This document discusses the promise and challenges of using genetically engineered microbes for bioremediation. It provides two examples where genetically engineered microbes were designed for bioremediation but never deployed, including an "oil-eating microbe" created in the 1970s and a microbe designed to degrade the toxic Agent Orange. Regulatory hurdles, including the EPA's reluctance to approve field trials due to containment concerns, have prevented the commercialization of these microbes. The document argues that risk-based regulation and technical safeguards could help translate such research from the lab to practical field applications.

1. Genetically engineered oil-eating microbes for bioremediation: Prospects
and regulatory challenges
Obidimma C. Ezezika*, Peter A. Singer
McLaughlin-Rotman Centre for Global Health, University Health Network and University of Toronto, Toronto, ON, Canada M5G 1L7
Keywords:
Bioremediation
Genetic engineering
Microbes
Microorganisms
Oil
Remediation
a b s t r a c t
The use of genetic engineering to enhance the natural capacity of microorganisms for
remediation has become very promising with new scientific discoveries occurring every
year. Unfortunately, the application and commercialization of this technology has not kept
pace with these research discoveries. This article uses two examples of genetically engi-
neered microorganisms that were designed but never deployed in the clean-up of wastes
to show how the application of genetically engineered microbes for bioremediation has
not progressed in line with other biotechnological innovations. We argue that a more risk-
based regulatory environment that fosters commercialization is important. In addition, we
show how scientists could foster the commercialization of genetically engineered
microbes for bioremediation through the use of technical safeguards and the consideration
of regulatory challenges at the onset of their research. The lessons provided by these
challenges could be applicable to current biotechnological innovations that face similar
regulatory challenges.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Bioremediation is one of the “Top 10 Biotechnologies to
Improve Global Health” [4]. Large amounts of toxic chem-
icals are released into the environment, either deliberately
as in the application of pesticides, or accidentally as in
the case of oil spills. A variety of microorganisms capable
of efficiently degrading toxic compounds and xenobiotics
in the environment have either been isolated or engi-
neered. However, the actual application of such microor-
ganisms in bioremediation has not progressed with the
same momentum as their invention, or as other innova-
tions in the biotechnology arena.
Microbial bioremediation is defined as the process by
which microorganisms like bacteria degrade or transform
hazardous organic compounds into non-toxic substances.
Such hazardous compounds include benzene, toluene,
PCBs, dioxins, and nitro-aromatics. There have been major
advances in the research and design of genetically engi-
neered microbes for bioremediation [12,5,17] and many
bioremediating microorganisms have been isolated.
Since naturally occurring microorganisms are not
capable of degrading all toxic chemicals, especially xeno-
biotics, genetically engineered microorganisms have been
tendered as the sine qua non for bioremediation, and
genetic manipulation has advanced. However, there have
been very few field trials for the use of genetically engi-
neered microorganisms for bioremediation [14].
Although there has been a boom in the commerciali-
zation of genetically modified drugs, crops, and other
biotechnological innovations over the last two decades,
genetically engineered microbes for bioremediation have
not been commercialized [14,18]. Some have speculated
that cost, complexity, and a burdensome regulation may be
a reason for the lack of commercialization [18]. However,
that explanation does not seem to present the full picture.
Since the United States Environmental Protection Agency
(EPA) started regulating genetically engineered microbes
* Corresponding author.
E-mail address: obidimma.ezezika@mrcglobal.org (O.C. Ezezika).
Contents lists available at ScienceDirect
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Technology in Society 32 (2010) 331–335

2. about 30 years ago, there has been no commercialization
of any genetically engineered microbe for bioremediation,
except in demonstration projects in closed reactor systems
[18,16].
What happened to the promise of genetically modified
microorganisms for bioremediation? We present an anal-
yses of the research-commercialization gap using examples.
We evaluate this gap from both a regulation and research-
based perspective, taking into account the containment
technologies that have been developed. In this study, we
reviewed recent literature on the development of geneti-
cally engineered microbes for bioremediation and inter-
viewed Ananda Chakrabarty, the first scientist to patent
a living organism (the “oil-eating microbe”). Through our
analysis of the interview and literature review, we found
that the inability of scientists to adapt their research to the
prevailing regulatory environment, lack of a risk-based and
evolving regulatory framework, and inadequate support by
government agencies to help bioremediation researchers
bring their products to the market could be an explanation
for this gap.
We suggest that risk-based regulation and the design
of genetically engineered microbes with technical safe-
guards could bring about the important translation of this
kind of bioremediation research from the lab to the field,
where it is most needed.
2. The potential of a genetically engineered microbe
for bioremediation
During the 1980s and 90s, there was a spark in research
in the development of genetically engineered microorgan-
isms for bioremediation [20]. The era held promise: many
bioremediation companies were born and researchers
in genetic engineering and microbiology increased the
intensity of their research in this newand emerging field [3].
However, due to the regulatory hurdles and high technical
cost required to satisfy regulation, many of these companies
went out of business and experiments on genetically engi-
neered microorganisms were confined to research institu-
tions. The research moved from the agenda of companies
to those of academies.
The first genetically engineered microbe was created by
an Indian-born microbiologist and genetic engineer,
Ananda Chakrabarty, in 1971 [15]. The patent was approved
in 1980 by the United States Supreme Court. The microbe
was a variant of the genus Pseudomonas and was capable
of breaking down the constituents of crude oil.
Chakrabarty showed that four strains of the common
Pseudomonas bacteria contained enzymes that enabled
them to break down different hydrocarbons. He first
determined that the genes for oil-degrading enzymes were
carried not on the microorganism’s chromosome, but rather
on other extra-chromosomal elements known as plasmids.
He combined these plasmids into a strain of Pseudomonas.
Unfortunately, due to regulations and public concerns of
using the microbe for bioremediation, Chakrabarty’s break-
through microbe still sits on a shelf, unused. At the time, the
new superbug created by Chakrabarty was said to have the
potential to degrade oil 10–100 times faster than other non-
genetically engineered independent strains [15].
This oil-eating microbe created by Chakrabarty is not
an isolated example; there are other cases of genetically
engineered microorganisms that have been designed but
not applied in bioremediation [12,7,19]. For example,
a bacterium, Deinococcus radiodurans, which is the most
radiation-resistant organism known, was successfully
engineered to degrade toluene. However, it has not been
applied or commercialized for bioremediation [9]. Several
factors may be associated with the failure of advancement
from research to commercialization.
3. The regulatory challenge of genetically engineered
microbes for bioremediation
The regulatory environment plays a central role in
either advancing or stifling the application of novel
biotechnological inventions. On the heels of the oil-eating
microbe created by Chakrabarty companies who desired to
commercialize genetically engineered microorganisms in
the 1980s and 90s were decelerated by the regulatory
framework in the United States [20]. The regulatory hurdles
were partly premised on the inadequacy of biotechnolog-
ical inventions to thoroughly contain genetically modified
bacteria once released into the environment.
For example, a genetically engineered microbe was
created to effectively degrade Agent Orange1
, a toxic defo-
liant used by the United States military during the Vietnam
War. The engineered microbe was produced from a strain
of Burkholderia cepacia and was designed for the removal
of Agent Orange at the U.S. Air Force in Pensacola, Florida,
where it was stored prior to its shipment to Vietnam. Agent
Orange has been linked to increased cancer cases [10,1].
According to Charkrabarty, the research on the Agent
Orange-degrading microbe was partly funded by National
Institute of Health and partly by the EPA, the EPA was
reluctant to approve its use based on concerns regarding its
potential impact on the environment. The EPA wanted
assurance that the toxic chemical-degradative genes would
not be transferred by natural gene exchange mechanisms
to neighboring pathogenic bacteria, and that such patho-
genic bacteria would not be able to feed on the pollutant.
According to Charkrabarty, “These are tricky issues to be
resolved through laboratory experimentations and require
massive field trials in isolated sites.”
The EPA has the authority to regulate the release of
genetically engineered microorganisms under Section 5
of the Toxic Substance Control Act (TSCA). Manufacturers of
genetically engineered microbes are required to submit
a Microbial Commercial Activity Notice (MCAN), which
states inter alia the environmental fate, health effects
data, and physical and chemical properties of the proposed
modified microorganism. Genetic modification of microor-
ganisms is presumed as high risk in the regulatory frame-
work of the EPA [16]. However, given the application of
genetic engineering in the pharmaceutical and agricultural
sectors, mere genetic manipulation should not necessarily
be considered high risk but should be dependent on the
1
1:1 mixture of two phenoxy herbicides, 2,4-dichlorophenoxyacetic
acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).
O.C. Ezezika, P.A. Singer / Technology in Society 32 (2010) 331–335
332

3. specific modification and use. The EPA, in regulating the
genetically engineered microbes, does not take into account
risk stratification among different applications. In addition,
the Act is not focused on regulating high risk organisms or
their uses, rather the guidelines are based on the assump-
tion that all genetic manipulation is high risk, making
the approval process quite burdensome. In addition, to
a more risk-based regulation, it is important that the EPA
have programs that support bioremediation companies to
bring their products to the market.
4. Mitigating risks through technical safeguards
There is high public resistance to unleashing recombi-
nant microbes for bioremediation due to a variety of risks
[2]. A notable risk is the issue of containment. How can
microorganisms be contained following completion of the
bioremediation process? The issue of containment
concerns both the public and scientists [8]. These concerns
stem from the potential of genetically engineered micro-
organisms to disrupt the existing ecological framework,
if they persist in the environment after the pollutant has
been depleted. There have been recent attempts to design
and track genetically engineered microorganisms including
the development of a set of criteria for the use of genetically
engineered microorganism. This was proposed in Annex 5
of the International Technical Guidelines for Safety in
Biotechnology for Micro-organisms.
“Using organisms with impaired ability to grow or
persist in the environment; minimizing gene transfer
by: using organisms that do not contain known self-
transmissible mobilizable or transposable genetic
elements; ensuring that introduced traits are stably
located on the chromosome.”2
A solution to the regulatorychallenge can come by placing
the burden and incentive on scientists to design genetically
engineered microorganisms that can be contained.
Although the idea of containment has been conceptual-
ized by the design of “suicidal genetically engineered
microorganisms” [8,13], the use of such technology has not
been applied. The solution for successful containment
involves programming the death of genetically engineered
microorganisms after the depletion of the concerning
pollutant. A genetic design model was proposed by Paul
et al. [11] that accounts for the uncertainty of the fate of
genetically engineered microorganisms after they have
accomplished their task. This involves introducing killer–
anti-killer genes, which are induced by the pollutant. When
the pollutant is depleted the killer genes are expressed,
which exterminate the genetically engineered microor-
ganism. The use of killer genes on plasmid eliminates the
microbial recipient if a transfer occurs, and the concern
of horizontal gene transfer is mitigated. Such a design
removes the risks, and associated concerns of use, generally
associated with introducing genetically engineered micro-
organisms into the environment [11]. This is an avenue by
which the uncontrolled proliferation of genetically engi-
neered microorganisms could be mitigated and the risks
associated with developing these microorganisms for
successful bioremediation minimized.
Unfortunately, many of these containment technologies
have evolved but not been taken into account in the
regulatory framework or applied by scientists in the design
of genetically engineered microbes for bioremediation.
5. Technical safeguards coupled with risk-based
regulation is key
We argue that a risk-based regulatory framework,
coupled with a novel scientific innovation mindset that
incorporates technical safeguards in the design of geneti-
cally engineered microorganisms, is crucial to harness their
potential for bioremediation (see Fig. 1).
The applicable and safe use of microbes for bioremedi-
ation are more valuable than mere construction of geneti-
cally engineered microorganisms for bioremediation that
cannot be applied. Scientists need to design microbes
that take into account the regulatory framework of the EPA.
In addition, it is important that all introduced DNA are well
characterized in order to facilitate the EPA approval
process. For example, it can be argued that in the design
of the Agent Orange-degrading bacterium, the delay of use
and associated environmental health risks could have been
avoided if the recipient strain, Burkholderia cepacia
(implicated in fatal infections in cystic fibrosis patients),
was not used but rather a different bacterium already
exempt from tough regulatory oversight by the EPA.
Conversely, the regulatory framework should be encour-
aging to those working on bioremediation: stratified and
risk based.
To resolve the failure of the research to commercializa-
tion cycle in the use of genetically engineered microorgan-
isms for bioremediation, we propose the following:
Fig. 1. Improved bioremediation through innovation and an encouraging
regulatory framework.
2
EPA (1995). International Technical Guidelines for Safety in Biotech-
nology. Biotechnology, UNEP, Nairobi, Kenya.
O.C. Ezezika, P.A. Singer / Technology in Society 32 (2010) 331–335 333

4. We also believe that these challenges to the commercializa-
tion of genetically engineered microbes for bioremediation
can provide important lessons for other life science projects
where genetic engineering is involved and where there are
similar regulatory challenges. For example, ineffective regu-
latory systems were cited as the most important constraint
to GM crop development and commercialization in devel-
oping countries [6]. In general, the regulation of biotechnol-
ogies should be stratified and should encourage innovation.
Bio-safety regulations should be continually updated to
integrate the rapidly evolving field of genetics. Regulations
should be risk based and not unnecessarily burdensome. In
addition, scientists working on technologies should be aware
of the bio-safety laws and regulations at the onset of their
research in order to avoid loss of resources, in time and
money, when developing biotechnologies which may not be
applied or commercialized due to high regulation hurdles, as
observed in the case of genetically engineered microbes for
bioremediation.
Acknowledgements
We are grateful to Jocalyn Clark for her comments on
earlier drafts of the manuscripts.
Funding
This project was funded by the Bill & Melinda Gates
Foundation and supported by the McLaughlin-Rotman
Centre for Global Health, an academic centre at the
University Health Network and University of Toronto. The
findings and conclusions contained within are those of
the authors and do not necessarily reflect official positions
or policies of the foundation.
References
[1] Ambrus JL, Islam A, Akhter S, Dembinski W, Kulaylat M, Ambrus CM.
Multiple medical problems following agent orange exposure. Jour-
nal of Medicine 2004;35(1–6):265–9.
[2] Broda P. Using microorganisms for bioremediation: the
barriers to implementation. Trends in Biotechnology 1992;10
(9):303–4.
[3] Chen W, Bruhlmann F, Richins RD, Mulchandani A. Engineering of
improved microbes and enzymes for bioremediation. Current
Opinion in Biotechnology 1999;10(2):137–41.
[4] Daar AS, Thorsteinsdottir H, Martin DK, Smith AC, Nast S, Singer PA.
Top ten biotechnologies for improving health in developing coun-
tries. Nature Genetics 2002;32(2):229–32.
[5] Dua M, Singh A, Sethunathan N, Johri AK. Biotechnology and
bioremediation: successes and limitations. Applied Microbiology
and Biotechnology 2002;59(2–3):143–52.
[6] James C. Global status of commercialized biotech/GM crops. Itaca,
NY: ISAAA; 2009.
[7] Jiang JD, Gu LF, Sun JQ, Dai XZ, Wen Y, Li SP. Construction of multi-
functional genetically engineered pesticides-degrading bacteria by
homologous recombination. Chinese Journal of Biotechnology 2005;
21(6):884–91. Sheng wu gong cheng xue bao.
[8] Kolata G. How safe are engineered organisms? Science (New York,
N.Y.) 1985;229(4708):34–5.
[9] Lange CC, Wackett LP, Minton KW, Daly MJ. Engineering
a recombinant Deinococcus radiodurans for organopollutant degra-
dation in radioactive mixed waste environments. Nature Biotech-
nology 1998;16(10):929–33.
[10] Marwick C. Link found between agent orange and chronic
lymphocytic leukaemia. BMJ (Clinical Research Edition) 2003;326
(7383):242.
[11] Paul D, Pandey G, Jain RK. Suicidal genetically engineered micro-
organisms for bioremediation: need and perspectives. BioEssays:
News and Reviews in Molecular, Cellular and Developmental
Biology 2005;27(5):563–73.
[12] Pieper DH, Reineke W. Engineering bacteria for bioremediation.
Current Opinion in Biotechnology 2000;11(3):262–70.
[13] Ramos JL, Andersson P, Jensen LB, Ramos C, Ronchel MC, Diaz E,
et al. Suicide microbes on the loose. Biotechnology 1995;13(1):35–7
(Nature Publishing Company).
[14] Sayler GS, Ripp S. Field applications of genetically engineered
microorganisms for bioremediation processes. Current Opinion in
Biotechnology 2000;11(3):286–9.
[15] Environment: oil-eating bug. Time Magazine 1975. Time.
[16] Timian SJ, Connolly DM. The Regulation and Development of Biore-
mediation [September 7, last update 2010] [Homepage of Franklin
Pierce Law Center], [Online]. Available: http://www.piercelaw.edu/
risk/vol7/summer/timian.htm [2009, June/05], http://law.unh.edu/
risk/vol7/summer/timian.htm.
[17] van der Meer JR, de Vos WM, Harayama S, Zehnder AJ. Molecular
mechanisms of genetic adaptation to xenobiotic compounds.
Microbiological Reviews 1992;56(4):677–94.
[18] Watanabe ME. Can bioremediation bounce back? Nature Biotech-
nology 2001;19(12):1111–5.
[19] Yang C, Xu L, Yan L, Xu Y. Construction of a genetically engineered
microorganism with high tolerance to arsenite and strong arsenite
oxidative ability. Journal of Environmental Science and Health. Part
A, Toxic/Hazardous Substances & Environmental Engineering 2010;
45(6):732–7.
[20] Zwillich T. Hazardous waste cleanup. A tentative comeback for
bioremediation. Science (New York, N.Y.) 2000;289(5488):
2266–7.
Obidimma Ezezika is a Program Leader in Ethics at the McLaughlin-
Rotman Centre for Global health at the University Health Network and
University of Toronto. His research is focused on the intersection of science
and policy in the development of biotechnology initiatives in developing
countries. Obidimma graduated from the University of Lagos in Nigeria
with a Bachelor of Science and completed a master’s degree in environ-
mental management at the Yale School of Forestry and Environmental
Studies focusing on environmental policy, economics and law. Obidimma
pursued a PhD in Microbiology at the University of Georgia in the United
States where he focused on structural biology and the molecular genetics
of bioremediation with lead-author publications in the journals of
Molecular Biology, Applied and Environmental Microbiology, and Acta
Crystallograhica.
Proposal
1. The EPA should emphasize risk stratification in
the use and application of genetically engineered
microorganisms for bioremediation in its TSCA
regulations.
2. Scientists working on bioremediation research
should take note of existing regulatory frame-
works at the onset of their research in order to
facilitate their application and commercialization
of their research products.
3. The EPA should not presume that all genetic
manipulation is high risk and focus their regula-
tory efforts on high risk organisms and their uses.
4. Regulations should continually be updated by to
the EPA to take into account the rapidly evolving
containment technologies and new information in
genetic engineering development.
5. Scientists should focus efforts on the application
of technical safeguards in the design of GM
microbes for bioremediation.
6. The EPA should initiate programs that support
start-up bioremediation companies to try their
product in field trials.
O.C. Ezezika, P.A. Singer / Technology in Society 32 (2010) 331–335
334

5. Obidimma has worked in various capacities for a variety of national
and international organizations including a chartered construction cost
and consulting firm in Nigeria, the University of Georgia River Basin
Center, the Yale Department of Molecular, Cellular and Developmental
biology, the Grenada permanent mission to the United Nations, the
United Nations Development Program in New York City, where he
reviewed environmental projects worth $14.2 million awarded by the
Small Grants Program of the Global Environmental Facility, working with
45 UNDP coordinators in Africa, Asia, Middle East, Latin America and
Europe.
O.C. Ezezika, P.A. Singer / Technology in Society 32 (2010) 331–335 335