and using TerraZyme (Oppenheimer Biotechnology) in enhancing
biodegradation of a heavy oil spilled from Nakhodka in Japan (Tsutsumi et al., 2000). However,
the success of these studies was based on either visual observation (i.e. the Mega Borg study) or
digital photographic image analysis (i.e., the Nakhodka study). No comprehensive monitoring
program was used to verify the oil was indeed removed through enhanced biodegradation. The
two products basically contain the same bacterial cultures and nutrients (Hozumi et al., 2000).
The observed visual effects might have been due to physical or chemical processes such as
surfactant action associated with the products (Swannell etal., 1996) or sinking.
All these peer-reviewed journal articles show that even though the addition of
microorganisms may be able to enhance oil biodegradation in the laboratory, the effectiveness of
bioaugmentation has not been convincingly demonstrated in the field. Actually, most field
studies indicated that bioaugmentation is not effective in enhancing oil biodegradation in inland,
estuarine, and marine environments. It appears that in most environments, indigenous oil-
degrading microorganisms are more than sufficient to carry out oil biodegradation if nutrient
levels and other adverse environmental conditions do not limit them.
2.3 Application of Biostiniulation Products
Biostimulation involves the addition of rate-limiting nutrients to accelerate the
biodegradation process. In most shoreline ecosystems that have been heavily contaminated with
hydrocarbons, nutrients are likely the limiting factors in oil biodegradation. The one exception is
wetlands. If oil has penetrated wetland or marsh sediment to any appreciable extent, the impact
zone is anoxic or anaerobic, and oxygen limitation will be the predominant mechanism
precluding effective treatment. Theoretically, approximately 150 mg of nitrogen and 30 mg
phosphorus are consumed in the conversion of 1 g of hydrocarbon to cell material (Rosenberg
and Ron, 1996). Therefore, a commonly used strategy has been to add nutrients at concentrations
that approach a stoichiometric ratio of C:N:P of 100:5:1. However, the practical use of these
ratio-based strategies remains a challenge. Particularly, in marine shorelines, maintaining a
certain nutrient ratio is impossible because of the dynamic washout of nutrients resulting from
the action of tides and waves. A more practical approach is to maintain the concentrations of the
limiting nutrient or nutrients within the pore water at an optimal range (Bragg et al., 1994;
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Venosa et a/., 1996). It is overwhelmingly evident from the literature that common agricultural
fertilizers would be the first choice of nutrient additives since they are both inexpensive and
readily available. However, because water-soluble nutrients are amenable to rapid washout,
attempts have been made to design nutrient delivery systems that overcome the washout
problems and to enhance nutrient availability for oil biodegradation. As a result, a few
commercial biostimulation products have been developed. Field studies on both common
agriculture fertilizers and commercial biostimulation agents are reviewed in the following
section.
2.3.1 Common agricultural fertilizers
2.3.1.1 Water-soluble fertilizers
Commonly used water-soluble nutrient products include mineral nutrient salts (e.g.
KNO3, NaNO3, NH4NO3, K2HPO4, MgNH4PO4), and many commercial inorganic fertilizers.
They are usually applied in the field through the spraying of nutrient solutions or spreading of
dry granules. This approach has been effective in enhancing oil biodegradation in many field
trials (Swannell et a/., 1996; Venosa et a/., 1996). One of the early field trials using common
commercial fertilizers was carried out in Spitsbergen, Norway in 1976 (Sendstad, 1980).
Forcados unweathered crude oil was released at a rate of 10 L/m2 on each of two 10 m2 plots.
One plot served as an oiled control, and the other was treated with an unspecified commercial
fertilizer at a concentration of 0.1 kg/m . A marked increase in microbial respiration rate was
observed in the fertilized plot compared to the control plot, suggesting that the application of
fertilizer stimulated oil degradation. However, the conclusion was questionable due to the
inadequate control and the lack of replicate plots in this study (Venosa, 1998).
Researchers from Fisheries and Oceans-Canada (Lee and Levy, 1987; Lee and Levy,
1989; Lee and Levy, 1991; Lee and Trembley, 1993; Lee et a/., 1995; and Lee et a/., 1997)
conducted a series of field tests to investigate the effect of different types of fertilizer and
different delivery strategies in a low energy, sandy beach or in a salt marsh. Their studies
demonstrated that biostimulation using periodic addition of inorganic fertilizers (ammonium
nitrate and triple super phosphate) increased the rate of oil removal from beaches as measured by
changes in oil composition relative to conserved biomarkers such as C2-chrysene and/or the
decline in the w-Cn/pristane and w-Cig/phytane ratios (Lee and Levy, 1987 and 1989). Another
study involved periodic addition of water-soluble fertilizer granules (ammonium nitrate and
triple super phosphate) in an attempt to enhance biodegradation of waxy crude oil in a low-
energy, sandy beach and in a salt marsh (Lee and Levy, 1991). Two concentrations of the
NH4NOs were tested (0.34 and 1.36 g/L sediment). The oil used was Terra Nova crude at two
different levels (0.3 and 3.0%). Results from the sandy beach showed that at the lower level of
oil contamination, no enhancement by fertilizer was achieved. However, at the higher oil
contamination level, substantial oil degradation occurred in the fertilized plots compared to the
unfertilized ones. Results in the salt marsh were the exact opposite. Enhancement by fertilizer
was significant at the 0.3% contamination level, but no enhancement occurred at 3% oil
contamination, which was attributed to the penetration of oil into the anaerobic zone where little
degradation is expected. Another field study conducted by Lee et al. (1995) compared the
performance of inorganic nutrients with organic fish bone-meal fertilizer. These results showed
that the organic fertilizer had the greatest effect on microbial growth and activity due to the
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presence of a readily biodegradable form of carbon in the bone meal, while the inorganic
nutrients were much more effective in stimulating crude oil biodegradation.
Recent studies found that the oil biodegradation rate depends on the nutrient
concentrations in the pore water of the sediments, which provides important guidance for
nutrient applications (Bragg et al, 1994; Venosa et al, 1996). This finding may also explain
why some earlier trials failed to demonstrate the effectiveness of nutrient application since the
nutrient concentrations in the interstitial pore water had not been monitored and controlled in
most of these studies. As mentioned earlier, the Delaware field study compared the effectiveness
of biostimulation with inorganic mineral nutrients with that of microbial inoculation in
enhancing the removal of crude oil. Venosa et al. (1996) found that maintenance of a threshold
nitrogen concentration of 1-2 mg N/L in the interstitial pore water would result in close to
maximum hydrocarbon biodegradation in a sandy beach. Another important conclusion from this
study was that background nutrient concentrations at the contaminated site should be a
determining factor in the decision to apply bioremediation. The background nitrogen
concentration at the Delaware beach was high enough to permit close to maximum hydrocarbon
biodegradation without the need to apply additional fertilizer despite the enhancement observed
from nutrient addition. The enhanced effect, although statistically significant, was not substantial
enough to have warranted a decision to implement bioremediation on a full-scale basis had there
been a real spill at this site. This demonstrates that nutrient amendment might not always be
necessary if sufficient nutrients are naturally present at a spill site in high enough concentrations
to perform natural cleanup.
This conclusion is mitigated somewhat by the need to consider the resources at risk. For
example, every spring in the Delaware Bay, horseshoe crabs come ashore to mate and lay eggs in
the intertidal zone of the beaches. Migratory birds making their way from South America to
Arctic Canada stop at this location to feed on the horseshoe crab eggs deposited in the sand. If an
oil spill were to take place a few weeks before this feeding season occurs, biostimulation might
be warranted despite the already high natural concentrations of nutrients present in the bay. Any
amount of acceleration of the disappearance of oil in order to protect these sensitive bird
populations would be justified. If a similar spill occurred in July, however, then bioremediation
would likely not be justified since the natural attenuation rate is expected to be high enough to
allow sufficient rates of biodegradation to take place with little likelihood of exposure of
sensitive species to the oil spill.
Field studies conducted in wetland environments showed that biostimulation was
ineffective in treating certain oil-contaminated salt marshes or freshwater wetlands due to
oxygen limitation (Garcia-Blanco et al, 2001; Shin et al, 1999; Venosa et al., 2002). In 1999
and 2000, a field study was conducted on the shoreline of the St. Lawrence River (Garcia-Blanco
et al, 2001; Venosa et al, 2002). The experimental design was similar to the one used on the
marine shoreline in Delaware Bay (Venosa et al, 1996). The four oiled treatments included: (A)
a natural attenuation control plot with no amendments; (B) a plot receiving ammonium nitrate
and orthophosphate nutrients but with the wetland plants continually cut back to ground surface
to suppress photosynthetic activity and growth; (C) a plot receiving the same nutrients as
Treatment B but with the plants left intact, and (D) a plot similar to Treatment C but with only
nitrate (no ammonium) serving as the nitrogen source. The results demonstrated that with respect
to biodegradation of total alkanes and PAHs during the first 21 weeks of the investigation as
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measured by GC/MS analysis, only about 35% biodegradation occurred in all treatments on
average, and no significant differences among any of the treatments were observed (p > 0.05).
The study also found that better biodegradation occurred in surface samples in plots where the
plants had been removed than in any of the core samples because of the oxic nature of the
surface and the lack of competition for nutrients by the plant species. The authors concluded that
nutrient amendment of an oil-contaminated freshwater wetland where significant penetration of
oil has taken place into the sediment has limited potential for enhanced cleanup of the
contamination. A similar result was obtained from a field trial conducted in a Louisiana salt
marsh (Shin et a/., 1999), in which natural attenuation of crude oil was as effective as nitrogen
(NH4NC>3) amendment, and oxygen availability appeared to control the oil biodegradation
process in salt marshes.
All these results suggest that the success of biostimulation is case specific, depending on
oil properties, the nature of the nutrient products and the characteristics of the contaminated
environments. When oxygen is not a limiting factor, one of keys for the success of oil
biostimulation is to maintain an optimal nutrient level in the interstitial pore water. To achieve
this under field conditions, especially in many estuarine and marine environments, frequent
nutrient applications are required when using water-soluble fertilizers, therefore, resulting in
more labor-intensive, costly, and physically intrusive operations.
2.3.1.2 Slow-release fertilizers
Use of slow release fertilizers is one of the approaches used to overcome washout
problems and provide continuous sources of nutrients to oil contaminated areas. Slow release
fertilizers are also readily available nutrient products normally in solid forms that consist of
either relatively insoluble nutrients or water-soluble nutrients coated with hydrophobic materials
such as paraffin or vegetable oils. This approach may also cost less than adding water-soluble
nutrients due to the need for less frequent applications. Slow release fertilizers have shown some
promise in oil bioremediation studies and applications. For example, following the Exxon Valdez
accident, a slow-release granular fertilizer, Customblen (Sierra Chemical Co.), was chosen as
one of the bioremediation agents to apply over 120 km of the oil-contaminated shorelines during
1989 and 1990. This fertilizer consists of vegetable oil coated calcium phosphate, ammonium
phosphate, and ammonium nitrate (N:P:K ratio 28-8-0). The results showed that Customblen
performed well on some of the shorelines of Prince William Sound, particularly in combination
with an oleophilic fertilizer, Inipol EAP22 (see next section) (Atlas, 1995; Pritchard et a/., 1992;
Swannelle/a/., 1996).
Several field studies have been carried out to evaluate the effectiveness of slow-release
fertilizers on enhancing oil biodegradation. A field test was carried out to evaluate the
performance of bioremediation by nutrient amendments for treating a mixture of Forties Crude
Oil and Heavy Crude Oil stranded on Bullwell Bay, Milford Haven, UK, after the grounding of
the Sea Empress in 1996 (Swannell et a/., 1999a&b). A randomized block design with triplicate
treatments was used to test the efficacy of two biostimulation amendments: a weekly application
of an inorganic nutrient solution (NaNOs and KH2PO4) and a single application of a slow-release
fertilizer. The slow release fertilizer pellets consisted of a mixture of inorganic nutrients (15-4.8-
13) with a coating derived from soya oil. Oil components were measured using GC/MS, and
hopane was used as a biomarker. Results showed that the addition of both liquid inorganic
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nutrients and the slow release fertilizer significantly stimulated natural oil biodegradation. After
two months, the oil in the nutrient-amended plots was 37% more degraded than that found in the
control plots. Because the slow-release fertilizer was much less labor-intensive to apply, the
study concluded that the application of slow-release fertilizers might be a cost-effective method
for treating low-energy, oil-contaminated shorelines.
A field study conducted in a tropical marine wetland in Australia also showed promise
for the application of slow-release fertilizers (Burns et al., 2000). In this study, four oiled
treatments (two types of oils with and without the bioremediation treatments) and two unoiled
controls (enclosure and ambient controls) were tested. Four replicate plots were used for each
treatment in the salt marsh plots. The bioremediation treatment consisted of sprinkling Osmocote
Tropical fertilizer at 0.15 kg m"2 40 hours after oiling. Other than total petroleum hydrocarbons
(TPHs), individual alkanes were also analyzed using GC-FID, and phytane was used as a
biomarker. The results indicated that although the predominant oil removal processes were
evaporation and dissolution, the addition of the fertilizer to the salt marsh plots stimulated the
degradation of the medium range crude oil (Gippsland), resulting in about 20% more oil loss as
compared with the untreated plots after 9 months. However, the nutrient amendment did not
significantly affect the rate of loss for the heavier Bunker C oil, confirming that the efficacy of
bioremediation is somewhat dependent on the type of oil to be treated.
Another field trial involving the application of a slow-release fertilizer was carried out on
an Arctic beach (Prince et al., 1999). Four treatments (tilled, tilled & fertilized, fertilized, and
oiled control) were evaluated on four unreplicated plots along a gravel shoreline contaminated
with a fuel oil (IF-30) near Sveagruva, Norway. A series of applications of various nutrient
products were performed for both fertilized plots during the first two months of study. These
nutrient products included a mixture of water-soluble fertilizers, yeast extract, and a slow-release
formulation (Inipol SP1, CECA, Paris La Defense), which contained 18% NH4 -N and 1% P as
P2O5. By 399 days after the first application of the fertilizers, changes in the chemical oil
compositions (ratio of phenanthrene to the dimethyl- and ethyl-phenanthrenes) suggested to the
investigators that biodegradation was significant. However, the extent of this preferential
removal of phenanthrene was not different among the two fertilized plots and the oiled control
(no statistical analysis was possible because of the lack of replicate plots). The authors concluded
that the biostimulation application was effective in enhancing oil biodegradation based only on
increased microbial activity (oxygen consumption) and biomass growth. This conclusion, which
is based on indirect evidence, may be somewhat questionable due to lack of replication in the
experimental design and the attempt to determine too many factors in a limited number of tests,
resulting in the confounding of different effects (e.g., the effect of yeast extract on enhancing
oxygen consumption).
The efficacy of biostimulation also depends on environmental factors such as
temperature, shoreline energy, substrate, and background nutrient concentrations. A field study
conducted by Lee et al. (1993) indicated that the effectiveness of specific nutrient formulations
might be influenced by temperature conditions. The study investigated the efficacy of water-
soluble inorganic fertilizers (ammonium nitrate and triple super phosphate) and a slow release
fertilizer (sulfur-coated urea) to enhance the biodegradation of a waxy crude oil in a low energy
shoreline environment. The results showed that at temperate conditions above 15ฐC, the slow-
release fertilizer appeared to be more effective in retaining elevated nutrient concentrations
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within the sediments and enhancing oil degradation than water-soluble fertilizers. However,
lower temperatures were found to reduce the permeability of the coating on the slow-release
fertilizer, and as a result nutrient release rates were suppressed. Water-soluble fertilizers, such as
ammonium nitrate, were recommended under these temperature conditions.
Oudot et al. (1998) evaluated the influence of a slow-release fertilizer (Max-Bac, Grace-
Sierra International) on the biodegradation rate of an Arabian Light crude oil contaminating an
estuarine environment in the bay of Brest, France. A randomized block design with five replicate
plots was used to examine the effects of two treatments (oiled control and fertilizer addition).
The slow-release fertilizer (10% NO3-N, 12% NH4-N and 13% P2O5) was applied monthly over
the 9 months field test. On average, 40% of the total oil, 83% of the aliphatics, and 55% of the
aromatics were biodegraded in all the plots at the end of the experiment. No significant
difference in oil biodegradation rates was observed between fertilized and non-fertilized plots
based on GC/MS analysis using norhopane as the normalizing biomarker. These results were
attributed to the high background levels of N and P at the study site. It was proposed that
bioremediation by nutrient enrichment would be of limited use if background interstitial pore
water levels of N exceed 1.4 mg/L, which is close to the levels found to be near optimum by
Venosaetal. (1996).
The physical forms of fertilizers are also important in selecting appropriate nutrient
products. Field trials conducted following the Exxon Valdez spill evaluated two types of slow
release nutrients: isobutylidene diurea (IBDU) briquettes and Customblen granules. The
application of the briquettes was problematic in regards to buoyancy of the briquettes and
redistribution by tide and wave action (Glaser, 1994; Glaser et al., 1991). The method used
during the Exxon Valdez spill involved packing the briquettes in mesh bags tethered to steel bars
driven into the beach subsurface. The poor distribution problem occurred by channeling of
nutrients vertically down the beach rather than lateral spreading. In contrast, Customblen
granules were evenly applied using a commercial broadcasting fertilizer spreader. Within two
weeks after the fertilizer application, the area of cobble beach treated with Customblen appeared
to be visibly cleaner than the untreated area (Pritchard et al., 1992).
The major challenge for the application of slow-release fertilizers is how best to control
the release rates so that optimal nutrient concentrations can be maintained in the pore water over
long time periods. For example, if the nutrients are released too quickly, they will be subject to
rapid washout and will not be a lasting source. On the other hand, if they are released too slowly,
the concentration will never build up to a level that is sufficient to support rapid biodegradation
rates, and the resulting stimulation will be less effective than it could be. The field trials on of the
shorelines of Prince William Sound showed that on certain beaches, Customblen granules were
apparently washed away before any significant enhancement of bioremediation was recorded
(Swannell et al., 1996). A recent mesocosm study by Sveum and Ramstad (1995) showed that a
slow release nutrient (Max Bac) failed to demonstrate enhancement of oil degradation because
the nutrient release rate was too low to affect oil biodegradation. Clearly, proper application of
slow release fertilizers could be a promising bioremediation strategy for stimulating oil
biodegradation.
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2.3.2 Commercial biostimulation agents
Compared to microbial products, very few nutrient additives have been developed and
marketed specifically as commercial bioremediation agents for oil spill cleanup. It is probably
because common fertilizers are inexpensive, readily available, and have been shown effective if
used properly. However, due to the limitations of common fertilizers (e.g., being rapidly washed
out due to tide and wave action), several organic nutrient products, such as oleophilic nutrient
products, have recently been evaluated and marketed as bioremediation agents. Four of the 15-
bioremediation agents listed on the NCP Product Schedule fall into this category (Table 2.1). The
rationale for using oleophilic organic nutrients is that oil biodegradation mainly occurs at the oil-
water interface, and since oleophilic fertilizers are able to adhere to oil and provide nutrients at
the oil-water interface, enhanced biodegradation should result without the need to increase
nutrient concentrations in the bulk pore water. This approach can also be used to overcome the
problem of water-soluble nutrients being rapidly washed out. Field evaluation results for some
of these products have been available in the peer-reviewed literature.
2.3.2.1 Inipol EAP22
Inipol EAP22 (Societe, CECA S.A., France) is currently listed on the NCP Product
Schedule as a nutrient additive and probably the most well-known bioremediation agent for oil
spill cleanup due to its use in Prince William Sound, Alaska. This nutrient product is a
microemulsion containing urea as a nitrogen source, sodium laureth phosphate as a phosphorus
source, 2-butoxy-l-ethanol as a surfactant, and oleic acid to give the material its hydrophobicity.
The claimed advantages of Inipol EAP22 include: 1) preventing the formation of water-in-oil
emulsions by reducing the oil viscosity and interfacial tension; 2) providing controlled release of
nitrogen and phosphorus for oil biodegradation; 3) exhibiting no toxicity to flora and fauna and
good biodegradability (Ladousse and Tramier, 1991).
Following the Exxon Valdez spill, Inipol EAP22 was chosen as one of the nutrient
products to use in the cleanup, and approximately 50,000 kg of nitrogen and 5,000 kg of
phosphorus were applied over 120 km of the oil-contaminated shorelines during 1989 and 1990.
Inipol EAP22 was selected also because it was the only commercially available bioremediation
agent with large production capacity at the time other than common agricultural fertilizers
(Pritchard et al., 1992). Visual observation seemed to suggest that the bioremediation agent
worked (Pritchard and Costa, 1991). However, the "window pane effect" observed within 2
weeks after application of Inipol to the beach was simply the result of oil having been lifted from
the cobble and re-deposited in the interstitial sand matrix between and under the cobbles.
Using hopane as the biomarker, Bragg et al. (1994) showed that fertilizer application
accelerated the rate of oil removal by a factor of approximately five-fold compared to natural
attenuation. However, conclusions on the effectiveness of bioremediation in the Exxon Valdez
experience are somewhat questionable, in part because the flawed experimental design was not
based on sound statistical principles (Venosa, 1998). Major flaws included the lack of
replication, inadequate sampling procedures, unequal treatment of controls and treated plots, and
an attempt to determine too many factors in a limited number of tests, resulting in the
confounding of different effects. The lessons learned from the Exxon Valdez project led to the
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replacement of "post Exxon Valdez excitement" with more scientifically-valid approaches
(Mearns, 1997).
Extensive studies have been carried out under various field conditions on the efficacy of
Inipol EAP 22 and have produced mixed results (Lee and Levy, 1989, Ladousse and Tramier,
1991; Sveum and Ladousse, 1989). Lee and Levy (1989) conducted a field trial to investigate the
effect of different types of fertilizers on enhancing the biodegradation of Scotian Shelf
condensate and Hibernia crude oil. The study occurred in the intertidal zone of a low-energy
sandy beach in Nova Scotia, Canada. The two nutrient products tested were Inipol EAP22 and a
mixture of 10:1:0 agricultural fertilizer. The study demonstrated that biostimulation using
periodic addition of the inorganic fertilizer increased the rate of oil removal from beaches as
measured by changes in oil composition relative to conserved biomarkers such as C2-chrysenes
and/or the decline in the w-Cn/pristane and w-Cig/phytane ratios. In contrast, the addition of the
oleophilic fertilizer, Inipol EAP 22, did not enhance oil degradation.
The effectiveness of Inipol EAP22 also depends on the characteristics of the
contaminated environment, such as action of wave and tide, and the effect of different sediment
types. Based on several field studies on the effectiveness of Inipol EAP22, Sveum et al. (1994)
indicated that this oleophilic fertilizer appeared to be more effective than water-soluble fertilizers
when the spilled oil resided in the intertidal zone. But they have no advantages in enhancing oil
biodegradation in the supratidal zone where water transport is limited. Inipol EAP 22 was found
to be more effective in coarse sediments than in fine sediments due to the difficulty in
penetration for the oleophilic fertilizer in fine sediments (Sveum and Ladousse, 1989), although
stronger evidence is needed to confirm this suggestion. Variable results have also been produced
regarding the persistence of oleophilic fertilizers. Some studies showed that Inipol EAP 22 could
persist in a sandy beach for a long time under simulated tide and wave actions (Santas and
Santas, 2000; Swannell et al. 1995). Others found that Inipol EAP22 was rapidly washed out
before becoming available to hydrocarbon-degrading bacteria (Lee and Levy, 1987; Safferman,
1991).
2.3.2.2 BIOREN
Researchers from European EUREKA BIOREN program recently conducted a field trial
in an estuary environment to evaluate the effectiveness of two bioremediation products
(BIOREN 1 and 2) (Le Floch et a/., 1997 and 1999). The EUREKA BIOREN project was an
international effort to develop commercial nutrient products able to enhance hydrocarbon
biodegradation on contaminated shorelines. The two nutrient products are derived from fish
meals in a granular form with urea and super phosphate as nitrogen and phosphorus sources and
proteinaceous material as the carbon source. The major difference between the two formulations
was that BIOREN 1 also contained a biosurfactant. To reduce the effect of physical removal of
oil and nutrients, the study was conducted on a sheltered sandy beach in a small estuary in
Brittany, France. A light Arabian crude oil was used as the model contaminant in this field trial.
Four treatments (unoiled control, oiled control, BIOREN 1, and BIOREN 2) were randomly
assigned to four experimental plots (5m x 5m), and four smaller oiled control plots (2m x 3m)
were also set up along side the treated plots. The nutrient products were applied twice over the 4-
month study (once immediately after oiling and again two weeks after). The results showed a
"starter effect" for the BIOREN 1 formulation: biodegradation was significantly enhanced during
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the first five weeks of the experiments based on the analysis of the nCn/pristane and
nCig/phytane ratio. However, after five weeks, the enhancement was reduced and "significant
differences" were no longer observed between treatments at the end of the test. The authors
concluded that the BIOREN 1 hold promise for accelerating microbial activity immediately after
an accidental oil spill.
The results seem to suggest that the biosurfactant in BIOREN 1 was the most active
ingredient that contributed to the increase in oil degradation rates since BIOREN 2, which
contained no surfactant, was not effective in that respect. The biosurfactant could have
contributed to greater bioavailability of hydrocarbons to microbial attack. It would have been
better if the investigators had used replicate treatments because this would have enabled them to
calculate experimental error. Results would have been stronger in support of conclusions made.
Other studies on the effect of similar organic fertilizers derived from natural products
also yielded mixed results. A field trial conducted by Lee et al. (1995a) compared the
performance of inorganic nutrients with an organic fish bone-meal fertilizer on the
biodegradation rates of Venture Condensate in a sandy beach environment. The results showed
that the organic fertilizer had the greatest effect on microbial growth and activity, while the
inorganic nutrients were much more effective in stimulating crude oil biodegradation. One of the
problems with these types of fertilizers is that they contain organic carbon, which may be
biodegraded by microorganisms in preference to petroleum hydrocarbons (Lee et al., 1995a;
Swannell et al., 1996), which may lead to undesirable anoxic conditions (Lee et al., 1995b;
Sveum and Ramstad, 1995).
2.3.2.3 Oil Spill Eater IIฎ (OSE II)
Oil Spill Eater IIฎ (Oil Spill Eater International, Corp.) is another nutrient product listed
on the NCP Schedule (U.S. EPA, 2002). This product is listed as a nutrient /enzyme additive
and consists of "nitrogen, phosphorus, readily available carbon, and vitamins for quick
colonization of naturally occurring bacteria". A field demonstration was recently carried out at a
bioventing site in a Marine Corps Air Ground Combat Center (MCAGCC) in California to
investigate the efficacy of OSEII for enhancing hydrocarbon biodegradation in a fuel-
contaminated vadose zone (Zwick et al., 1997). The selection of OSEII was base on the findings
from a previous microcosm study, in which various amendments were evaluated by monitoring
microbial respiration using soils collected from the site. The results suggested that fertilizer
amendment, not bioaugmentation, might be cost-effective for accelerating biodegradation rates at
this field site (Alleman and Foote, 1997). Although groundwater environments and subsurface
hydrocarbon contamination are generally not within the scope of this review, this article was
included because it is the only peer-reviewed paper available on a NCP-Schedule-listed
bioremediation nutrient additive, other than numerous publications on Inipol EAP 22.
At beginning of the test, air was pumped into vent wells for 36 hrs to achieve oxygen
concentrations of over 20% at the site. Groundwater was injected at 20 ft below ground surface
(bgs), while OSE II solution was pumped to 30-40 ft. bgs through two monitoring points. OSE II
was also injected into two monitoring points in an uncontaminated area of the site to act as
product controls. An On-line Environmental Monitoring System (OEMS) was used to conduct
in-situ measurement of O2 and CO2 at depths of 10, 20, 30, and 40 ft bgs. Hydrocarbon
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degradation rates (as mg hexane per kg of soil per day) were calculated based on oxygen
utilization rate, and CC>2 data were used to verify biodegradation. The effect of OSE II was
monitored for about three months after the initial application and the results were compared to
those obtained before the OSE II injection when irrigation was conducted for over a year.
The oxygen and CO2 data showed that an increase in respiration rates occurred shortly
after the addition of OSE II, especially at the 30-ft bgs level, indicating an increase in microbial
activity as a result of OSE II addition. The extent of the rate increase was much higher than that
measured at the uncontaminated background site receiving OSE II, suggesting the BOD
associated with the product may not be the main cause for the increase in the respiration rates at
the contaminated site. However, the respiration rates at both 10 ft bgs (oiled-control) and 20 ft
bgs (groundwater irrigation) levels at the contaminated site were also higher than the pre-test
levels, suggesting that factors other than the OSE II addition might also be responsible for the
increase in oxygen utilization rates. These factors could be the pre-aeration at the beginning of
the study (the control site had not been pre-aerated as the test site was), changes in
environmental conditions, or differences in geology at the various depths., No statistical analysis
was carried out to test the significance of the findings in this study. Although respiration rates
may be an indirect measure of product effectiveness, proof of effectiveness comes from
measurement in decline of hydrocarbons, which was not discussed in the report. Although this
field trial suggested that irrigation and OSE II addition might have enhanced microbial activities
in the deeper soils at the site, the report was inconclusive in regards to direct evidence that
hydrocarbons were degraded.
In summary, the effectiveness of these various types of nutrient formulations will depend
on the characteristics of the contaminated environment and of the formulations themselves.
Slow-release fertilizers may be an ideal nutrient source if the nutrient release rates are well
controlled. Water-soluble fertilizers are likely more cost-effective in low-energy and fine-grained
shorelines where water transport is limited. Oleophilic fertilizers may be more suitable for use in
high-energy and coarse-grained beaches or less accessible rocky outcroppings. Successful
application of bioremediation products will always require appropriate experimental design,
testing, and evaluation based on the specific conditions of each contaminated site.
2.4 Summary
Peer-reviewed literature on the use of bioremediation products has clearly indicated that
biostimulation, if used properly, could be a cost-effective treatment tool for cleaning certain oil-
contaminated environments. Important findings and lessons learned from these studies are
summarized as follows.
Bioaugmentation appears to have little benefit for the treatment of spilled oils in an open
environment. Microbial addition has not been shown to work better than nutrient addition
alone in many field tests. However, application of bioaugmentation products could still
have some potential in the treatment of specific oil components or isolated spills in
confined areas, although more evidence is still required to verify this notion.
Bioremediation with addition of nutrient products has been proven to be an effective tool
to treat certain aerobic oil-contaminated marine shorelines. Typically, it is used as a
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polishing step after conventional mechanical cleanup options have been applied, although
it could also be used as a primary response strategy if the spilled oil does not exist as free
product and if the contaminated area is remote enough not to require immediate cleanup or
not accessible by mechanical equipment.
Effectiveness of biostimulation is also highly site-specific. When oxygen is not a limiting
factor, one of the key factors for success is to maintain an optimal nutrient level in the
interstitial pore water. In other words, background nutrient concentrations at the
contaminated site should be a determining factor in the decision to apply bioremediation,
and biostimulation might not always be necessary if sufficient nutrients are naturally
present at a spill site in high enough concentrations to permit effective microbial treatment.
Availability of oxygen is often the limiting factor in wetland environments, where addition
of nutrient products has not been successful in enhancing oil biodegradation. If the oil is in
the aerobic zone of a wetland sediment (upper few mm) and if background nutrients are
low, then biostimulation may still be an effective cleanup strategy. Even if oil has
penetrated into the anaerobic zone, biostimulation may at least allow for faster recovery of
the wetland vegetation.
Nutrient products have shown variable effectiveness, depending on oil properties, the
nature of the nutrient products, and the characteristics of the contaminated environments.
In general, commercial oleophilic nutrient products have not shown clear advantages over
common agricultural fertilizers in stimulating oil degradation.
As this review has pointed out, many field tests have not been properly designed, well
controlled, or correctly analyzed, leading to skepticism and confusion among the user
community when selecting response options (Venosa, 1998). Future field studies should
devote more energy and investment to adopting scientifically valid approaches and
acquiring the highest quality data possible.
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3 Assessment of Oil Bioremediation Products: Non-Peer Reviewed Literature
As indicated in the previous chapter, only limited field studies and applications of
bioremediation products have been reported in the peer-reviewed literature. In an attempt to
better document the potential and understand the scope of the actual use of bioremediation
products, a thorough search of non-peer-reviewed and 'gray' literature pertaining to the use of
bioremediation agents in response to oil spills in inland, estuarine, and marine environments has
also been conducted. A comprehensive review of this information is presented in this chapter,
which includes the review of government agency reports, vendor reports, and vendor client
reports. Section 3.1 presents a thorough review of field trials of bioremediation products based
on various government agency reports. Section 3.2 summarizes the results of an information
collection effort from manufacturers and vendors of bioremediation products and provides an
assessment of the efficacy of some bioremediation agents based on the gathered information.
The potential of using bioremediation products in the areas of non-point source such as storm
water runoff countermeasures and bilge oil treatment will be discussed in Section 3.3.
3.1 Government Agency Reports
Many government agencies have been involved in various aspects of oil spill cleanups.
At the federal level, oil spill response planning is coordinated through the U.S. National
Response Team (NRT, www.nrt.org). This is an interagency group made up of 16 federal
agencies and co-chaired by the EPA and the U.S. Coast Guard (USCG), each with
responsibilities and expertise in various aspects of spill response. For example, EPA is in charge
of coordinating oil spill response in inland environments; the USCG coordinates oil spill
prevention and response in the coastal zone; and National Oceanic and Atmospheric
Administration (NOAA) as well as EPA provide national coordination with respect to scientific
support. In this project, many of the governmental agencies that have likely been involved in
evaluating and using bioremediation approaches in oil spill responses were contacted and/or their
publication websites were searched. These governmental agencies include U.S. EPA Oil Spill
Office, USCG, NOAA, Department of Energy, Department of Interior, the U.S. Navy, U.S.
Army Corps of Engineers, and various state agencies. From this search, a number of government
reports pertaining to the use of commercial bioremediation products were obtained. Although
some of this literature has already been covered in other reviews, such as Swannell et al. (1996),
Venosa (1998), and Zhu et al. (2001), they will still be discussed within the context of
effectiveness of commercial bioremediation agents. Again, all this literature will be reviewed
based on the two main bioremediation approaches, bioaugmentation and biostimulation.
3.1.1 Application of bioaugmentation products
Several field studies or applications on the use of commercial bioaugmentation agents
have been published in government agency reports with mixed results. Mearns (1991) reported
on a bioaugmentation field test of an oiled marsh in an estuary environment of upper Galveston
Bay, Texas. The spill occurred in August 1990, when a collision occurred between three Apex
Barges and the Greek tanker Shinoussa. The test was conducted 8 days after the spill, which
resulted in the release of approximately 700,000 gallons of catalytic feedstock (a partially refined
oil). Four plots were used in selected areas of Marrow Marsh: two treated with a commercial
bioaugmentation product (Alpha BioSea, Alpha Environmental, Inc.), and two left untreated
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as controls. The product consisted of a bacterial culture in a cornstarch carrier and a
nutrient/micronutrient mixture. It was prepared by premixing with ambient brackish water, and a
diluted stock solution was sprayed on the two marsh test plots. Oil constituents were determined
using GC/MS and the extent of biodegradation was measured by the decline in the n-
C18/phytane ratio in this study. During only a 4-day monitoring period, results of the chemical
analysis indicated no apparent difference in the extent of oil biodegradation between treated and
untreated plots, although no statistical analysis was performed. Mearns speculated that either the
oil had too low a content of degradable components or insufficient bioremediation agent was
used. However, the major deficiency of this study was that 4 days were insufficient for reaching
any convincing conclusion on the effectiveness of bioremediation. Biodegradation is a relatively
slow process, and usually weeks or months are needed before significant microbial activity may
take effect. Mearns et al. (1993) later summarized some lessons learned from this experiment,
which included that bioremediation is not a rapid response tool, experimental design should meet
basic statistical requirements, and more comprehensive monitoring is needed to demonstrate the
efficacy of treatment.
In another NOAA report, Hoff (1991) described a bioaugmentation project along a
California shoreline. On October 31, 1990, a well blowout offshore of Seal Beach, CA, released
approximately 400 gallons of crude oil resulting in the contamination of 2-3 acres of marsh
grasses in the Seal Beach National Wildlife Refuge. A bioremediation treatment that consisted of
the hand spraying of a microbial product used in wastewater treatment plants (INOC 8162) and a
commercial fertilizer (Miracle Gro 30-6-6) was carried out one week after the incident, followed
by an application of the fertilizer alone two weeks later. Oil degradation was monitored based on
the measurements of 14C-mineralization and most probable number counts of bacteria. No
difference was observed between treated and untreated salt marsh plots 35 days after the initial
treatment, suggesting neither bioaugmentation nor biostimulation with nutrient addition worked
in this case. However, no reporting of nutrient concentrations was provided, so it was difficult to
determine if sufficient nutrients were available to allow biodegradation to take place.
Nonetheless, the result is consistent with the finding by others that oxygen availability is likely
the limiting factor for oil biodegradation in wetland environments (Shin et al., 1999; Simon et
a/., 1999; Venosa etal., 2002).
A pilot-scale project was carried out recently to test the efficacy of a bioremediation
procedure in treating soils contaminated by petroleum oils and lubricants (POLs) at an Army
installation at Fort Hood, TX (U.S. Army, 1999). POLs are common contaminants on
Department of Defense (DoD) installations, and the U.S Army is increasingly being asked to
comply with more stringent regulations for disposal of these wastes. The treatment procedure
tested in this study included the addition of a commercial bioremediation agent, BET BioPetro
(BioEnviroTech, Inc.), at 1 lb/ydJ of contaminated soil and an agricultural fertilizer (24-8-8) at a
rate of 0.5 lb/ydJ. After the addition of both microbial and nutrient products, the contaminated
soil was tilled and then watered at a rate of 1.5 in/week. BET BioPetro is one of the
bioremediation agents listed on the NCP Product Schedule (Table 1.1). According to the
vendor's description, BET BioPetro is "a powder containing granules of bacterial product
formulated to provide performance in the bioremediation of heavy refined and crude
hydrocarbon contaminants in both soil and water environments" (U.S. EPA, 2002). In this field
trial, the bioremediation procedure described above was tested for six months at three sites
contaminated by JP8 fuel oil, and the results were successful in terms of oil degradation based on
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TPH analysis. For example, six months after the initial treatment was applied, TPH
concentrations decreased dramatically from the initial values of 10,100 - 13,100 mg/kg to the
final results of 195 - 1,170 mg/kg at Site 2. Treated soils were able to meet the reuse
requirements for the final disposal to sanitary landfills. Unfortunately, no control was set up
during this study; therefore, no conclusion could be reached in regard to whether this apparent
drop in oil concentration was due to the addition of BET BioPetro or any other treatments (i.e.,
nutrient addition, tilling, or watering). The lesson from this project again demonstrates how
critical a proper experimental design is in testing the effectiveness of bioremediation.
Bioremediation experience at U.S. Navy's Naval Facilities Engineering Service Center
(NFESC) also suggested that bioaugmentation is generally not needed for treating fuel/oil
contaminated soils because of the ubiquity of hydrocarbon degraders in nature. However,
according to personnel at the NFESC, microbial amendment may have limited use for treating
specific contaminants or in specific environments. An example was given of the potential
usefulness of a specific microbial product. A bioremediation project that involved adding a
commercial bioaugmentation agent to a biopile was conducted at a naval air station (NAS) in
Fallen, Nevada in 2002 (personal communication with personnel at the NFESC). The soils were
contaminated with a mixture of fuels (mostly aged gasoline and JP-5). Sulfate and sulfide levels
in these soils were also very high. When aeration was imposed by means of a vacuum pump, the
soil temperature increased dramatically to about 70 C, probably due to high oxidation rates of the
sulfides. The naval investigators surmised that because of the high temperatures caused by the
forced aeration conditions, bioaugmentation might be helpful to enhance hydrocarbon
biodegradation in a subsequent treatment step. A bioaugmentation treatment was carried out by
Pintail Systems (Denver, CO), which involved the application of a product consisting of a
mixture of microbial isolates from soils at the site plus some cultures isolated from an acid mine
drainage site. Three months after the application of the microbial product, TPH concentrations in
the soils dropped dramatically from pre-treatment levels of about 2,000 mg/kg to about 200
mg/kg. Again, no control pile was established to help demonstrate the effectiveness of the
bioaugmentation treatment. Therefore, this result in no way proves or even suggests that the
bioaugmentation product used was effective. It is common that compost and biopiles can be
highly effective in treating organic waste products, and temperatures that are attained can reach
very high levels.
3.1.2 Application of biostimulation products
Several government agency reports of field experience with evaluation and use of
commercial nutrient products are available. Included among them are several articles that cover
bioremediation experience following the Exxon Valdez oil spill (Bragg et a/., 1992; Pritchard et
a/., 1991; Venosa et a/., 1990). Field studies on the use of nutrient products have produced
mixed results. Hoff (1991) summarized a field trial conducted by Exxon Research & Engineering
Company with technical support from DuPont Environmental Remediation Services, on Frail's
Island, New Jersey. The study was carried out between September and December 1990 to assess
the effectiveness of a slow-release fertilizer (Customblen, Sierra Chemicals) following a pipeline
leak of No.2 fuel oil on a beach at the Frail's Island bird sanctuary in January 1990. The slow-
release fertilizer was placed in two shallow trenches about 4 to 6 inches bellow the surface in the
intertidal zone. A no-treatment control plot was set up next to the treated zone. According to
Hoff (1991), the results were inconclusive, in part due to the high variability in the TPH levels
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within the treated and control areas and the lack of replicated plots. Hoff also suggested that
either cross contamination of nutrients might have occurred between the two treatment zones or
high background nutrient levels might have masked effects of nutrient addition. The ammonium
concentration in the interstitial pore water at the control plot was about 1 mg/L, which is close to
the minimum nitrogen level in pore water that would permit close to maximum hydrocarbon
biodegradation (Venosa et a/., 1996). It should also be noted that in a company report, prepared
by Exxon Research & Engineering Company and DuPont Environmental Remediation Services
(Madden et a/., 1991), a different conclusion was reached based on the same experimental data.
The company report indicated that "fertilizer addition clearly accelerated the rate of
biodegradation", although it admitted that the controls were not sufficient to provide a clear side-
by-side comparison between treated and untreated zones. The conclusion was based mainly on
the marked decrease in average TPH within the treated plots three weeks after the nutrient
amendment. Unfortunately, neither of these two reports provided any statistical analysis in
regard to the experimental data to support their conclusions. Of course, statistical analysis would
have been impossible anyway since neither the control plots nor the treated plots were replicated
and randomly situated in the intertidal zone. Again, this controversy demonstrates the importance
of proper experimental design and data interpretation in bioremediation evaluation. For a
detailed description of proper protocol for oil bioremediation field studies and evaluation,
readers may refer to EPA's Guidelines for the Bioremediation of Marine Shorelines and
Freshwater Wetlands (Zhu et a/., 2001).
In a manual on treating oil spills in tundra environments published by Alaska Department
of Environmental Conservation, Athey et al. (2001) compiled dozens of case studies on the
experience of oil spill cleanup in Alaska. Among these cases, five involved the use of
bioremedial approaches, mostly biostimulation with agricultural fertilizers. The results showed
that nutrient amendments have generally been successful in reducing hydrocarbon levels in the
tundra soil. For example, in August 1989, a crude oil spill from a leaking valve on a production
line occurred in the North Slope oilfields. After pooled oil was vacuumed, fertilizers (8-32-16 at
215 Ibs/acre and 34-0-0 at 185 Ibs/acre) were applied during spring/summer of 1990 and 1991,
respectively. Following the nutrient treatment, the mean soil TPH concentration decreased 92%
by 1993 and 96% by 1996. Similar results were obtained in two other cases that used the same
fertilizers. In another case, a field trial was carried out to evaluate different revegetation methods
following a crude oil spill from a check valve station near Prudhoe Bay. Five treatments were
established: 1) cover with clean material, 2) remove contaminated material and replace with
clean material, 3) till with a concrete rake, and 4) apply an unidentified oil degrading bacterial
product, 5) no treatment. These plots were then seeded with vegetation and fertilized with a 14-
30-14 agricultural fertilizer. The study found that the most effective treatment in terms of both
oil degradation and grass yield was the combination of tilling and fertilization. The result also
suggested that biostimulation with nutrient addition alone was more effective than microbial
seeding in this arctic tundra environment.
3.2 Vendor's Reports
To better understand the scope and potential of the use of bioremediation products,
efforts were made to collect information from vendors of bioremediation agents and their service
providers. A summary of the results of this information collection and a comprehensive review
of these non-peer reviewed articles are presented in this section.
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3.2.1 Information collection
In February 2002, 70 vendors of bioremediation agents that are listed either on the NCP
Schedule or on 20* International Oil Spill Control Directory (Oil Spill Intelligence Report,
2000) were contacted through emails or letters in regard to their interest in participating in our
case study review (see Appendix A, Initial Letter Calling for Information). A follow-up letter
listing the detailed information requested was then issued to each company that positively
responded to our initial letter (see Appendix B, Follow-Up Letter to Participating Companies). A
total of eight vendors of bioremediation products were willing to participate in this investigation
and submitted at least some the information requested. The name, major bioremediation
products, and contact information of these participating companies are listed in Table 3.1.
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Table 3.1 List of Companies That Participated in This Review
Manufacture or Vendor
Bioremediation Product(s)
Contact Information
Enviro-Zyme, Inc.
BR, formerly
ENVIROZYME BR
P.O. Box 169
Stormville, NY 12582
Tel: 1-800-882-9904
info@envirozyme. com
Forrester Environmental
Technologies Corp. (FET
Group)
BioCATalystIOS-500
P.O. Box 3652
Vero Beach, FL 32964
1-407-758-9033
envirotec@earthlink.net
Garner Environmental Services,
Inc.
Petro-Clean
1717 West 13th Street
Deer Park, TX 77536
1-800-424-1716
wsbiosolve@> aol. com
Industrial Wastewater Solution
IOS-500
P.O.Box 157
Sebastopol, CA 95473
1-707-824-1282
IWS@somc.net
Medina Agriculture
Products Co., Inc.
Medina Microbial Activator
Bio-D Nutrients
Hydrocarbon D-Grader
P.O. Box 309
Hondo, TX 78861
1-830-426-3011
medina@medinaag. com
Petrol Rem, Inc.
PRP (Petroleum Remediation Product)
(a/k/a WAPED)
2275 Swallow Hill Road
Bldg 2500
Pittsburgh, PA 15220
1-800-246-2275
info@petrolrem.com
Verde Environmental, Inc.
Micro-Blaze
7309 Schneider
Houston, TX 77093-8501
1-800-626-6598
bscogin@micro-blaze.com
WMI International, Inc.
WMI-2000
4901 Milwee Suite 109
Houston, TX 77092
1-800-460-45074
wmi@wt.net
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3.2.2 Summary of case studies submitted by vendors
A complete list of materials submitted by the eight vendors is shown in Table 3.2. A
brief description of their major bioremediation product(s) and a summary of selected case studies
for each company are presented here, following the alphabetical order of the companies' names.
It should be noted that these cases do not necessarily reflect all cleanup efforts of each company.
They are selected based on relevancy to the scope of this report, sufficient information provided,
and scientific merit.
3.2.2.1 Enviro-Zyme, Inc.
Product(s)
BR (formerly ENVIRO-ZYME BR) is the major oil bioremediation product
manufactured by Enviro-Zyme, Inc. This product was listed on the NCP Product Schedule as a
biological addictive when this report was written. According to the company, "BR" contains
sufficient types of microorganisms enriched to degrade oil, aliphatic, and aromatic chemical
pollution in soil and aqueous environments. BR also contains nutrient additives and a surfactant.
It is a dry solid product with a shelf life of 1 year. BR should be mixed with water and applied
through a low-pressure spray nozzle.
Case studies
Information on several pilot-scale and full-scale applications of BR was provided by the
company for each case, which included the use of BR to treat wastewater containing oil and
grease in two full-scale cases and to treat a BTEX-contaminated soil in one pilot study. A
summary of these relevant case studies is described as follows.
Bioremediation of BTEX: A pilot test was conducted in central Florida to evaluate the
effectiveness of BR in treating a surface soil contaminated by a hydrocarbon waste from a
dye manufacturer. The primary contaminants in this waste were benzene, toluene,
ethylbenzene, and xylene (BTEX). The results showed that 7 months after the application
of BR, the removal efficiencies for the benzene, ethylbenzene and toluene were essentially
100%, while the xylene reached 88% removal. No control was reported in this study.
Bioaugmentation treatment of oil and grease: A wastewater from a bus installation in
Washington D.C. contained high levels of grease and oil. The average concentration of
grease and oil in the waste holding tank was 21,800 mg/L, which was an unacceptable
discharge to the municipal sewage system. BR was then added daily to the grease and oil
holding tank in conjunction with aeration. After six weeks of the treatment, the grease and
oil level decreased to an average of 1,200 mg/L. Similarly, BR was also used to assist in
treating a wastewater containing oil and grease from a New York railroad yard. The
industrial wastewater was normally treated in an aeration lagoon. However, during the
winter season, treatment efficiency declined, and the lagoon could no longer meet the State
Pollution Discharge Elimination System (SPDES) Permit limits. The wastewater used to
be bypassed to a holding basin until spring. However, with the addition of BR to the
influent of the aeration lagoon at a rate of 2 pounds per day during the winter season, the
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effluent has successfully met the SPDES permits for BOD, TSS, and grease & oil, and has
been allowed to discharge into the Hudson River all year-round.
3.2.2.2 Forrester Environmental Technologies Corp. (FET Group)
Product(s)
BioCATalyst (a.k.a. Sheen Solution, Bio Cat 2001, BIOCAT VFB) is a bioremediation
product custom manufactured for FET Group by Biocat VFB Solutions Company, Inc., Toccoa,
GA. This product is a kelp extract modified by a surface-active agent and other natural materials.
The main active ingredient is a cytokinin extract, which is a plant hormone known to be a plant
growth enhancer.
According to a FET Group's technical bulletin, BioCATalyst "works in two ways on oil
spills": dispersion and biostimulation. The product contains a natural plant surfactant and
emulsifier, which acts to break up oil into droplets, suspending the droplets into the water
column. Indigenous bacteria are claimed to be biostimulated by the cytokinin such that their
metabolic processes are greatly enhanced, resulting in much quicker biodegradation of
substrates. BioCATalyst is normally applied as a fine spray. It is also recommended to be mixed
on site with nutrient solutions supplied by the manufacturer.
Case studies
Chemical analysis reports on two full-scale applications of BioCATalyst were provided
by FET Group, which involved treating petroleum hydrocarbon contaminated water in Florida.
One of the reports came with a one-page case description, which is summarized as follows.
At an orange grove in central Florida, a leak of fuel tanks used for orange blossom heaters
led to heavy soil contamination by diesel and gasoline. After the removal of the
contaminated soil, groundwater flowed into the excavation and also became contaminated
with petroleum hydrocarbons. The 163,000 gallons of water was first treated with the
chemical oxidant potassium permanganate. However, this approach was unable to meet the
Florida DEP requirements. BioCATalyst was then applied at a rate of approximately 10
gallons per day over a period of 14 days. The analysis results (EPA method 610/8100)
showed that hydrocarbon concentrations decreased dramatically after the treatment, and
the site was able to meet the required State levels. For example, the concentrations for
benzene, xylene, and naphthalene were reduced from 42, 335, and 22 (ig/L to 1, 1 and 5
|j,g/L , respectively, in 14 days. Similar results were obtained using BioCATalyst for the
treatment of another open pit of contaminated aquifer water in a second analytical report.
3.2.2.3 Garner Environmental Services, Inc.
Product(s)
Garner Environmental Services, Inc. is a primary distributor of "Petro-Clean", a product
manufactured by Alabaster Environmental Corp., Pasadena, TX. According to the product
catalog, Petro-Clean contains surfactant, nutrients, and hydrocarbon degrading bacteria. This
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product is currently listed on the NCP Product Schedule as a surface-washing agent based on one
of its main active ingredients - a surfactant. On the other hand, it is also marketed as a
bioremediation agent by vendors since it also contains active microbes (estimated by the
manufacturer at approximately 50 billion per gallon). The main microbial species are naturally
occurring Bacillus spores. Petro-Clean is normally applied through power washers or even
garden type sprayers in diluted solution. More information in regard to this product can be found
on the web site of EPA Oil Spill Program (http://www.epa.gov/oilspill).
Case studies
The manufacturer provided information on four full-scale applications of Petro-Clean.
Three of these cases involved the cleanup of petroleum hydrocarbon contaminated soil, and the
other concerned the treatment of creosote-contaminated soil. TPH or creosote concentrations
before and after Petro-Clean treatments were provided for all the cases, which showed apparent
reduction of the contamination levels after the treatments. One of the case studies that involved
the cleanup of petroleum hydrocarbons and included a detailed sampling layout is summarized as
follows.
Bioremediation of a Petroleum Compressor Station: A severe petroleum hydrocarbon spill
occurred due to a leak from three compressor units. The soil/gravel site was approximately
six acres with heavy contamination on three acres. The bioremediation treatment involved
the following techniques: 1) wet vacuum removal of surface liquid contamination; 2)
surface cleaning with Petro-Clean; 3) subsurface injection with Petro-Clean; and 4) tilling
in absorbents with Petro-Clean. Three sampling locations that covered various
contamination levels were set up to monitor the performance of the treatment. Before the
bioremediation operation, TPH concentrations for the three sampling sites were 11,000,
147, 000, and 130,000 mg/L, respectively. The results showed that within 2 to 6 weeks
after the treatment, the levels at all three sites were reduced to a permitted TPH level,
which was less than 10,000 mg/L.
3.2.2.4 Industrial Wastewater Solutions Corp.
Product(s)
Industrial Wastewater Solutions Corp. (ISW) is a corporate spinoff from International Organic
Solutions (IOS), which is the manufacturer of a microbial product, called IOS-500 (US Patent
#5.531.898). According to materials provided by ISW, which consults for IOS on
bioremediation projects, IOS-500 is a blend of facultative bacterial species that use organic
compounds as a primary food source and is effective in both aerobic and anaerobic
environments. Based on the patent description (Wickham, 1995), IOS-500 also contains an
enzyme mixture and an organic nutrient source. This product has been used for treatment of
contaminated soils, remediation of contaminated bodies of water, and in agricultural, industrial
and domestic wastewater treatment systems. The product is normally mixed with water for
about 6 to 48 hours at ambient temperature to produce an acclimated mixture, and then applied to
the contaminated environment.
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Case studies
The manufacturer provided a table that lists 28 remediation cases using IOS-500 for
treating hydrocarbon-contaminated soils. The information in the table includes clients' names
and/or locations, types of oil/hydrocarbons, duration of the treatment, amount of the
contamination, and the TPH levels before and after treatment. The package submitted by IWS
also contained more detailed information on three cases that were conducted in Mexico for oil-
spill cleanup using IOS-500, which included a one-page description for each case, pictures from
the field, and letters of acceptance/acknowledgement from the customers. A brief summary of
these three cases is presented as follows:
Case #1: This case study was a demonstration at the request of a State-owned Oil
Company in Mexico to prove the ability of the IOS product and process to provide rapid
degradation of heavily contaminated areas. The project involved the remediation of 25
cubic yards of 50-year-old weathered crude oil in mud and soil. After 21 days of IOS-500
treatment, the TPH level was reduced by 89%, although no concentration data and
sampling plan were described.
Case #2: IOS-500 was used to cleanup 5,000 ydJ of wetland soils contaminated by a 50-
year-old spill of crude oil and tar. The contaminated soil was first excavated from the
wetland and then mixed with IOS-500 using small front-end loaders, a water pump, and
hoses. The TPH concentration for the untreated soil was 436,000 ppm on average (number
of samples and replication unknown). After 42 days of treatment, the soil TPH level was
reduced to 4500 ppm.
Cases #3: This case study involved the remediation of 10,000 ydJ clay, sand and mud type
soil contminated by a fuel oil. Using similar land treatment procedure as in Case #2, TPH
concentrations were reported to decrease from 80,000 to 452 ppm within 60 days. It was
also noted that this job was completed during the rainy season.
3.2.2.5 Medina Agriculture Products Co., Inc.
Product(s)
Medina bioremediation products include Medina Microbial Activator or Soil Activator,
several nutrient formulations and microbial blends. Medina Microbial Activator is a liquid
formulation derived from a controlled fermentation process and contains compounds that
stimulate microbial activities. Nutrient products include NP-1000 and MS 100-plus-NP for water
phase bioremediation, and Bio-D for soil remediation. Bio-D is a liquid product containing
organic humic substances, as well as inorganic nitrogen, phosphorus and potassium. Microbial
products include a granular formulation (Hydrocarbon D-Grader) and a liquid formulation
(HCD). Medina also offers a "Spill Response and Bioremediation Kit", which includes Medina
Microbial Activator, Bio-D nutrients, Bio-C organic solvent, Hydrocarbon D-Grader, Petrosorb,
plastic bags and sheet, and manual.
A general procedure (material concentrations unspecified or not reported) for using the
kit to treat oil-contaminated soil was provided, which includes: 1) spray Bio-C solvent on the
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affected area if the spill is old; 2) till the soil to the depth of the oil penetration; 3) mix
Hydrocarbon-D-Grader with water and spray over affected area, 4) mix Medina Microbial
Activator and Bio-D together with water, spray over affected area, tilling the treated area, 5) add
water on a weekly basis to keep treated area moist.
Case studies
Information on several pilot and full-scale applications of Medina bioremediation
products was provided, which include two journal articles (Shafer 1991 & 1992) and three
"Bioremediation Reports" (a two-page company fact-sheet) on the performance of Medina Soil
Activator, and an abstract of a company report on a soil bioremediation project using an organic
nutrient product. A summary of these case studies is described as follows.
Pilot study on effectiveness of Medina Soil Activator: In October and November 1989, a
seven week pilot study was carried out by Woodward-Clyde Consultants (Denver, CO) on
a site in Oakland, CA to evaluate the effectiveness of three bioremediation methods for
treating an oil contaminated soil. The three treatments included the addition of 1) Medina
Soil Activator, 2) an emulsifier plus a multiple-nutrient fertilizer, and 3) an emulsifier, a
fertilizer, and proprietary hydrocarbon-degrading microorganisms. The contaminated
material was placed in three wooden frame plots (6.5 cubic yards per pile) for testing the
three treatments respectively. No untreated-control was set aside in this study nor were any
replicate plots set up. After the applications of these bioremediation products, each plot
was tilled three times for the first four weeks. Two composited samples were taken from
each plot for TPH analysis at 2nd, 4th, and 7th weeks after the treatment. The results showed
that all three treatment methods were able to reduce TPH concentrations from 605 mg/kg
on average to at or below 200 mg/kg level. The Medina Soil Activator plot achieved the
highest hydrocarbon removal with an average TPH concentration of 145 mg/kg by week 7.
Because Medina Soil Activator was also the least expensive product, it was proposed as
the choice of the treatment for the cleanup of this site.
Bioremediation of diesel contaminated soil and tundra: In the early 1980s, a diesel fuel
pipeline ruptured at a U.S. Air Force station in arctic Alaska. During the summers of 1989
and 1990, two bioremediation approaches were applied by Woodward-Clyde Consultants
using Medina Microbial/Soil Activator to remediate the contamination. The first approach
was to clean up the contaminated soil using a land treatment unit (LTU). The diesel-
contaminated soil was excavated and moved to a nearby LTU. The average initial TPH
concentration was 11,500 mg/kg (number of sample replicates unknown). A surfactant
solution was first applied to the soil to make the oil more available. Then a diluted Medina
soil activator was applied once every two weeks. The soil was also turned once a week to
aerate the LTU. At the end of the six-week period, TPH in the soil was reduced by 42
percent. The treatment was resumed in the summer of 1990. The land treatment process
achieved 75% TPH removal during these two treatment periods. To reduce the disturbance
to natural vegetation in the arctic tundra, an in-situ treatment approach was also tested
during the same period, which involved surface-spraying a surfactant and Medina soil
activator. Overall TPH levels were reduced from an average of 19,000 mg/kg to 8,300
mg/kg (57% removal) based on analytical results of four composite samples from
randomly selected locations in the affected area.
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Soil bioremediation using an organic nutrient product: Approximately 100 cubic yards of
diesel-contaminated soil was treated using a Medina organic nutrient product in a Coca-
Cola distribution facility in California. The contaminated soil was treated with this
unspecified Medina nutrient product five times between November 2, 1990 and April 23,
1991 in a land treatment unit. TPH concentrations were reduced from an average of 240
ppm to less than 10 ppm (EPA method 418.1).
3.2.2.6 Petrol Rem, Inc.
Product(s)
Petrol Rem is the manufacturer of PRP (a/k/a WAPED), a bioremediation product listed
as an Enzyme Additive on the current NCP Product Schedule. According to materials submitted
by Petrol Rem, PRP consists of tiny spheres of treated wax, which contain nutrients. As
mentioned earlier in Chapter 2, PRP also contains nonpathogenic bacteria within wax particles
although they are mostly not oil degraders (Lee et al, 1997; NET AC, 1993b). When PRP is
sprayed as a loose powder, it can absorb twice its weight of oil and form a physical matrix that
floats on water, thereby preventing the pollutants from sinking and limiting the transport of oils
to more sensitive areas. The matrix then provides an environment that uses naturally occurring
microorganisms in the water to degrade the pollutant as well as PRP itself. The mechanisms of
enhancing oil biodegradation for this product, however, are still not well understood (NETAC,
1993b).
PRP is available in three forms: 1) powder form for treating open water spills, 2)
BioBoom for oil spills that require containment, and 3) BioSok for treating spills in enclosed
areas such as boat bilges.
Case studies
Information on several pilot and full-scale applications of PRP products was provided,
including a 50-page report from the National Environmental Technology Applications Center
(NETAC, 1993b) on the effectiveness of PRP in a mesocosm field study, a report from GMS
Technologies (1999) on the evaluation of the BioSok product in a microcosm study, and
company reports of two field trials. A summary of these case studies is described as follows.
Bioremediation Product Evaluation: Mesocosm Field Study PRP Formulation #1 (NETAC,
1993b): A 21-day mesocosm study was conducted in a Petrol Rem testing facility to
evaluate the performance a PRP product. Three treatments were tested in three equal size
tanks (10x3x4 ftJ), where a gallon of fresh diesel fuel was added to each tank, and water
from a natural source was pumped through continuously to simulate a flowing fresh water
stream. The three treatments were 1) absorbent control (two one-pound sleeves of
polypropylene absorbent), 2) no treatment control, 3) BioBoom + BioSok + PRP powder.
GC/MS was used for hydrocarbon analysis, and the ratio of C17/pristane used as the
measure of biodegradation effectiveness. The study concluded that PRP significantly
stimulated the degradation of the hydrocarbon slick as compared to the absorbent and no
treatment controls. Evidence of biodegradation was observed based on the slight decrease
in C17/pristine ratio in the PRP tank. However, the report also pointed out that the PRP
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mode of action for the enhanced biodegradation (whether due to the added nutrients or
beeswax material) was still unclear. One deficiency of this study was the lack of treatment
replicates (only three tanks were used to test the three treatments). An inspection of the
GC/MS data shown in the report revealed that the disappearance of all hydrocarbons
appeared to take place at the same rate, suggesting that biodegradation may not have been
the primary cause for the disappearance of the hydrocarbons. Support for this conclusion
comes from a 3-D histogram of the data, from which it was very difficult to read
concentrations.
Evaluation of the BioSok product for boat bilge treatment: This study was conducted to
test the effect of three treatments (untreated control, mini-BioSok with encapsulated oil-
degrading Bacillus sp., and mini-BioSok without addition of bacteria) on remediation of
bilge water using diesel fuel and bench-scale Bilge Model Reactors. Although this study
was not conducted under field conditions, it is mentioned here because the test did help to
understand the role of microorganisms in PRP. The results showed that the overall oil
removal efficiencies were 71%, 84%, and 77% on average in four weeks for the cases of
natural biodegradation, PRP without microbes, and PRP with encapsulation of microbes,
respectively. A statistical analysis (Student's T-test) indicated that there was a significant
difference between both PRP formulations and the no treatment control (p < 0.10), but no
significant difference between the two PRP formulations (p > 0.10). However, since the
treatments were not replicated, the statistical analysis was flawed since such an analysis
requires replicate treatment units in order to calculate experimental error. Even if the
results were statistically significant, the question remains if the incremental 13% benefit
from using PRP justifies the cost of treatment compared to natural attenuation. The study
further demonstrated that PRP relied on indigenous bacteria, not encapsulated ones, to
perform the oil biodegradation.
Field applications of PRP: Petrol Rem provided two real-world case studies. In both cases,
only visual observation was used to monitor the performance of PRP treatment. In one case
study, a barge carrying 4,000 metric tons of fuel oil sank in January 1998, resulting in
fouling beaches and a mangrove along the Persian Gulf in Abu Dhabi. PRP was tested on
site and visual observations (10 pictures enclosed in the report) found that the treated
sections of the contaminated beach and mangrove showed marked improvement compared
to untreated areas. Another case involved heavy oil spilled into a lagoon in Mexico in
1994. PRP was used to treat the water surface and the shore. After 26 days, it was
estimated that approximately 75% of the original oil had been degraded based on visual
examination of oil coverage on the lagoon surface (6 pictures enclosed in the report).
However, use of visual observations with no attendant chemical analysis is not a reliable or
scientifically sound method of assessing the treatability of a site by any technology.
3.2.2.7 Verde Environmental, Inc.
Product(s)
Verde Environmental, Inc. is the manufacturer of Micro-Blaze", another oil
bioremediation product listed on the NCP Product Schedule. Micro-Blaze is a liquid formulation
of several microbial strains (Bacillus spores), surfactants, and nutrients designed to metabolize
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organics and hydrocarbons in soil and water (http://www.epa.gov/oilspill). Its commercial name
is also called Micro-Blaze Emergency Liquid Spill Control. When in use, Micro-Blaze should be
diluted with water to a certain percentage as determined by the type of contamination and
applied through normal spray devices.
Case studies
Information on over a dozen full-scale applications of Micro-Blaze was provided in the
Handbook for Suggested Uses and Applications of Micro-Blaze Emergency Liquid Spill Control,
with one to a few pages of description for each case. The applications include hydrocarbon spill
control, remediation of leaking underground petroleum storage tanks, wastewater treatment, and
cleanup of firefighting training fields and food preparation sites. Two case studies that are
relevant to this document are summarized as follows.
Spill responses in a refinery plant: One anonymous major refinery plant frequently uses
Micro-Blaze to cleanup its petroleum spills. For example, Micro-Blaze was used to
remediate a crude oil spill in a tank-field in June 1997. TPH levels dropped from the low
thousands to low hundreds of mg/kg in approximately eight weeks. In another case, a
diesel spill in a pipe conduit occurred in November 1997. After the free product was
pumped out, Micro-Blaze was used to finish the cleanup. In approximately four weeks,
TPH concentrations dropped from a range of 25,000 - 57,000 mg/kg to a range of 600 -
9,900 mg/kg. Again, no controls were reported nor were treatments replicated.
Gasoline spill clean up: A major gasoline spill (approximate 6000 gals) occurred in
Conroe, Texas, on January 27, 1994, resulting in the contamination of the city sewage
system. The wastewater treatment plant was shut down in the early morning and the
gasoline-contaminated wastewater was diverted to a holding pond. Micro-Blaze
Emergency Liquid Spill Control was sprayed to the basins and the lagoon in the
wastewater treatment plant. Within ten minutes, the fumes disappeared. The wastewater
treatment plant was able to operate at half capacity in the early afternoon and went back to
normal by midnight. No analytical data, however, were presented in this case to
demonstrate the effectiveness of Micro-Blaze as well as to explain the mode of action for
this product. It is known that bioremediation is too slow acting to account for such a quick
a hydrocarbon spill. It is likely that, since the spill was gasoline, most if not all of the
treatment occurred as a result of volatilization.
3.2.2.8 WMI International, Inc.
Product(s)
WMI International's major oil bioremediation product is WMI-2000. This product is also
listed on the NCP Product Schedule as a microbial additive. WMI-2000 is a dry power that
contains microbial cultures specifically selected for remediation of petroleum products and other
contaminants. The product may be applied as a dry powder or activated in water for 2 hours and
applied to the surface of oil spills in water, pits, or ponds. It is also recommended by the
manufacturer to apply nutrients with WMI-2000 microbes and maintain nitrogen and phosphorus
concentrations in the treated water at 5-20 mg/L and 1-5 mg/L, respectively.
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Case studies
Information on several pilot and full-scale applications of WMI-2000 was provided,
which includes brief descriptions of three case studies and a company report with detailed
analytical results on bioremediation of bilge water. A summary of two of these case studies is
described as follows.
Treatment of a hydrocarbon-contaminated storm water storage pond: WMI-2000 was used
to clean up a hydrocarbon contaminated storm water storage pond for a major U.S.
railroad. The pond was aerated with WMI Fine Bubble Diffusers and inoculated with
WMI-2000 microbes. Visual observation was used to monitor the treatment performance.
The results were illustrated using a series of pictures, which showed the reduction of oil
coverage and the improvement of water clarity from Day 1 through Day 7 of the treatment.
Again, since this study relied on visual observations for measurement of effectiveness,
results must be discounted.
Bioremediation of hydrocarbon contaminated water in ballast and bilge tanks: MWI-2000
was used to treat the ballast tank of a ship that was contaminated with coatings of heavy
hydrocarbons, especially on the interior walls. During the first week of the treatment, 36
Ibs. of WMI-2000 and 100 Ibs of commercial fertilizer (12-24-0) were added in the ballast
tank with a capacity of 179,000 gallons. Another 8 Ibs of WMI-2000 each week was
applied between the 2nd and 4th weeks. Forced air aeration of the bilge water was also
applied continuously as part of the treatment. Analytical results using GC-FID showed that
during the six weeks of the treatment, the concentrations of the various hydrocarbons were
reduced from a range of 0.2- 4.3 mg/L to ND or 0.2 mg/L (detection limit), although no
data was reported about the oil adhered to the interior walls of the vessel.
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Table 3.2 Summary of materials submitted by participating companies
Manufacturer
or Vendor
List of submitted material
Enviro-Zyme,
Inc.
Product line cards: description, direction and MSDS about all Enviro-
Zyme Products (BR, C, COMP, DOC, EZ, GT, L, LGD, M, N, O, P,
POUCH, R, SEP, T)
A copy of "BR Composite" that includes:
Detail information about BR (primary ingredients, pH range, nutrient
requirement, etc.)
General considerations and summaries of application for the use of
bioaugmentation products for assisting the cleanup of oil spills or soil
contamination.
Brief description of four case studies on the use of BR
A journal paper promoting the use of bioaugmentation: Jensen, R.A.
(1996) Bioremediation using bioaugmentation, Environmental
Technology, 11/12, 1996
Forrester
Environmental
Technologies
Corp. (FET
Group)
Information about BioCATalyst --a biostimulation product from botanical
extraction.
FET Group technical bulletins about BioCATalyst
BODS test results from BSC (Biocatalyst Solution Company)
MSDS
Letter of acceptance from The Bureau of Petroleum Storage System,
Department of Environmental Protection, Florida, for injection type of
aquifer remediation
Brief description of two case studies on the use of BioCATalyst with the
attachment of hydrocarbon analysis reports
Garner
Environmental
Service, Inc.
A catalog on Petro-Clean microbial products that includes:
Petro-Clean products facts
Brief description of four case studies
Direction for use of Petro-Clean
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MSDS
Industrial
Wastewater
Solutions Corp.
Medina
Agriculture
Products Co.,
Inc.
A detailed letter of qualifications through email, which describes:
Company background and its product IOS-500
A list of field applications and treatment results
Areas of applications
A CD-ROM containing further information about IOS-500:
Introduction of IOS-500
Brief descriptions of three case studies
Letters of acceptance/acknowledgement from customers
Pictures from the field
Letter of recognition of non-pathogenicity for IOS-500 from Health
Care Service of Alameda County, CA
Brochures regarding Bioremediation Division of the company, its
products and services.
A more detailed Medina Bioremediation Catalog, which includes:
Procedure for using bioremediation products and an example of a land
farming project
Bioremediation compatibility testing
Product guide
List of support service and price quotes
Domestic customer list
MSDS
Summaries of three case studies on the effects of Medina Soil Activator
Detailed abstracts of two company reports on a lab treatability study and a
field trial on Medina bioremediation products.
Two technical journal articles that involved the use of Medina
bioremediation products:
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Shafer, R. (1992). "Bioremediation of a California Land Site: a cost-
effective way to treat oil contamination." Our California Environment,
winter 1992.
Shafer, R. (1991). "Cleanup old problems: Bioremediation of diesel
contaminated soil and tundra." Land and Water, Nov/Dec, 1991
PetrolRem, Inc
A PRP portfolio, including a PRP Bioboom-Biosock Product Video CD
Reports on PRP from governmental agencies and Petrol Rem's partners
Review worksheet on PRP by U.S. Coast Guard's Alternate Response
Tool Evaluation System (ARTES) program
National Environmental Technology Applications Corporation
(NETAC) (1993b). "Bioremediation Product Evaluation: Mesocosm
Field Study PRP Formulation #1."
QMS Report (1999). "Evaluation of the BioSokฎ product for boat bilge
treatment and the reduction of non-point pollution," a research report by
QMS Technologies to USEPA under EPA contract No. 68-D-98-138
Larry Lawson (1999). Excerpt from the Report to GMS Technologies on
Commercialization Assessment of Improved Bio-Sok by Foresight
Science & Technology under contract to the EPA
Company reports on two case studies
Mexico field study results
Abu Dhabi field test results with a letter of recognition by the customer
Verde
Environmental,
Inc.
Three introduction and training videos
Micro-Blaze microbial products for use in wastewater/septic systems
Micro-Blaze emergency liquid spill control (training video)
Micro-Blaze Out Microbial fire fighting agent (training video)
Handbook for suggested uses and applications, which include several
documents and case histories, as well as regulatory agency acceptance
letters
Acceptance letters from the states of Florida and Ohio that have been
received since the manual was put together
A manual of technical papers, which include bioremediation background
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information and several biodegradability studies
A manual of toxicity, bioremediation tests, and chemical analysis, all of
which was required by the EPA for Micro-Blaze to be placed on the EPA
NCP product Schedule as a bioremediation agent
Various promotional flyers.
WMI
International,
Inc.
Summary of three case studies on the effects of WMI-2000 microbial
products
A company report: Bio-Remediation of Hydrocarbon Contaminated water
in Ballast and Bilge Tanks, which include
Treatment schedule in ballast tanks
Hydrocarbon analysis report
MSDS of WMI-2000
Toxicity test of WMI-2000
Letters from EPA and Department of Health & Human Services
3.2.3 Review of vendor reports
As shown in the previous section, the amount and quality of the submitted information
was highly variable. Case study information mostly ranged from a few sentences to one to two
pages, although there were also a few detailed technical reports of up to 50 pages. It is
impossible to give a comprehensive review of each case based on this limited information.
Therefore, a summary of important findings and general critique on the technical merit of all
these reports are presented here.
Bioremediation products have been applied to clean up petroleum hydrocarbon
contamination in various ecosystems and under a wide range of environmental conditions.
Their applications include in-situ remediation of hydrocarbon contaminated marine
shorelines, soil environments, surface water, groundwater, and bilge water, and ex-situ
treatment of hydrocarbon contaminated soil (e.g., using a land treatment unit) and water
(e.g., in a bioreactor). However, most of these cases involved treating relatively small-scale
petroleum hydrocarbon contamination in somewhat confined environments (e.g., lagoons,
land treatment units, ships, etc). The bioremediation applications were used as either a
primary response strategy or a secondary polishing step after conventional mechanical
cleanup options had been applied to remove free oil products. Oil and hydrocarbon
contamination were observed to be reduced based on TPH analysis and visual
observations. The submitted materials seem to suggest that these bioremediation products
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have satisfied many customers and have been able to meet regulatory requirements for
their clients.
A major limitation of these vendor case studies is that due to the confounding of different
effects, it is impossible to determine whether the claimed enhanced oil biodegradation, if
true, resulted mainly from the addition of microbial cultures, nutrients, enzymes, oxygen,
or any combination of above. Among the ten bioremediation products described in the
vendors' reports, six were bioaugmentation agents that contain hydrocarbon degraders (i.e.,
BR, Petro-Clean, IOS-500, Medina Hydrocarbon D-Grader, Micro-Blaze, and WMI-2000)
and four were biostimulation agents containing either enzymes or nutrients but no active
hydrocarbon degraders (i.e., BioCATalyst, Medina Microbial Activator, Bio-D Nutrients,
and PRP). All of these microbial products contained nutrients and surfactants or required
applying with nutrient products. In most of the reported field applications
(bioaugmentation or biostimulation), certain types of oxygen amendment were used as
well, such as tilling of contaminated soil or forced air aeration of polluted water.
Therefore, no conclusion can be made solely based on these reports in regard to the
determination of the limiting factors (microbes, nutrients or enzymes) for oil
biodegradation, although in some cases there was some evidence that microbial
amendments did not enhance oil biodegradation better than biostimulation (GMS Report,
1999; Shafer, 1992).
The technical merit of these company reports was generally not sound in terms of
providing strong or even suggesting moderate scientific evidence for demonstrating the
effectiveness of bioremediation products. As described in Chapter 2, evidence for the
effectiveness of oil bioremediation in terms of oil biodegradation should include: (1) faster
disappearance of oil in treated areas than in untreated areas, and (2) a demonstration that
biodegradation was the main reason for the increased rate of oil disappearance. To obtain
evidence of increased rate of disappearance, a set-aside untreated area or a control should
be used, which has similar physical and biological conditions as the treated site. However,
no controls were used in most of the reported case studies. Although oil spill responders
prefer not to set aside any oiled untreated sites, it is difficult to assess the true impact of a
treatment without control or set-aside areas. To effectively distinguish biodegradation from
abiotic loss, specific oil components or analytes should be analyzed occasionally using
GC/MS techniques. These analytes should be normalized to a conserved biomarker, such
as hopanes and/or alkyl-substituted chrysenes. Again, except for the report on the
mesocosm test of PRP (NETAC, 1993b), none of these cases used GC/MS analysis. In
most of the cases, oil/hydrocarbon concentrations were evaluated by simple TPH analysis.
For others, visual observation was the only method of monitoring the treatment
performance. Since all TPH techniques are severely affected by spatial heterogeneity, it is
essential that a well-thought out sampling plan be designed according to valid statistical
principles involving randomization and replication of treatments in order to ensure that
monitored results reflect reality in such a highly heterogeneous environment,.
Unfortunately, very little if any statistical analyses were conducted to support the
conclusions and claims made by the vendors and writers of those reports. Considering
current regulatory requirements and the cost of the hydrocarbon analysis, it may not be
realistic for oil spill responders to conduct detailed GC/MS analyses during oil spill
bioremediation applications (except perhaps an occasional sample). However, in order to
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provide more convincing evidence of product effectiveness, the oil bioremediation industry
can and should do more to acquire the highest quality data possible within budget
restraints, such as following sound statistical principles of experimental design and
adopting a well-designed sampling plan.
3.3 Bioremedial Approaches for Controlling Petroleum Hydrocarbons in Stormwater
and Bilge Water.
One of the objectives of this review was to evaluate and assess the use of bioremediation
technology for the cleanup of hydrocarbon contamination from storm water discharges and boat
bilges. After a thorough search of the literature, however, little information was available on the
field experience pertaining to the use of bioremediation agents for treating petroleum
hydrocarbons in stormwater and bilge water. Therefore, the potential of using bioremediation
products in these non-point sources can only be briefly discussed as follows.
3.3.1 Bioremediation of hydrocarbon contamination from storm water discharges
It is estimated that about 0.12 metric tons of petroleum hydrocarbons are released into the
world's water through urban runoff every year, which make up approximately 4% of oil input
into the oceans (NAS, 1985). Urban runoff is also the leading source of impairments to surveyed
estuaries according to recent National Water Quality Inventory reports (U.S. EPA; 1996).
Technologies that are currently used as Best Management Practices (BMPs) for
controlling hydrocarbons in stormwater include oil and grease trap devices, wet detention ponds,
wetland systems (phytoremediation), and filter systems (Botts et al. 1996, Shutes et al., 1997,
Schueler, 1987, U.S. EPA, 1997). Trap devices for removing oil and grease from stormwater
include various mechanical oil-water separators, oil and grease skimmers, and water quality
inlets. The skimmer is often used at the outlet of a sediment basin. Water quality inlets consist of
a series of chambers or basins that remove sediment, screen debris, and separate free oil from
storm water. Wet detention ponds maintain a permanent pool of water in addition to temporarily
holding stormwater, and they provide both quality and quantity control of storm water.
Hydrocarbons in stormwater are degraded through natural attenuation. Constructed or restored
wetlands are also effective means of controlling low-level hydrocarbon contamination, which use
green plants and their associated rhizosphere microorganisms to degrade and contain pollutants.
Nix et al. (1994) investigated the performance of a wetland system for treating storm runoff
containing diesel fuel and found that 96% of the total extractable hydrocarbons were removed
after only five hours retention. Another bioremedial technology for treating stormwater is
compost stormwater filters (CSF). This innovative system removes contaminants from
stormwater by allowing water to pass through layers of specially tailored compost. A CSF can
typically remove over 90% of all solids and 85% of oil and grease (U.S. EPA, 1997).
Although the approach of adding biostimulation and/or bioaugmentation agents has not
been selected as BMPs for the treatment of hydrocarbon contamination in stormwater, limited
information gathered suggests that application of bioremediation agents could be a promising
approach, especially used in conjunction with other stormwater countermeasures, such as wet
detention ponds. For example, BR (Enviro-Zyme, Inc) was used to assist in treating an industrial
runoff containing oil and grease in an aeration lagoon during winter seasons, when the existing
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treatment system could not meet the regulatory standard (see Section 3.2.2.1). WMI-2000 also
treated hydrocarbon-contaminated stormwater in a wet detention pond, but this reported success
was based on visual observation (see Section 3.2.2.8). However, further field tests are still
required to evaluate the effectiveness of this strategy, particularly to determine whether addition
of microbial cultures is necessary or effective in enhancing oil biodegradation in stormwater.
3.3.2 Bioremedial approaches for treating bilge oil
Bilge oil discharge is another major source of petroleum contamination for the world's
navigable waters. It is estimated that bilge oil comprises 9% of oil input into the oceans (NAS,
1985). Currently international regulation does not allow ships to discharge bilge water containing
more than 15 mg/L of oil. Some of the BMPs for reducing the amount of oil from boat bilges
entering marinas and surface waters include: promoting the installation and use of fuel/air
separators on air vents; avoiding overfilling fuel tanks, maintaining proper engine performance
and routinely checking for fuel leaks; promoting the use of materials that capture or digest oil in
bilge water; extracting used oil from absorption pads if possible; and prohibiting the use of
detergents and emulsifiers on fuel spills (U.S. EPA, 2001).
Commonly used methods for in-situ treatment of bilge water include oil water separators,
such as centrifuges, for large vessels and absorption devices, also called "bilge socks" or "bilge
pads", for smaller recreational boats. Some of these products are also combined with bioremedial
processes. For example, a "bio-mechanical" oil water separator, trademarked as PetroLiminator,
was developed recently by Ensolve Biosystems, Inc. (Raleigh, NC). This device is a non-
pressurized three-stage vessel: Stage 1 allows for initial separation of heavy or pure oil. The
retained emulsified oil is then biodegraded in a separate biofilm reactor at Stage 2. Stage 3
consists of a final clarifier for solids removal, and the effluent is then discharged. PetroLiminator
has been tested in sea trials (MarineLog.com, 2001) and has been type-approved by U.S. Coast
Guard (USCG) and the International Maritime Organization (IMO).
Another bioremedial method for treating bilge oil is the use of absorption pads that may
also contain bioremediation agent(s). BioSok is one such product. According to the
manufacturer, PetrolRem, each BioSok, which measures 9 inches in length by 3 inches in
diameter, contains eight ounces of PRP. It is purported to absorb up to one pound of contaminant
and degrade much more oil over time. As mentioned previously, BioSok achieved about 80%
diesel removal in a microcosm study, slightly better than the performance of natural attenuation.
However, no scientifically verifiable field data in regard to the rate and capacity of oil
biodegradation for this product is available.
Considering the simplicity of use and environmental friendliness of the processes,
products like BioSok could be a promising solution for bilge water treatment. However, this type
of product also faces tough competition from other absorption devices. Tests conducted by
consumer-oriented organizations often emphasize the oil absorption capacity and the firmness of
the oil binding (Boat U.S. Foundation, 2001; Costa, 2000), which normally is not the strength of
the bioremedial type of bilge pads. Therefore, more work needs to be done in terms of both
market improvement and technical demonstration of these bioremedial sorbents for them to reach
their potential in the area of bilge oil treatment.
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4 Summary and Findings
Bioremediation is a process that attempts to accelerate natural biodegradation processes.
The success of oil spill bioremediation depends on our ability to optimize various physical,
chemical, and biological conditions in the contaminated environment. There are two main
approaches to oil spill bioremediation: 1) bioaugmentation, in which oil-degrading
microorganisms are added to supplement the existing microbial population, and 2)
biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of
nutrients or other growth-limiting cosubstrates and/or habitat alteration. Bioremediation agents
are also classified as bioaugmentation and biostimulation agents based on these two main
bioremedial approaches. Since the objective of this document was to conduct a thorough
assessment of bioremediation products by a comprehensive review of their actual use in real
world cases, it was hoped that documented field experiences would be able to provide the more
convincing argument for the effectiveness of bioremediation technology. Literature reviewed
included peer-reviewed journal articles, company reports, government reports, and actual reports
by cleanup contractors engaged in the response to spills in inland, estuarine, and marine
environments. The key findings of this literature review are summarized bellow:
Bioremediation products have been applied to clean up petroleum hydrocarbon
contamination in various ecosystems and under a wide range of environmental conditions.
Their applications include in-situ remediation of hydrocarbon contaminated marine
shorelines, soil environments, surface water, groundwater, and water, and ex-situ treatment
of hydrocarbon contaminated soil (e.g., use of land treatment units or other types of reactor
systems such as compost piles, biopiles, slurry reactors, etc.) and water (e.g., in a
bioreactor). Bioremediation technology is typically used as a secondary polishing step after
conventional mechanical cleanup options have been applied to remove free oil product.
However, many case studies have demonstrated that bioremediation can also be used as a
primary response strategy, especially for the cleanup of environmentally sensitive areas
that are not amenable to conventional cleanup techniques and/or low-level petroleum
hydrocarbon contamination.
According to the peer-reviewed literature, bioaugmentation appears to have little benefit
for the treatment of spilled oil in an open environment. Microbial addition has not been
shown to work better than nutrient addition alone in many field trials. However, case
studies provided by vendors seem to suggest that application of bioaugmentation products
could still have some potential in the treatment of specific oil components, isolated spills in
confined areas, or certain environments where oil-degrading microorganisms are deficient.
Unfortunately, the evidence for such a conclusion is not strong and in most cases
scientifically deficient.
Biostimulation has been proven to be a promising tool to treat certain aerobic oil-
contaminated marine shorelines. One of the key factors for the success of oil
biostimulation is to maintain an optimal nutrient level in the interstitial pore water. In other
words, background nutrient concentrations at the contaminated site should be one of the
primary determining factors in the decision to apply nutrients, and biostimulation might
not always be necessary if sufficient nutrients are naturally present at a spill site to supply
non-limiting concentrations to the degrading populations. However, effects of nutrients are
44
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also highly site-specific. For example, the availability of oxygen rather than nutrients is
often the limiting factor in wetland environments, where addition of nutrient products has
not been successful in enhancing oil biodegradation (although it has been successful in
accelerating the restoration of the affected plant biomass to an abundant and rich recovery).
Different nutrient products have shown variable effectiveness, depending on oil properties,
the nature of the nutrient products, and the characteristics of the contaminated
environments. Based on limited field trials, it appears that slow-release fertilizers may be
an excellent choice if the nutrient release rates are balanced against physical loss rates;
water-soluble fertilizers may be more cost-effective in low-energy shorelines and fine-
grained sediments where water transport is limited; and oleophilic fertilizers may be more
suitable for use on hard, rocky shorelines, although further research is still required to
confirm this suggestion. In general, commercial oleophilic nutrient products have not
shown clear advantages over common agricultural fertilizers in stimulating oil
biodegradation.
Bioremedial approaches may have a role in treating hydrocarbon contamination for non-
point sources. Limited available information appears to suggest that application of
bioremediation agents could show promise for the treatment of hydrocarbon contamination
in stormwater, especially used in conjunction with other stormwater countermeasures, such
as wet detention ponds. Bioremediation agents may also be effective for the treatment of
bilge water, although, due to the lack of any systematic investigation into its effectiveness,
it is still uncertain whether this approach could compete with other existing technologies.
Further field tests are needed to provide stronger evidence on the potential of this strategy.
The extreme uncertainty associated with the efficacy of bioremediation agents is due in
large part to the poorly designed field tests that have been conducted to demonstrate
efficacy. Much of the reported literature lacked proper controls and treatment
randomization and replication, or the data were incorrectly analyzed. If there is any hope
for advancement of commercial bioremediation for the environments described in this
report, especially estuaries, experiments based on sound scientific principles are needed.
Unfortunately, resources for field-testing commercial bioremediation agents are scarce,
and field studies are extremely expensive to carry out. That's why it is best to rely on
laboratory microcosm or mesocosm studies to provide needed data to support this
technology. When spills occur and the on-scene coordinator in conjunction with the
Regional Response Team decides to implement commercial bioremediation for cleanup,
they should try to set aside control areas if at all possible to allow a more effective
evaluation of treatment success. If this practice is carried out, a true advancement in
knowledge will be possible.
If there is any hope for advancement of commercial bioremediation, especially estuaries,
experiments based on sound scientific principles are needed. Unfortunately, due to the
extreme resource intensiveness of field studies, the benefit accruing to testing one
bioremediation agent is only applicable to the one product being tested, not to the overall
science of bioremediation. Testing products in the field is not within the purview of the
federal government unless such a test has the potential of advancing science in terms of
general microbiological and engineering principles.
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Appendix A: Initial Letter Calling for Information
Dear Madam/Sir:
I am a research associate with the University of Cincinnati's Department of Civil and Environmental Engineering,
working on a contract with the U.S. Environmental Protection Agency's Office of Research and Development. I am
conducting an in-depth literature review on the efficacy of commercial bioremediation products for cleaning up oil-
contaminated environments, with special emphasis on estuaries. The objective of this project is to conduct a
thorough assessment of the use and effectiveness of commercial bioremediation products by reviewing actual field
cases where bioremediation has been used.
Based on information from EPA's Oil Program Center, the National Contingency Plan Product Schedule, and the
Oil Spill Intelligence Report (Cutter Information Corp.), it is my understanding that your company has been
involved in producing and marketing bioremediation products for oil spill cleanup. Since we intend to make our
review as inclusive and fair as possible, the field experience of your company on bioremediation agents is extremely
valuable to us. Therefore, I would like to obtain some technical information and experience in regard to your
bioremediation product(s). The information that is of particular interest to me includes the following:
Principle ingredients of your bioremediation products (without your divulging
confidential business information).
Technical publications regarding the effectiveness of your bioremediation products in
actual case studies.
Client response and contact information.
The purpose of this letter is to see whether your company is willing to participate in this inquiry. Please understand
that your participation in this endeavor is strictly voluntary. If you wish to respond, please drop me a quick note via
e-mail, and I will contact you via telephone at your convenience and will provide you more detailed information
about my request. If you do not wish to participate, please also let me know so that I may strike you from my list.
Due to the time constraint of this project, I would be most appreciative if you would reply within two weeks. Thank
you for taking the time to consider this request, and I am very hopeful to receive a positive reply from you.
Sincerely yours,
Xueqmg Zhu, Ph.D., P.E.
Department of Civil and Environmental Engineering
University of Cincinnati
Cincinnati, OH 45221-0071
Tel: 513-556-3638, Fax: 513-556-2599
E-mail: zhuxi@email.uc.edu
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Appendix B: Follow-up Letter to Participating Companies
Dear Madam/Sir:
Thank you for your willingness to participate in this information collection effort as a part of EPA's investigation of
the efficacy of bioremediation products. I am writing to you to provide more information about this project and what
I need from you. The objective of this project is to conduct an in-depth literature review on the efficacy of
commercial bioremediation products for cleaning up oil-contaminated environments. The scope of this review is
limited to the use of bioremediation agents (i.e., microbial additives and nutrient additives) for cleanup of surface oil
spill (inland, estuarine, and marine environments, but not groundwater). The performance of other oil spill control
agents and approaches may be included only as a point of reference and comparison. The information that is of
particular interest to me is listed as follows:
(1) Principle ingredients of your bioremediation products.
Without your divulging confidential business information, can you tell me whether your products include any living
organisms (bacteria, fungi, etc.), nutrients, enzymes, exogenous hydrocarbons, sorbents, surfactants, or anything else
that characterizes your product and its primary mechanism of action?
(2) Efficacy for oil spill cleanup in the field.
Do you have any technical publications, client reports, third-party reports, and/or company reports regarding the
effectiveness of your bioremediation products in actual case studies? Information that would be most helpful
includes:
Type of spilled oil and extent of contamination;
Effectiveness data on hydrocarbon destruction or removal;
Analytical methods used to support your conclusions;
Detailed sampling plan design;
Any statistical analysis done;
Environment in which the product was used;
Anything else you would deem important to my investigation.
(3) Client contact information.
If you don't mind, it would be most helpful if you could reveal the names, addresses, and telephone numbers or
email addresses of your clients so that I could contact them personally to interview them for their view on how well
bioremediation worked in their instance.
Our final report to EPA is due in September. I would be most appreciative if you would provide the above
information by the end of March. I will be happy to send a copy of our completed report when it is finished, peer-
reviewed, and cleared by the Agency, should you desire one. Thanks again for your help. If you need any additional
information about this request, please do not hesitate to contact me.
Sincerely yours,
Xueqmg Zhu, Ph.D., P.E.
Department of Civil and Environmental Engineering
University of Cincinnati
Cincinnati, OH 45221-0071
Tel: 513-556-3638, Fax: 513-556-2599
E-mail: zhuxi(3),email.uc.edu
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