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Home Bioengineered Threats Bioengineered Threats Genetically Modified Organisms for the Bioremediation of Organic and Inorganic Pollutants

Genetically Modified Organisms for the Bioremediation of Organic and Inorganic Pollutants

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New http://www.centerforfoodsafety.org/blog/3159/the-choice-is-simple-choose-organic-apples#

QUOTE from Center for Food Safety:

That’s exactly what occurred earlier this month when the National Organic Standards Board (NOSB)[1] voted to end the allowance of streptomycin, an antibiotic used to fight a persistent bacteria that threatens organic apple and pear orchards.

Antibiotics have never been permitted in organic agriculture with the singular exception of apple and pear production. Since the inception of OFPA, meat and dairy producers have been forbidden to use any antibiotics of any kind. Yet, early in the development of organic regulations, policy makers temporarily allowed both streptomycin and tetracycline as the exception to the organic rule to combat fire blight, a destructive bacteria that attacks tree blossoms, limbs, and shoots. Last year, tetracycline was banned and now, with the NOSB’s latest vote to rid organic of streptomycin, all uses of antibiotics in organic have been completely eliminated.








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AND 'WE' WERE TOLD TO STOP FLUSHING ANTIBIOTICS DOWN THE TOILET.

[see following]

 

 

FINALLY, the light bulb has been turned on (or the powers to be are finally revealing the truth)! "More and more people are beginning to understand that what we PUT ON OUR FOOD, LAND AND WATER ENDS UP IN OUR BODIES." (quote from Center for Food Safety in an email sent to me from the Center for Food Safety. < This e-mail address is being protected from spambots. You need JavaScript enabled to view it >)

Stated:  "antibiotics, USDA temporarily allowed them in apple and pear orchards". and "With your help, tetracycline was banned for use in organic apple and pear orchards last year and now, with the National Organic Standards Board's (NOSB) latest vote to ban streptomycin, all uses of antibiotics in organic have been completely eliminated!"

Listed IS tetracycline; which is OBTAINED FROM BACTERIA of the genus STREPTOMYCES. Additionally, the mold Penicillium has been used for years as the antibiotic Penicillin. Streptomyces and Penicillium have been used as biological fungicides on our crops.

See HUMANS / ANIMALS & CROSS - INFECTIONS from PLANTS, SOILS AND INSECTS MANY are the “ACTIVE INGREDIENT” in BIO-CONTROL PRODUCTShttp://issuu.com/biotechharm/docs/finished.full.cross.over.insects.pl_10ff47f1c3cdb4

STREPTOMYCES is the "active ingredient" IN FUNGICIDES (see Table S-3 [pages 42-43] STREPTOMYCES HUMAN HEALTH RELATED: See Chart on page 23. PENICILLIUM is the "active ingredient" IN FUNGICIDES; See Table P-3 [page 36] PENICILLIUM HUMAN HEALTH RELATED See Chart on page 20.

This information above on the link, is just a very "small" example of what "naturally found" microorganisms, which are being used as the "active ingredient[s]" in bio-control products, and what are 'some' of the human health implications.

You may also be interested in watching Anne K Vidaver’s presentation “Cross-infective microbes: from plants to humans” by Anne Vidaver; Enhancing Regulatory Communication Workshop in November 2006. This workshop lasted for three days and was presented before numerous governmental agencies; including the National Institute of Health, Center for Disease Control, Environmental Protection Agency, United States Department of Agriculture ... to name just a few. Additionally, numerous Universities were represented, as well as one ‘private’ Research and Development Biotech Company, which searches the world for novel microorganisms; as candidates for being the “active ingredient[s]” in bio-control products.

As you will see/hear in Dr. Anne K Vidaver's Abstract & Video presentation of “Cross-infective microbes: from plants to humans” she exposed the dangers of some of the “naturally found” bacteria & fungi being used for bio-control products because of the hazards to human health. NOTE: Professor Vidaver seemed to have been sabotaged prior to the workshop as many of her references that were sent in with her abstract before the workshop ended up “missing” . [see footage 00:01:38.0 “I should also say that a couple of the references that I’ve provided with my abstract are missing.“]

VIDEO alone: http://biopesticide.ucr.edu/video/assets/MOV00F_Vidaver.wmv
TRANSCRIPTION with video link: http://issuu.com/biotechharm/docs/vidaver.cross.infective
























Genetically Modified Organisms for the Bioremediation of Organic and Inorganic

Pollutants

Final Report

March 2002
WS Atkins Environment
Woodcote Grove, Ashley Road, Epsom, Surrey KT18 5BW

Tel: ]01372] 726140@Fax: ]01372] 740055
Genetically Modified Organisms
for the Bioremediation of Organic
and Inorganic Pollutants
Final Report
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PREFACE
This study was carried out under DEFRAs Genetically Modified Organisms (GMO)
Research Programme, which aims to underpin Government policy on the environmentally
safe use of genetically modified organisms.
This report represents the findings of a desk study on the current and future uses of
genetically modified organisms (GMOs) for the bioremediation of organic and inorganic
pollutants, and an assessment of the risks of such uses to the environment and human health.
The report was compiled by Dr Colin Cartwright and Helen Folkard-Ward of WS Atkins
Environment; Dr Ian Thompson, Dr Michelle Barclay and Prof Mark Bailey at the Centre for
Ecology and Hydrology, Oxford; and Prof Andrew Smith from the Department of Plants
Sciences, University of Oxford.
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CONTENTS
PREFACE I
CONTENTS II
EXECUTIVE SUMMARY VI
1. INTRODUCTION 1-2
Background to the Project 1-2
Aims and Structure of the Study 1-4
2. REVIEW OF THE CURRENT AND POTENTIAL FUTURE USES OF GMOs
IN BIOREMEDIATION 2-6
The Use of Microorganisms for the Bioremediation of Pollutants 2-8
General Strategies for the Optimisation of Bioremediation Applications 2-9
Improvements in transcription of the gene sequences 2-10
Improving translation 2-13
Improving protein stability and activity 2-14
Extending the scope of existing catabolic pathways 2-15
Construction of recombinant microorganisms 2-17
Use of Bacteria for the Bioremediation of Organic Pollutants 2-18
Cytochrome P450s for the bioremediation of organic pollutants 2-19
Chlorinated compounds 2-20
Polychlorinated biphenyls 2-24
Hydrocarbons 2-29
Nitroaromatic compounds 2-36
Use of Bacteria for the Bioremediation of Inorganic Pollutants 2-36
Metallothioneins 2-37
Other general strategies for the bioremediation of heavy metals 2-40
Mercury 2-41
Nickel 2-44
Use of Fungi for the Bioremediation of Pollutants 2-44
The Use of Microorganisms for the Monitoring of Pollutants 2-47
The Use of Plants for the Bioremediation of Pollutants 2-51
Approaches and Advantages to Phytoremediation 2-52
Types of Phytoremediation 2-54
Phytoextraction 2-55
Rhizofiltration 2-55
Phytostabilisation 2-55
Phytovolatilisation 2-55
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Phytodegradation 2-55
Phytoremediation of Metals 2-56
Phytoextraction 2-57
Phytostabilisation 2-69
Rhizofiltration 2-70
Phytovolatilisation 2-71
Phytoremediation of Organic Pollutants 2-75
Phytodegradation 2-77
Phytostabilisation 2-78
Technical Problems Encountered with Transgene Expression in Plants 2-79
Combined Strategies for the Bioremediation of Pollutants 2-81
Multi-plant Strategies 2-81
Plant-microbial Strategies 2-81
Multi-microbial Strategies 2-84
3. ASSESSMENT OF THE RISKS OF THE USE OF GMOs FOR THE
BIOREMEDIATION TO THE ENVIRONMENT AND HUMAN HEALTH 3-85
The Use of Microorganisms 3-87
Transfer of Genetic Material 3-90
Potential for genetic material to be transferred 3-90
Effect of transfer of genetic material to other organisms or biological processes 3-92
Accumulation of Toxic Compounds 3-93
Production of Toxic Metabolites or By-products 3-94
Disruption of Other Organisms and Biological Processes 3-96
The Use of Plants 3-97
Transfer of Genetic Material 3-97
Accumulation of Toxic Compounds 3-98
Production of Toxic Metabolites 3-99
Disruption of Other Organisms and Biological Processes 3-101
4. POTENTIAL MANAGEMENT STRATEGIES FOR THE USE OF GMOs IN
BIOREMEDIATION 4-103
Microorganisms 4-103
Transfer of Genetic Material 4-104
Biological Containment Systems 4-106
Attenuation based biological containment systems 4-107
Controllable suicide systems 4-107
Application of Microorganisms to the Contaminated Site 4-111
Plants 4-112
Transfer of genetic material 4-112
Accumulation of toxic compounds 4-112
Production of toxic metabolites 4-114
Disruption of Other Organisms and Biological Processes 4-114
5. REPORT OF THE WORKSHOP 5-115
Introduction 5-115
Prospects and challenges for bioremediation with GMMs 5-116
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Presentation by Professor Kenneth N Timmis 5-116
Questions 5-119
Reporter gene based biosensors – risk-based management
support for remediation of contaminated land 5-120
Presentation by Professor Ken Killham 5-120
Questions 5-124
Field release of P. fluorescens HK44:. Long term persistence
and field performance of a bioremediation bioluminescent bioreporter 5-124
Presentation by Professor Gary S Sayler 5-124
Questions 5-128
Metal accumulation by plants 5-129
Presentation by Professor Andrew Smith 5-129
Questions 5-134
Phytoremediation of toxic chemicals in our environment 5-134
Presentation by Professor Richard B Meagher 5-134
Questions 5-139
Defusing the environment: engineering transgenic plants to degrade
explosives 5-139
Presentation by Dr Neil Bruce 5-139
Discussion on the use of GMOs for the Bioremediation of Pollutants 5-142
Chaired by Professor Chris Leaver 5-142
Incorporation of plant and microbial based strategies 5-142
Secondary effects of organisms used in bioremediation 5-143
Perception of risk by the general public 5-144
Specific advantages of GMOs for bioremediation 5-146
What information is required? 5-147
6. CONCLUSIONS 6-149
7. RECOMMENDATIONS FOR FUTURE WORK 7-153
8. REFERENCES 8-156
9. APPENDICES 9-175
Appendix A - Abstracts of Workshop Presentations 9-176
Reporter gene based biosensors – risk-based management support for
remediation of contaminated land 9-176
Professor Ken Killham, Department of Plant & Soil Science, University of Aberdeen,
Aberdeen. 9-176
Phytoremediation of toxic chemicals in our environment 9-178
Professor Richard B. Meagher, Department of Genetics, University of Georgia,
Athens, GA 30602, USA. 9-178
Field release of P. fluorescens HK44:. Long term persistence and field
performance of a bioremediation bioluminescent bioreporter 9-183
Professor Gary S Sayler, Center for Environmental Biotechnology,
University of Tennesse, Knoxville, Tennesse, USA 9-183
Prospects and challenges for bioremediation with GMMs 9-185
Professor Kenneth N Timmis, National Research Centre for Biotechnology,
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Braunschweig, Germany. 9-185
Metal accumulation by plants 9-187
Professor J.A.C Smith, Department of Plant Sciences, University of Oxford, Oxford 9-187
Defusing the environment: Engineering plants to degrade explosives 9-190
Dr Neil C Bruce, Institute of Biotechnology, University of Cambridge 9-190
APPENDIX B - LIST OF WORKSHOP DELEGATES 9-193
APPENDIX C - WORKSHOP PROGRAMME 9-195
List of Tables
Table 4.1 - Alternative selectable genetic markers [37] 4-104
List of Figures
Figure 2.1 - Construction of bioluminescent reporter plasmid pUTK21) [91] 2-30
Figure 2.2 - Schematic diagram of the lysimeter containing PAH contaminated soil 2-32
Figure 2.3 - Aerial photograph of lysimeters in the field. Photograph taken prior to
filling the lysimeters and demonstrates the scale of the operation 2-32
Figure 2.4 - Cadmium ions entering the cell activate PC synthase that catalyses the
transformation of GSH to PC [103] 2-59
Figure 2.5 - Possible structure of a phytochelatin (n=3, X=Gly) binding 2-59
Figure 2.6 - Regulation of GSH/PC biosynthesis in plants [17] 2-61
Figure 2.7 - The bacterial enzymes MerA and MerB catalyse the detoxification of methyl and
ionic mercury respectively to produce volatile Hg(0) [19] 2-72
Figure 2.8 - A proposed model for the selenium flow in Indian mustard plants [211] 2-76
Figure 4.1 - Elements and functioning of a biological containment system [261] 4-109
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EXECUTIVE SUMMARY
This report constitutes a review of the use of genetically modified organisms (GMOs) for the
bioremediation of organic and inorganic pollutants. The report covers the current and future
applications of GMOs in bioremediation, an assessment of the risks posed to the environment
and human health, and the management strategies available to reduce the likelihood of any
risks from being realised. The information presented in this report was compiled from details
published in peer-reviewed journals, from information presented at a one-day workshop held
as part of the project and the expertise of the project consortium.
Genetic modification technology is reported to offer a wide variety of current and potential
applications for use in the bioremediation of pollutants. To date, three main types of GMOs
have been developed and are currently undergoing field trials or already being used in
commercial applications. These types are genetically modified microorganisms (GMMs)
designed to degrade organic pollutants; genetically modified (GM) plants designed to
hyperaccumulate or volatilise metal pollutants; and GMMs used as biosensors to detect the
presence and toxicity of particular pollutants on site. No releases of GMOs into the
environment have taken place in the UK for bioremediation applications, although contained
applications of lux modified GMMs have been used at a commercial level to map the
distribution of pollutants in contaminated sites.
All of the applications of GMOs for bioremediation have used bacteria or plants as the
modified organism. Although fungi have the capability to degrade a wide range of often
highly recalcitrant compounds, these organisms have not yet, to date, been genetically
modified for use in bioremediation. Possible explanations for this include the relative
complexity, at the genetic level of the degradation of organic pollutants by fungi compared to
bacteria, and the identification of many fungi as plant pathogens, and therefore unsuitable for
release into the environment.
In addition to their application in biosensors, the use of GMMs for bioremediation has
focused on the degradation of organic pollutants, largely chlorinated compounds and
hydrocarbons. In contrast, GM plants have been developed predominantly for the treatment
of metal pollutants. Although GMMs have the potential to bioremediate metal pollutants,
and GM plants are being developed to target organic contaminants such as nitroaromatic
compounds, there is currently a distinction between applications for GMMs and GM plants.
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This has arisen as a consequence of the existing levels of knowledge of the genes and
metabolic systems involved, and as a result of the particular characteristics of the organisms
themselves.
The genetic basis of the activity of microorganisms towards pollutants is less complicated
than similar systems in plants. Consequently more information is available on the genes
involved and the structures of the metabolic pathways. In bacteria, many of the processes
involve only a few genes arranged on a single operon. This makes the identification and
transfer of bacterial genes to other organisms relatively straightforward, certainly when
compared to a similar approach with plants. However, although GM plants that incorporate
bacterial genes have been developed, differences in guanine cytosine (GC) content and codon
bias between microorganisms and plants mean that any bacterial genes used in plants may
require some modification for them to function effectively.
The objective of the processes designed to bioremediate organic pollutants is the complete
mineralisation of the compound. GMOs have been designed that are able to either mineralise
the pollutant, or to perform one stage of the catabolic pathway that may otherwise prevent the
biodegradation of the compound by the natural flora and fauna. Because metals cannot be
biodegraded, their bioremediation requires the sequestration and/or accumulation of the metal
by the organism. Whilst microorganisms are capable of sequestering metals, any
bioremediation strategy using such organisms also requires systems to remove the
metal/microbial complex from the environment. The advantage of using plants for the
bioremediation of metals is that the metal pollutants can be accumulated in the plant, which
can ultimately be removed from the contaminated site. GM plants have been developed to
bioremediate metals, by the modification of plants with bacterial genes, for example the merA
and merB bacterial genes used to bioremediate mercury contaminated sites; and also by the
modification of faster growing high biomass plants with plant genes isolated from natural
metal-accumulating plant species.
The types of risks posed by the use of GMOs for bioremediation are similar for both GMMs
and GM plants, although the level of risk that might be realised differs between the two
groups of organisms. The four hazards common to all GMOs (with the exception of GMMs
used in contained systems for biosensor applications) are the transfer of genetic material, the
accumulation of toxic compounds, the production of toxic metabolites and the disruption of
other organisms and biological processes by the GMO.
The likelihood of each of these hazards is dependent on the characteristics of the individual
GMO and the environment in which the organism is intended to be used. However, based on
the types of GMOs that are reportedly being developed for bioremediation applications, the
general consensus is that for GMMs the hazards most likely to be realised are the transfer of
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genetic material and the disruption of other organisms or biological processes. For GM
plants the transfer of genetic material and the accumulation of toxic compounds are identified
as the most significant risks. However, in assessing the significance of the risks and the
requirement of management strategies to minimise or reduce them, the unique characteristics
of the contaminated site should be addressed.
Unlike the application of GMOs in agriculture, the contaminated sites that may potentially be
bioremediated by GMOs usually have poor biodiversity and are often so contaminated that
they do not support any vegetation. Therefore, the implications of the transfer of genetic
material from GMOs to other organisms on the site, or the disruption on natural processes are
likely to be low considering the limited number and diversity of other organisms present.
Many of the modifications designed to bioremediate pollutants are also reported to offer the
recipient organism no selective advantage outside the contaminated site, and may even offer a
selective disadvantage to the organism in the absence of the specific pollutant.
Although the accumulation of toxic compounds has been identified as a hazard of using
GMOs designed to sequester metal compounds for example, it should be noted that this is the
intended purpose of the bioremediation strategy and the genetic modification. It should also
be recognised that the use of plants to phytoaccumulate metals may be less hazardous to the
environment and human health than alternative physical or chemical based remediation
technologies.
Management systems are however available to minimise many of the risks identified. The
insertion of the recombinant genes into the host organism by transposon mediated
modification, rather than on a mobilisable plasmid, should reduce any subsequent transfer of
those genes to other organisms. With GM plants, various horticultural techniques are
available to prevent gene transfer, most of which are common to all GM plants. Examples of
these include the use of sterile plants and species that reproduce vegetatively.
Restricting the GMO to the contaminated site by employing biological containment systems
will reduce any impact the GMOs may have on the environment outside the contaminated
site. Any bioremediation strategy (involving GM or non-GM organisms) will affect the
organisms present in the contaminated site, as the strategy will, by design, alter the physical
and chemical conditions present. The remediation of the site using physical or chemical
processes will also have a significant impact on the organisms present, and this should be
considered when assessing the risks posed by the use of GMOs.
Whilst a number of GMOs that have been designed for bioremediation are undergoing field
trials, many of the developments are still at the laboratory or early field test stage.
Improvements in the understanding of the genetic basis of the processes involved in the
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biodegradation and bioaccumulation of pollutants, and as importantly, information on the
basic mechanisms that determine how plants and microorganisms behave and interact in the
environment, will lead to the development of a wide diversity of potentially very powerful
applications to bioremediate pollutants. These have the potential to be able to target even the
most recalcitrant pollutants in inhospitable environments at relatively low cost.
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1. INTRODUCTION
BACKGROUND TO THE PROJECT
1.1 Anthropogenic activities such as heavy industry, mining and some agricultural
practices have resulted in the contamination of the environment with a large number
of organic and inorganic xenobiotic compounds. Such compounds are defined for the
purposes of this report as compounds released through anthropogenic activities at
concentrations that are higher than in the natural environment [1]. Xenobiotic
compounds have the potential to harm environmental ecosystems directly, through
their inherent toxicity [2], or through the disruption of important ecological processes
[3]. Although improved practices have reduced the present level of chemical
contamination of the environment, accidental releases of chemicals still occur, and a
large number of contaminated sites still exist [4]. The total area of land in the UK
contaminated by xenobiotic compounds has been estimated to range from 50,000 to
200,000 hectares (equivalent to almost 1 percent of total UK land area), and may
include up to 100,000 contaminated sites [5].
1.2 Because of the potential for xenobiotic compounds to harm the environment and
human health, and also the increasing demand for the re-use of contaminated
(brownfield) sites for non-industrial purposes such as recreation or housing, there has
been increased pressure to clean-up or remediate contaminated sites. The purpose of
the remediation process is to prevent any subsequent exposure of the xenobiotic(s) at
the contaminated site to the end-users of the site or to the flora and fauna present.
1.3 Remediation of contaminated sites involves treating the site in such a way that the
contaminant is either removed from the site, converted into a less toxic form, or
rendered immobile, so that subsequent exposure cannot occur. A wide variety of
strategies have been proposed and/or demonstrated to remediate contaminated
environments [6], with the type of pollutant to be removed and the characteristics of
the site, including the location and distribution of pollutant, the principle determining
factors. If the pollutant is present in discrete localised areas of a site, then those areas
may be simply physically removed from the site and disposed of to landfill (so-called
‘dig and dump’ technology). This is a relatively inexpensive remediation strategy, but
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does depend on a suitable receptor being available to receive the contaminated
material. Where the contamination occurs over a wide area then removal of the
contaminated material may not be feasible, and some form of on-site strategy is likely
to be required. This may involve a physical based process such as air-sparging, or a
biological/chemical based process to remove or degrade the pollutant.
1.4 The use of biologically-based processes to remediate environmental pollutants is
described as bioremediation [7]. This type of strategy can offer a more suitable
alternative to physical and chemical methods of dealing with contaminated sites. In
particular, bioremediation can be less expensive [8] and importantly can be used to
achieve the selective remediation of the target contaminant without incurring
significant collateral damage to existing flora and fauna at the contaminated site [6].
Bioremediation strategies can be applied to the contaminated material in-situ, whereas
removal of the polluted material to designated landfill sites, or extraction of the
contaminants using physical processes simply concentrates the contaminated material
in a different location [9].
1.5 Bioremediation is applicable particularly for the in-situ remediation of contaminants
present at low concentrations, and for the selective removal of individual pollutants
[4]. When applied to organic pollutants, bioremediation has the potential to achieve
the complete destruction of the contaminant [10]. The bioremediation strategy that is
employed may be as simple as the addition of nutrients to the contaminated site to
improve the ability of the indigenous microbial community to degrade the pollutant
(biostimulation) [11], or may be more complicated and involve the addition of
particular organisms to the site (bioaugmentation) [6]. Such organisms are usually
selected due to their capability to sequester, degrade or transform the pollutant and
thereby bioremediate the contaminated site.
1.6 The purpose of this report is to review the current and potential future applications for
the use of genetically modified organisms (GMOs) in bioremediation strategies.
GMOs are defined as those organisms whose genetic profile could not have occurred
naturally by mating and/or natural recombination. Although the capability to degrade
many xenobiotic compounds is possessed by a range of non-GM (genetically
modified) organisms, particularly microorganisms, the continuing persistence of some
xenobiotics in the environment, including those that are known to be biodegradable,
indicates that the biodegradative capacity of an environment’s resident flora and fauna
is not expressed completely or effectively [12]. Developments in molecular biology
have the potential to produce organisms with new combinations of traits that can
remediate previously unbiodegradable compounds [13], or degrade xenobiotics at a
higher rate and/or to a greater extent than naturally occurring organisms [8].
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1.7 The release of GMOs into the environment is regulated in the UK by DEFRA1.
Applications to release GMOs are only approved on the basis that the release poses
negligible risk to the environment and human health. Whilst no applications have
been received by DEFRA to date in relation to the use of GMOs in the bioremediation
of pollutants, the technology does exist to use GMOs in this way.
1.8 As a footnote to this review, it is recognised that the use of GMOs in the UK is the
subject of intense public and political interest. Whilst this study has focused entirely
on the scientific issues involved, it is recognised that the public can only benefit from
access to current, objective and comprehensive reviews of both the use of GMOs and
the likelihood of any risk to the environment and/or human health being realised.
AIMS AND STRUCTURE OF THE STUDY
1.9 The aims of this study were to review the use of GMOs for bioremediation and assess
the risk of these applications to the environment and human health. These aims have
been addressed through the completion of the following objectives:
(a) to identify and review current and potential future applications for the use of
GMOs in the bioremediation of organic and inorganic pollutants;
(b) to assess the risks, in scientific terms, of the use of GMOs in the
bioremediation of pollutants to the environment and human health; and
(c) to propose management strategies that could be employed to reduce any of the
risks identified.
1.10 Whilst the report is intended as a review of the use of GMOs in bioremediation, and
not the use of bioremediation strategies to treat contaminated sites, it is recognised
that, to date, the use of GMOs in the field to bioremediate pollutants is very limited.
Because applications of GMOs are likely to be developed to overcome the limitations
of non-GM organisms, then this review will include some discussion of the use of
bioremediation strategies involving non-GM organisms. This is particularly relevant
to the future uses of GMOs in bioremediation.
1.11 The review of the use of GMOs in bioremediation has been presented in this report in
four stages. These represent the principle types of GMOs that are, or have the
1 Under Part VI of the Environmental Protection Act 1990 and Genetically Modified Organisms (Deliberate
Release) Regulations 1992 (amended 1995 and 1997).
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potential to be used in bioremediation applications, and cover the use of plants,
bacteria and fungi. The fourth stage addresses the use of combinations of plants and
microorganisms in bioremediation strategies.
1.12 The information presented in the desk study was supported by the findings of a
workshop, held at Magdalen College, Oxford. The one-day workshop brought
together people with expertise in bioremediation and plant and microbial disciplines,
to discuss the use of GMOs in this field. The workshop provided the opportunity to
review specific bioremediation applications of GMOs in more detail, and also to
discuss the relative risks and benefits of the use of GMOs in bioremediation and the
potential management strategies available. The specific areas that were addressed in
detail during the workshop were:
• the underlying constraints affecting the application of microorganisms and
plants in bioremediation;
• the use of reporter gene based biosensors for diagnostic bioremediation;
• the use of GMMs for the bioremediation of organic pollutants and as an online
process monitoring tool during a long-term field release;
• the application of plants for the accumulation of metals;
• the use of GM plants for the phytoremediation of pollutants, particularly
mercury; and
• the development of GM plants for the degradation of explosives.
1.13 A full report of the presentations and discussion that took place at the workshop is
presented in Chapter 5 of this report.
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2. REVIEW OF THE CURRENT AND POTENTIAL FUTURE
USES OF GMOs IN BIOREMEDIATION
2.1 The purpose of this chapter is to review the current and potential future uses of GMOs
in bioremediation strategies. The chapter is divided into sections addressing the use
of bacteria, fungi and plants, and also strategies that employ a combination of
organisms.
2.2 Applications to use GMOs for the bioremediation of pollutants encompass a range of
strategies, for example, the development of organisms with the capability to degrade a
specific contaminant. Although the approach used is often tailored to the individual
characteristics of the xenobiotic and the contaminated site, most approaches can be
assigned to a particular type of bioremediation strategy.
2.3 The most direct application of GMOs in bioremediation is the development of a GMO
that can be added to the contaminated site and will degrade the xenobiotic in situ.
This approach however requires that the GMO is able to survive in the contaminated
environment and still function as required, and that the contaminant is available in a
state in which it can be biodegraded (GMOs can be designed to improve the
bioavailability of the target contaminant as well as degrade it, if bioavailability is
identified as a significant inhibitor of bioremediation) [14]. Many contaminated sites,
particularly those that are historically polluted by industrial activities are polluted
with a cocktail of both organic and inorganic compounds [10].
2.4 The presence of other contaminants, in addition to the target pollutant, may prevent
degradation of the target compound through inhibition of the catabolic process or as a
consequence of their toxicity to the introduced GMO [10]. For example, where a site
is contaminated with a mixture of chlorinated solvents and radionuclides, any GMO
that is introduced to degrade the chlorinated solvents must also be resistant to
radiation. Strains of the microorganism Deinococcus radiodurans are reported to be
applicable for use in such environments, and GM D. radiodurans strains are reported
to be capable of degrading 125 n mole ml-1 chlorobenzene in environments with
radioactivity levels of up to 60 Gy h-1 [15].
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2.5 The bioremediation of multi-contaminated sites must consider all the xenobiotics
present before the addition of possible bioremediating organisms. No single organism
or community is likely to degrade all the organic pollutants present [10]. Survival of
the inoculant in the contaminated environment may also depend on the concentration
of target compound present. This is particularly relevant to microbial degraders,
many of which use the target compound as a source of carbon, energy and/or nitrogen.
If the concentration of target pollutant is too low, then the degrader will not obtain
sufficient biochemical or metabolic benefit from utilising that compound, and
degradation will not occur. If the concentration of the target contaminant is too high,
then any toxic effect of the compound may inhibit the activity of the inoculant and
degradation may be retarded [10]. If necessary, a consortium of inoculants or a
combined strategy may be required where one or more of the inoculants present is
designed to support the degrader with alternative carbon and/or nitrogen sources.
Combined strategies using plants and rhizosphere competent microbial degraders
(fungi or bacteria) are particularly suitable in these situations.
2.6 The low solubility and high hydrophobicity of many organic pollutants means that
they are often not available for biodegradation. Biological reactions occur in or at the
interface of the aqueous phase [4], and the rate and extent of the biodegradation of a
pollutant is often limited if the contaminant partitions towards the particulate phase in
an environment [16]. Strategies to bioremediate a poorly bioavailable compound,
may require the addition of an organism able to improve the bioavailability of the
compound, rather than degrade it (the capability to degrade the xenobiotic may
already exist in the contaminated site, but may only operate at a low level). Such
organisms include those that produce biosurfactants, and may be inoculated directly
into the contaminated environment, or grown in vitro and the biosurfactant harvested
and added to the environment on its own. The high cost of producing biosurfactants
has, however, limited their application, and current efforts are reported to be directed
towards designing inoculants that have both the desired catabolic trait and the
capability to produce the required biosurfactant [14].
2.7 The ability of the natural flora and fauna to biodegrade a contaminant may also be
limited due to the presence of a single rate-limiting step in the degradation reaction.
The addition of an organism designed to optimise a particular stage of the degradation
process, such as the rate-limiting step, may provide an effective strategy for the
bioremediation of the pollutant.
2.8 Strategies that involve the degradation of the contaminant are of course restricted to
organic pollutants. Strategies to bioremediate sites polluted with metals involve the
use of organisms that can convert the metal into another form [17]. This may be less
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toxic, more mobile in the environment, with greater bioavailability or immobility.
Whilst strategies that involve the conversion of metal pollutants to a more immobile
state (stabilisation) do not result in the metal being removed from the site, they do
result in the metal becoming effectively ‘locked-up’ in the environment in a
biounavailable state where it is unlikely to have an impact on human health and/or the
environment [18]. Contaminants can be adsorbed or sequestered within the soil
matrix or within particular organisms. Certain plants for example are able to
bioaccumulate metals in above-ground tissues, or sequester them in their roots [19].
2.9 All applications of GMOs in bioremediation strategies are based on the exploitation or
utilisation of a particular interaction between the organism and the target pollutant. In
order to use that interaction effectively, its genetic basis must be understood. Current
applications of GMOs in bioremediation have been developed following the
identification and subsequent manipulation of certain genetic sequences. Future
applications are likely to arise from the modification of existing processes, and also
the use and manipulation of new genetic sequences that are now being identified.
THE USE OF MICROORGANISMS FOR THE BIOREMEDIATION OF
POLLUTANTS
2.10 The ability of bacteria and fungi to utilise a wide range of compounds make
microorganisms ideal candidates for use in the bioremediation of pollutants [8, 20].
Bacteria and fungi have a broad spectrum of metabolic capabilities [21, 22], which
have evolved to enable the microorganisms to utilise natural compounds as sources of
nitrogen, carbon and energy. Several hundred of the genetic systems encoding these
capabilities are reported to be useful in bioremediation applications [7, 23]. However,
because microorganisms have not been exposed to xenobiotic compounds for a
sufficient length of time to develop catabolic pathways that are targeted directly at
those chemicals, then many of the microorganisms that are able to utilise xenobiotic
compounds do so using existing metabolic systems [24, 25]. For example, the
enzymes used by Pseudomonas putida LB400 and Alcaligenes eutrophus H850 to
degrade biphenyl and lightly-chlorinated polychlorinated biphenyls (PCBs) are
reported to have evolved to degrade terpenes produced naturally by plants in the
rhizosphere [26].
2.11 Because the pathways used for the biodegradation of xenobiotic compounds are only
modifications of those employed for the utilisation of natural compounds [27], then it
is likely that the biodegradation of xenobiotics by microorganisms does not occur at
potentially the most optimum rate. This is proposed as one of the reasons for the
continuing persistence of some xenobiotics in the environment [4].
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2.12 Optimisation of the biodegradative pathway is therefore an important consideration if
the bioremediation process is to operate effectively, and be adopted as part of a
commercial strategy. The characteristics of the contaminated site such as the types
and concentrations of contaminants present (including the existence of any toxic or
inhibitory waste types) and the bioavailability of the target compounds are also
important considerations and will influence the rate, extent and success of the
bioremediation process [22].
2.13 GM technology is reported to offer some solutions to the problems of cocontaminants
and bioavailability constraints, and may also be used to develop
monitoring tools to detect for the presence of pollutants in the environment [22].
However, it is the use of GM techniques to alter the metabolic pathways used by
microorganisms to degrade pollutants that offers the greatest potential application of
genetic modification in bioremediation. Few naturally occurring microorganisms
possess the pathways required to mineralise the more recalcitrant xenobiotic
compounds such as pentachlorophenol (PCP) and PCBs [28]. GM technology has the
potential to improve existing catabolic pathways or to extend such pathways to
include additional target compounds that may otherwise not be degraded [10, 13], and
may also be applied to overcome the toxic or inhibitory effect of a particular pollutant
or a metabolite [7].
2.14 Extension of the scope of catabolic pathways through the introduction of additional
genetic sequences, or the alteration of existing genes is reported to offer the simplest
application of GM techniques to bioremediation strategies [20, 29]. Many of the
applications offer a generic approach, particularly in the optimisation of the
bioremediation process.
General Strategies for the Optimisation of Bioremediation Applications
2.15 The degradation of organic pollutants by microorganisms typically occurs in a series
of stages or steps [30]. Each stage of the catabolic pathway is mediated by enzymes
that are produced following the transcription and translation of specific genetic
sequences. Depending on the compound involved, the complete catabolic pathway
may be encoded by a single microorganism, or may require a consortium of
microorganisms, each performing one or more of the stages. Altering various aspects
of the gene sequences that make up the catabolic pathway can improve the efficiency
and efficacy of the pathway at each of these stages. Such alterations are not specific
to individual pathways and may be applied to the biodegradation of all xenobiotic
compounds, if sufficient information is available on the genetic basis of the
degradative pathway.
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Improvements in transcription of the gene sequences
2.16 The genes involved in the degradation of pollutants are usually arranged in operons
carried on wide-host range, conjugative or mobilisable plasmids. Arranging the genes
in operons ensures that transcription occurs in the correct sequence to produce the
required enzymes [27]. Transcription of the catabolic operons is controlled at an
individual operon level and also at a ‘whole cell’ level.
2.17 Individual operons are controlled by positively acting regulatory proteins which are
activated by substrates or metabolites (termed effectors) present in the catabolic
pathway. The control of transcription in this way ensures that the microorganism will
only operate the catabolic pathway in the presence of specific substrates, and will not
waste carbon and energy synthesising unnecessary proteins in the absence of those
substrates [10].
2.18 The amendment of contaminated sites, with compounds that act as effectors for
particular catabolic pathways, may improve the degradation of xenobiotics by
activating the respective catabolic pathway. The promoter Plac is activated by the
addition of isopropyl-ß-D-thiogalactoside (IPTG), and is reported to function well in
Escherichia coli under laboratory conditions [10]. The Ptac promoter also responds to
IPTG and is reported to work better than Plac in a variety of Gram-negative bacteria.
However, IPTG is relatively expensive, and this means that the development of
genetically modified microorganisms (GMMs) with promoter systems based on this
effector are unlikely to be commercially viable for field release applications [10]. The
Pm promoter of the meta-cleavage pathway of the TOL plasmid functions well in a
number of Gram-negative bacteria, and is induced when the XylS regulatory protein
is activated by the presence of benzoate and its derivatives [30, 31]. Unlike IPTG,
benzoate is relatively cheap and is likely to be produced in-situ during the degradation
of aromatic pollutants. Because of its existing use as a food preservative and its poor
persistence in the environment benzoate has been described as an ‘environmentally
friendly’ inducer [10]. Other systems that are reported to be applicable to field
release systems are the salicylate-induced Psal-NahR promoter-regulator, the T7
promoter and the XylR/Pu expression system which controls the upper pathway of the
TOL plasmid and is induced by effectors such as toluene, ethylbenzene and xylene
(present at many contaminated sites) [30, 32]. The T7 promoter has been
incorporated for use in a number of soil bacteria [33, 34]. Future developments in this
area are proposed to include expression systems based on responses to metals, such as
CadC (cadmium regulatory protein) and ArR (regulatory protein for the arsenic
resistance system) [35] and other environmental contaminants [30].
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2.19 Operons, at a ‘whole-cell’ level, are controlled by global regulatory circuits. These
are designed to ensure that the operons operate as part of the overall nutrient and
energy requirements of the microbial cell. The global regulatory circuit responsible
for the coordination of the transcription of catabolic operons is known as catabolite
repression and acts to repress the operons used for the catabolism of certain substrates
when other preferred substrates are available [10]. Therefore where the target
compound is not a preferred substrate, degradation of the target compound may be
blocked by catabolite repression.
2.20 Inactivation of the catabolite repression circuit through genetic modification may
however lead to a reduction in the competitiveness of the microorganism and for the
purposes of bioremediation is therefore undesirable. A preferred approach is to block
the catabolite repression of the specific operon used in the degradation of the target
compound. This can be achieved through the identification and elimination of the
sites in the operon promoter where repression is exerted [10].
2.21 Activation of the catabolic operons occurs when the relevant effector is present at a
sufficient concentration (termed the effector concentration threshold). For example,
the catabolic promoters of the TOL plasmid are activated at an effector threshold
concentration of 5-50 µM [36]. The Pm promoter of the TOL plasmid pWWO in
Pseudomonas putida is activated at a concentration of 1 ppm of benzoate [37]. The
Pm, Pu and Psal promoters, which are activated by alkyl- and halobenzoates, alkyland
halotoluenes, and salicylates respectively, have a broad host range and can
function in a number of genera [37]. However, if the aim of the proposed
bioremediation strategy is to degrade the target pollutant to a concentration lower than
the effector concentration threshold, then an alternative catabolic pathway may be
required. Otherwise, when the pollutant drops to a concentration below the effector
concentration threshold, transcription of the catabolic operon will not occur, and no
degradation will take place. If an alternative catabolic operon is not available then the
microorganism may be genetically modified to place the catabolic genes under the
control of a different promoter that is not activated by the target compound [10].
2.22 The use of alternative promoters means that the activation of the catabolic operon can
be effectively separated from the needs of the microorganism to use the pollutant as a
carbon source. In addition to enabling the pollutant to be degraded to below the level
required to support microbial growth, this approach can also be used to degrade
pollutants that would provide little or no benefit to the microorganism in terms of
carbon or energy, for example trichloroethylene (TCE) [10]. Where the
bioremediative function of the microorganism is separated from the requirement to
use the pollutant as a carbon source, the microorganism must be able to utilise other
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carbon sources to maintain its energy balance. One option that avoids having to add
supplementary carbon sources to the contaminated site is the introduction of the
desired catabolic trait into microorganisms that naturally colonise the rhizosphere [4,
37]. For example, Mondello (1989) [38] reported the genetic modification of a rootcolonising
strain of Pseudomonas with a bph gene cluster used in the biodegradation
of lightly chlorinated PCBs.
2.23 The application of genetic modification techniques to insert alternative promoters also
increases the range of effectors that may activate the biodegradative pathway, and
therefore offers greater flexibility to the bioremediation process [4]. Such effectors
may confer greater transcription of the catabolic operon and ultimately improve the
efficiency of the catabolic process. However, to ensure that the inserted trait works
well in the environment, it is important to use a promoter that is known to function in
the target environment, and then fit the modified genes to that promoter. This is
likely to be more successful than attempting to modify a promoter that works well
under laboratory conditions to operate in the field [37].
2.24 If suitable promoters are not available, then the modified genes can be inserted into
the host microorganism using a minitransposon vector. The location of the modified
genes adjacent to the terminus of the minitransposon will result in the genes being
controlled by a promoter sequence indigenous to the host microorganism. Screening
of the GM microorganism is subsequently required to identify the most suitable
mutant strain [37].
2.25 The use of an indigenous promoter sequence to control the inserted gene may confer a
greater level of expression than the gene’s own promoter sequence. For example,
insertion of the opd gene from Flavobacterium sp ATCC 27551 and from P diminuta
into other Gram-negative bacteria under the control of its own promoter sequence
results in the poor expression of the gene. Expression of the opd gene (which confers
the ability to degrade the pesticides parathion and methyl parathion) is much better if
it is placed under the control of the modified organism’s own promoter [20].
2.26 Expression of the catabolic genes in contaminated environments that are lacking in
sufficient nutrients can be improved if expression of the catabolic genes is linked to
starvation-induced promoters such as groEL [39, 40] and tra [41]. Under conditions
of environmental stress, GroEL can constitute 3-4 percent of total cell protein.
Starvation linked promoters, which provide a universal signal to activate heterologous
gene sequences, are particularly suited to environments where the nutrient conditions
are too low to support exponential growth of the microorganism [37]. Promoters
responsive to carbon, nitrogen, iron and phosphate starvation have been characterised
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in many Gram-negative bacteria [37]. A further advantage of using starvationinduced
promoters is that it avoids the problems (technical, biological and financial)
associated with the introduction of large quantities of nutrients into contaminated
sites. Matin et al., (1995) [42] used starvation promoters from E. coli to control
synthesis of toluene monooxygenase in pseudomonads, with a resulting 60-90 percent
reduction in nutrient demand for transforming a given amount of TCE compared to
wild type microorganisms.
Improving translation
2.27 Improving the translation of a genetic sequence is particularly useful in optimising the
rate-limiting step of a reaction. The rate-limiting stage is often due to the relative lack
of specific protein compared to the others required for the pathway. However, unless
the improvements in translation are targeted, any measures are likely to lead to the
increased production of all proteins and may therefore not necessarily address the lack
of a specific protein (unless that necessary gene is under the control of a separate
promoter).
2.28 Alteration of the translation initiation region (TIR) of the relevant gene by sitedirected
mutagenesis can be performed to ensure that more of the required protein is
synthesised in relation to the other proteins [10]. However, as TIRs are reported to
behave differently in different hosts, then site directed mutagenesis may not solve the
problem when applied from one taxon to another one. Translational enhancers can be
used to ensure that translation of the desired sequence occurs in a range of host
organisms, and will therefore avoid the potential problems of host specificity
encountered with TIRs [37]. Translational enhancers are short sequences (40-50 bp)
present in some plant viruses. The insertion of a translational enhancer from tobacco
mosaic virus enabled the gene for chloramphenicol acetyl transferase to be expressed
in E. coli, S. typhimurium, Erwinia amylovora, A. tumefaciens, A. rhizogenes,
Rhizobium meliloti and Xanthomonas campestris [43].
2.29 Stability of the mRNA will also influence the expression of the desired trait. The
mRNA of gene 32 of the bacteriophage T4 is extremely stable (due to structural
determinants at its 5’ end). The fusion of the native promoter/TIR region of gene 32
to various heterologous genes has been shown to result in the production of more
stable transcripts when expressed in heterologous hosts. The fusion of this transcript
to the xylE gene (encoding catechol 2,3 dioxygenase) has been reported to result in
the expression of high levels of the reporter product in Gram-negative bacteria such as
A. tumefaciens and X. maltophila [44].
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2.30 The introduction of DNA cassettes into the 5’ untranslated region of a gene of interest
is reported to improve the stability of the mRNA by introducing hairpin structures at
the 5’-end of the mRNA [30]. Because the hairpin-containing mRNA had a half-life
three times greater than that of equivalent mRNA with no hairpins, then the
introduction of hairpins increases the amount of mRNA and therefore protein that is
produced [30].
Improving protein stability and activity
2.31 Improvements in transcription and translation will result in an increased production of
the enzymes and proteins required for the biodegradation of the target contaminant.
However, poor stability and activity of the proteins and low substrate specificity may
still restrict the efficiency of the whole biodegradative process [10].
2.32 The stability of a protein may be improved by altering the sequence of its amino acids
[45]. For example, the aromatic ring cleavage enzyme catechol-2,3-dioxygenase is
slowly inactivated by its substrates oxygen and catechol derivatives such as 4-
ethylcatechol. Ramos et al., (1987) [46], reported that by modifying the catechol-2,3-
dioxygenase by two single amino acids caused the enzyme to be less susceptible to
inactivation by 4-ethylcatechol and more stable in the presence of this substrate.
2.33 The activity and substrate specificities of enzymes can be altered through the
development of hybrid gene clusters. These encode subunits of different enzymes to
produce a single enzyme with superior transforming capability. For example, the
degradation of TCE by an E. coli genetically modified to express a hybrid gene
cluster consisting of genes from the toluene metabolic tod operon and the biphenyl
metabolic bph operon, was much faster than non-GM E. coli cells expressing either
the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl
dioxygenase genes (bphA1A2A3A4) [47]. The hybrid cluster consisted of todC1
(encoding the large subunit of toluene terminal dioxygenase in P. putida F1), bphA2
(encoding the small subunit of biphenyl terminal dioxygenase in P. pseudoalcaligenes
KF707), bphA3 (encoding the ferredoxin in P. pseudoalcaligenes KF707) and bphA4
(encoding the ferredoxin reductase in P. pseudoalcaligenes KF707).
2.34 Hybrid enzymes have also been developed to produce a single enzyme system capable
of degrading benzene and tetrachlorobenzene. The enzyme TecA chlorobenzene
dioxygenase is able to dehalogenate partially 1,2,4,5-tetrachlorobenzene but has no
activity against benzene, whereas the TodCBA toluene dioxygenase can dioxygenate
benzene but has no activity against tetrachlorobenzene. The genetic modification of
E. coli to express a hybrid enzyme consisting of the large alpha-subunit of the
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TodCBA dioxygenase whose specific todC1 alpha-subunit subsequences had been
replaced by equivalent sequences of the tecA1 alpha-subunit, resulted in a GMM with
activity towards benzene and tetrachlorobenzene [48].
2.35 Improvements in protein activity can also be developed through ‘forced evolution’ in
which the genes of interest are subjected to random mutagenesis followed by selective
screening for desirable properties [7, 49] or by a more rational approach based on an
understanding of the three dimensional structure of the protein and its structuresequence
relationship [7, 10]. Site-directed mutagenesis of the bphA gene of
Pseudomonas sp LB400 for example increased the specificity of the expressed
biphenyl dioxygenase to include congeners not degraded by the non-GM strain [50].
2.36 However, the information required for the more rational approach exists for relatively
few degradative enzymes [7], including cytochrome P450cam, haloalkane
dehalogenase, dihydroxybiphenyldioxygenase and methane monooxygenase
hydroxylase [10].
Extending the scope of existing catabolic pathways
2.37 Extending the scope of existing catabolic pathways to degrade new compounds avoids
the requirement to develop wholly new degradative pathways and may therefore offer
the most immediate route to the bioremediation of previously non-degradable
xenobiotic pollutants. One route for improving the scope of a catabolic pathway is to
widen the types of effectors that can regulate that particular pathway. This may allow
the catabolic pathway to operate in contaminated sites that do not contain the effectors
used in the non-modified pathway. Studies with the XylS regulator of the catabolic
operon of the TOL plasmid found that although the pathway was activated by
benzoate, some other benzoate analogues such as 4-ethylbenzoate competitively
inhibited the effector-mediated activation of XylS. Mutation of the XylS regulator
resulted in all benzoate analogues being able to activate the system [31]. The
inclusion of new effector compounds may also confer a greater efficiency of
transcription [10].
2.38 Extension of the catabolic pathway to include more substrates is described as either
lateral or vertical. Lateral extension involves the incorporation of more analogues of
existing substrates, and vertical extension describes the addition of totally new
substrates into an existing pathway. Due to the modular nature of many catabolic
pathways, the substrate range of a pathway can often be extended by adding a
biochemically compatible module to the microorganism which enables a new
substrate to be channelled into an existing pathway [10]. For example, the addition of
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dehalogenase genes to microorganisms able to co-metabolise PCBs can extend
existing pathways beyond the chlorobenzoate intermediate and improve the
mineralisation of PCBs by single microorganisms [51]. Lateral expansion of a
pathway can be achieved through the utilisation of isofunctional routes for the
degradation of structurally related compounds, the preferential use of enzymes with
relaxed substrate specificities, and site-directed mutagenesis to alter the specificity of
proteins for their substrates [10].
2.39 The ease with which protein mutagenesis can be used to alter existing pathways to
include previously non-metabolised compounds depends on the number of proteins
that need to adapt before catabolism of the new target compound can occur. Where
only a single protein needs to be altered, the required changes can be achieved by the
direct selection of a mutant able to grow on the target compound [10]. However,
where multiple ‘non-permissive’ proteins require alteration, the necessary mutations
are likely to occur at too low a frequency to produce the desired microorganism. In
this case, the proteins that are non-permissive for the new target compound must be
identified and either sequentially modified to achieve the required specificity, or
replaced by ‘permissive’ proteins from other organisms [10].
2.40 With respect to the catabolism of aromatic compounds, four points have been
identified in the relevant catabolic pathways that are likely to require attention or
alteration if the scope of the pathway is to be increased (either laterally or vertically)
[10]. These are substrate/metabolite-activated transcriptional regulators, the mono- or
dioxygenases that mediate the initial attack on the substrate, the ring cleavage
enzymes and the enzymes that transform the substituted muconolactones to
oxoadipate.
2.41 The inability of Pseudomonas sp B13 to degrade 4-chlorobenzoate (4CB) or 3,5-
dichlorobenzoate (3,5DCB) was found to be due to the narrow specificity of the first
enzyme in the pathway, benzoate 1,2-dioxygenase. This enzyme only allowed the
pseudomonad to degrade 3-chlorobenzoate (3CB) [52]. Insertion of the genes
encoding the isofunctional enzyme toluate 1,2-dioxygenase enabled the bacterium to
degrade 3CB and 4CB. Toluate 1,2-dioxygenase has a much broader substrate
specificity than benzoate 1,2-dioxygenase, and includes all alkyl- and
chlorobenzoates. To degrade 3,5DCB the pseudomonad had to be further modified by
mutation of the XylS regulator, as the existing one was not activated by the
dichlorobenzoate [52].
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Construction of recombinant microorganisms
2.42 Although potentially less straight-forward than the (minor) modification of existing
pathways and/or microorganisms, the construction of recombinant microorganisms
has the potential to provide a number of benefits that may not be available from the
natural selection of non-GM microorganisms. Genetic modification has the potential
to enable both the construction of bacteria with multiple degradative pathways and the
creation of microorganisms that are able to degrade previously non-biodegradable
compounds.
2.43 The development of recombinant microorganisms with the capability to degrade
xenobiotic compounds completely may be preferable to the use of consortia of non-
GM microorganisms, where most stages of the catabolic pathway are conducted by
different microorganisms. The limitations with a consortium based approach to
biodegradation are that pathway intermediates may need to be transported between
cells resulting in loss of efficiency of catabolism [53], and that metabolites may be
misrouted into dead-end incomplete pathways by some members of the consortium, or
transformed into toxic metabolites that may subsequently destabilise the entire
process [10]. The use of genetic modification, to ensure that the entire catabolic
process is conducted by a single cell may have the potential to avoid these problems.
2.44 The biodegradation of chloro- or methylcatechol (formed during the degradation of
chloro- and methylaromatic compounds) is a well-studied example of the potential
problems associated with the misrouting of pathway intermediates [53, 54]. Although
many soil microorganisms possess the abilities to degrade chlorocatechol by the ortho
cleavage pathway and methylcatechol by the meta cleavage pathway, both pathways
are only expressed in the presence of the respective substrate. Very few strains of
microorganisms are able to grow effectively on mixtures of methylated and
chlorinated aromatic compounds [54].
2.45 Exposure to either group of compounds on their own does not result in the production
of dead-end or inhibitory metabolites. However, in contaminated sites the presence of
chloro- and methylaromatic compounds means that both pathways are likely to be
induced and this can lead to inefficient degradation [54]. Catabolism of
methylcatechol by ortho cleavage results in the formation of dead-end methyllactone
metabolites [54], and the compounds formed during the meta cleavage of
chlorocatechol inhibit the activity of the aromatic ring cleavage enzyme catechol 2,3-
dioxygenase [53, 55].
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2.46 To avoid the formation of potentially inhibitory metabolites, Erb et al., (1997) [53]
developed a GM pseudomonad (designated Pseudomonas sp B13 SN45RE) that was
able to degrade both chloro- and methylaromatic compounds by the ortho cleavage
pathway. Another pseudomonad (Pseudomonas sp B13 FR1(pFRC20P)), genetically
modified to utilise chloro- and methylaromatic compounds by a constructed ortho
cleavage pathway was able degrade a mixture of 3-chlorobenzoate and 4-
methylbenzoate (25 µM of each) in intact sediment cores with an overlying water
column throughout a 4 week period [56], highlighting the potential for GMMs to be
used in the environment to overcome problems inherent with non-GM
microorganisms.
2.47 Analysis of the degradation of 3CB and 4MB by Pseudomonas sp B13
FR1(pFRC20P) in water and in sediment microcosms found that the observed and
theoretical half-lives corresponded well, indicating that GM pseudomonad functioned
optimally in these environments [57]. The physiological characteristics of the GMM
and its performance in aquatic microcosms are assessed to make this microorganism a
good candidate for in situ bioremediation at sites contaminated with mixtures of
chlorinated and methylated aromatics [57].
2.48 Genetic modification can also be used to develop microorganisms that are more
tolerant to the environmental stresses likely to be encountered in a contaminated
environment. Although microorganisms indigenous to the contaminated site are
likely to be adapted to the stresses to some extent, stress factors such as poor oxygen,
water and nutrient availability, high concentrations of toxic pollutants and extreme pH
may be expected to have some restriction on the level of in situ biodegradation that
occurs. Genetic modification has the potential to confer improved tolerance or
resistance to the likely stresses, and consequently improve the performance of the
degradative microorganism [10]. The coupling of the catabolic genes to promoters
that are responsive to environmental stresses such as low pH enables the degradative
properties of the GM microorganism to be enhanced in environments where those
stresses are likely to be realised, such as leachate from acid mine waste. Placing the
catabolic genes under the control of such promoters also means that the expression of
those genes is restricted to the particular ‘stressed’ environments (see Chapter 4).
Use of Bacteria for the Bioremediation of Organic Pollutants
2.49 Applications to bioremediate organic pollutants that have been reported to date (GM
and non-GM) have focused on the use of bacteria rather than fungi. Although both
groups of microorganisms have a number of unique advantages that are applicable to
their use in bioremediation strategies, it is likely that the ease of culture of bacteria
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and the greater level of understanding of bacterial genetic sequences are the factors
that are ultimately responsible for the more widespread application of bacteria in
bioremediation strategies. Bacteria are also reported to be more amenable to genetic
modification and are capable of metabolising chlorinated organic compounds [20, 58].
2.50 With the exception of the application of cytochrome P450 systems, the use of GM
bacteria for the bioremediation of organic pollutants is presented in this report in
sections according to the target compound. Applications to use GM bacteria in the
environment are likely to be targeted at specific pollutants such as PCBs or
nitroaromatic compounds. Cytochrome P450 systems have a broad substrate
specificity and potentially have applications in the biodegradation of a wide range of
organic pollutants. The information presented reviews the current state of the science
regarding the use of GM bacteria to bioremediate organic pollutants, and where
possible, provides an indication of future developments in this field.
2.51 The most likely area of future development is the modification of microorganisms
with the genes currently being identified as important in the degradation of
xenobiotics by indigenous microorganisms. As the level of understanding of the
genetic basis of pollutant degradation increases so the potential for GMMs in
bioremediation applications will also increase. Other possible developments include
the use of genetic modification to provide ‘support’ to other bioremediation processes
(GM or non-GM). Examples include the use of GMMs to prevent shock loading of
natural microflora by toxic contaminants in wastewater treatment plants [53] and the
genetic modification of drought or desiccation tolerant microorganisms. In addition
to their use in dry environments, drought tolerant microorganisms may be more
suitable for use in commercial bioremediation applications where the microorganisms
are likely to be stored for a period of time until they are required in the field [59].
Cytochrome P450s for the bioremediation of organic pollutants
2.52 Cytochrome P450s are found in microorganisms, plants and animals and perform a
range of chemical reactions, many of which are central to the degradation of organic
pollutants, such as the cleavage of both ether and carbon-chlorine bonds [10, 49].
Microbial P450s are particularly applicable for the bioremediation of xenobiotics
because of their ubiquity and strong reductive and oxidative potential [49]. The
P450cam from P. putida for example is capable of dehalogenating halogenated
methane and ethane substrates such as hexachloroethane and pentachloroethane (to
tetrachloroethylene and trichloroethylene respectively) [49].
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2.53 The potential of P450s for bioremediation of organic pollutants has developed from
an improving understanding of the structure-function relationships in the various
cytochrome P450s that have been isolated and characterised to date. The significant
difference between the tertiary structure of individual microbial P450s is due to
variations in structural interactions that form the substrate-binding pocket of the
enzyme [49]. A number of reports have shown that by engineering changes in the
design and structure of the substrate-binding pocket, the substrate specificity, range
and catalytic efficiency of the enzyme can be altered [49, 60, 61].
2.54 Jones et al., (2000) [62] reported that reducing the volume of the active site of the
haem monooxygenase cytochrome P450cam enzyme expressed by P. putida through
the substitution of amino acids with bulkier side chains improved the enzymesubstrate
fit, resulted in extending the substrate range of modified microorganism
from chlorophenol to include polychlorinated benzenes. Reducing the size of the
active pocket of P450cam has also been found to increase the rate of dehalogenation of
1,1,1,2-tetrachloroethane by up to 150 percent compared to the non-modified wild
type (Sligar et al., 1996, cited by) [49]. Altering the substrate-binding pocket to
increase the space available is reported to confer a more relaxed substrate specificity
[63].
Chlorinated compounds
2.55 Chlorinated compounds have been detected as pollutants in many contaminated sites.
The presence of these compounds is a consequence of their use by the chemical
industry for a wide variety of applications, and also due to their formation during the
degradation of polychlorinated compounds such as PCBs [29]. The principal groups
of chlorinated compounds which have been targeted by bioremediation applications
are the chlorobenzoates (CBAs) and dichlorobenzoates (DCBs); tetrachloroethene
(PCE) and trichloroethylene (TCE); the chlorobenzenes (CB) and chlorinated
herbicides such as 2-methyl-4-chlorophenoxyacetate (MCPA), 2,4-
dichlorophenoxyacetic acid (2,4-D) and atrazine.
2.56 Many of the pathways involved in the biodegradation of chlorinated compounds share
similar metabolites, and single enzymes are often able to catalyse the degradation of a
number of different compounds. Chlorinated catechols for example are key
intermediates in the degradation of chlorinated aromatic compounds, and the enzyme
toluene 4-monoxygenase has activity against TCE, toluene, ethylbenzene, acetanilide,
2-phenylethanol and phenol [10, 64, 65]. The similarities of many of the catabolic
pathways for chlorinated compounds do provide some advantages in the development
of GMMs able to degrade a number of contaminants. However, the presence of a
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number of chlorinated compounds in a single contaminated site may inhibit the
degradation of particular compounds and result in the production of dead-end or toxic
metabolites [53].
2.57 Compounds such as TCE are particularly suitable as targets for bioremediation by
GMMs, as non-GM microbial degraders require the addition of specific substrates
such as methane, ammonia, cresol or toluene, which are themselves environmental
pollutants. The removal of TCE by chemical and/or physical-based processes is
expensive and does not result in the complete degradation of the compound,
respectively [66].
2.58 A number of studies have been conducted using E. coli and P. putida genetically
modified to express the toluene monoxygenase genes from Pseudomonas mendocina
KR1, and to degrade TCE in the absence of the types of substrate inducers described
above [67]. Winter et al., (1989) [68] constructed a GM E. coli to degrade TCE in the
presence of glucose. Because the inserted genes were placed under the control of the
temperature inducible lambda phage promoter (P1), the cloned toluene monoxygenase
genes were only expressed at a temperature of 42 ºC and above. The use of this
GMM was therefore confined to a bioreactor bioreactor-based facility, the inserted
genes would not be expressed if the GMM was released into the environment. The
use of temperature sensitive promoters therefore represents a form of biological
containment for GMMs.
2.59 E. coli and P. putida have also been genetically modified to degrade TCE through the
insertion of a range of phenol catabolic genes (pheA, pheB, pheC, pheD and pheR)
carried on plasmid pS10-45)2 isolated from P. putida BH [66]. Although this
microorganism is capable of degrading TCE in the presence of phenol, it is unable to
degrade phenol, as were the E. coli JM103, E. coli HB101 and P. putida KT2440 used
as host strains for the genetic modification. Modification of the E. coli strains with
plasmid pS10-45 enabled them to degrade TCE at a concentration of 20 ppm.
Comparison of the TCE degradation (1 ppm) by E. coli HB101, P. putida KT2440
and the non-GM P. putida BH showed that the recombinant E. coli strain removed the
TCE more efficiently and to a greater extent than the pseudomonad strains. This was
due to the inhibition of the pheA gene in the pseudomonads by the TCE metabolite
TCE-epoxide. The E. coli was apparently unaffected by this compound. The results
highlight the importance of selecting host organisms for genetic modification that are
2 Genes encoding phenol hydroxylase, catechol-2,3-dioxygenase, 2-hydroxymuconic semialdehyde
dehydrogenase, 2-hydroxymuconic semialdehyde hydrolyase and the positive regulation of the phenol catabolic
operon respectively.
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not sensitive to the formation of metabolites likely to be produced during degradation
of the target compound [66].
2.60 The addition of phenol to a contaminated site to induce degradation of TCE may
however be undesirable if the phenol itself has a toxic effect to the flora and/or fauna
present. GM technology has been applied to develop microorganisms that have a
similar capability to degrade TCE, but in the absence of potentially toxic substrate
inducers such as phenol. Krumme et al., (1993) [69] compared the degradation of
TCE by the non-GM P cepacia G4 that was able to degrade TCE in the presence of
phenol and tryptophan, with the GM P cepacia G4 5223 PR1 that expressed the
toluene ortho-monooxygenase constitutively. Both microorganisms degraded TCE in
groundwater microcosms from 50 µM to <0.1 µM in 24 h at a cell density of 108 cells
ml-1 and both strains were also able to survive in aquatic sediment microcosms for a
period of 10 weeks. In the environment the GM strain is therefore likely to behave in
a similar way to the non-GM strain, and should therefore survive and compete with
the natural microflora. However, the use of the GM strain may be preferred to the
phenol dependent non-GM strain in field based bioremediation applications, should
the use of phenol be of environmental or regulatory concern [70].
2.61 Where the GMM is unable or unsuited to survive in the field environment for a
sufficient period of time to degrade the target contaminant, then the microorganism
may be more suited to a bioreactor based bioremediation application, such as ‘pump
and treat’ where a more contained and regulated environment for the microorganisms
can be maintained [71, 72]. The ability of the microorganism to survive in the
environment in which it is intended to be used is as important a consideration as the
capability of the organism to degrade the target pollutant.
2.62 Many of the applications involving microorganisms in bioremediation (GM and non-
GM) use heterotrophic soil microorganisms [73]. Whilst these organisms are likely to
be reasonably well suited to survive in most soil and sediment environments, the poor
nutrient conditions that are characteristic of many aquatic environments may be more
suited to photoautotrophic microorganisms such as cyanobacteria. The use of such
organisms would avoid the addition of organic nutrients to the inoculated
environment, thereby reducing the costs incurred and maintenance required.
2.63 Two strains of cyanobacteria Anabaena sp and Nostoc ellipsosporum were genetically
modified by insertion of linA (from P. paucimobilus) and fcbABC (from Arthrobacter
globiformis) respectively. The gene linA controls the first step in the biodegradation
of lindane (γ-hexachlorocyclohexane), and fcbABC confers the ability to biodegrade
halobenzoates. The GM Anabaena sp showed enhanced degradation of lindane
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compared to the wild type strain, and the GM N. ellipsosporum was found to able to
degrade 4CB, a capability not expressed by the wild type strain [73].
2.64 Strong et al., (2000) [11] avoided the issue of survival of the GMM in the
environment by using an inoculum of killed Escherichia coli cells to remediate soil
contaminated with the s-triazine herbicide atrazine under field conditions. The E. coli
had been genetically modified to overexpress the atzA gene, isolated originally from
Pseudomonas ADP. The atzA gene encodes the production of the enzyme atrazine
chlorohydrolase that dechlorinates atrazine in a single step to hydroxyatrazine. This
product is non-toxic to plants [11].
2.65 GM E. coli cells were grown under laboratory conditions and then killed and crosslinked
by exposure to 0.3 percent glutaraldehyde. Cross-linking the cells was
reported to retain more than 50 percent of the cell’s enzyme activity, for up to eight
months storage in buffer at room temperature. This was reported to compare
favourably with the storage of purified enzymes [11]. Inoculation of the atrazine
contaminated soil with 0.5 percent (w/w) of killed E. coli cells reduced the atrazine
concentration by 77 percent (from 6700 ppm to 1450 ppm) in 8 weeks. This level of
reduction required the supplementation of the soil with phosphate (300 ppm),
although in the absence of the killed cells, the addition of phosphate had no
significant effect on atrazine levels.
2.66 The complete degradation of polychlorinated compounds such as pentachloroethane
and PCBs requires the sequential occurrence of anaerobic and aerobic reactions [25].
Degradation proceeds by an initial reductive dehalogenation reaction (under anaerobic
conditions), which converts the parent compound to the substrates susceptible to
attack by bacterial oxygenases produced under aerobic conditions [25]. The
requirement for two sets of degradation pathways poses a challenge for the
development of a single microorganism with the capability to degrade such
compounds completely. (The advantages of using a single microorganism to encode
the entire degradative process have been presented earlier in this report). Wackett et
al., (1994) [25] used the cytochrome P450cam and toluene dioxygenase (encoded by
the todC1C2BADE genes) systems to catalyse the consecutive reductive and oxidative
dehalogenation reactions (respectively) required to degrade pentachloroethane. In the
presence of camphor P. putida G786 (pDTG351) converted pentachloroethane to
TCE, and aeration of the culture with oxygen resulted in the reduction of TCE levels.
Utilisation of TCE by this GMM was improved by placing the tod genes under the
control of a hybrid tac promoters [25].
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2.67 The same system has also been applied to the biodegradation of polybrominated and
chlorofluorocarbon compounds [74]. Under anaerobic conditions, the GMM
(designated P. putida G786 (pHG-2)) [74] reduced 1,1,2,2-tetrabromoethane, 1,2-
dibromo-1,2-dichloroethane and 1,1,1,2-tetrachloro-2,2-difluoroethane to less
halogenated compounds. Under aerobic conditions, the GMM oxidised cis- and
trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene and 1,2-dichloro-1-
fluoroethene [74].
Polychlorinated biphenyls
2.68 The application of GMMs to the biodegradation of PCBs has only been reported for
the aerobic part of the degradation pathway, i.e the degradation of lightly-chlorinated
biphenyls. Dehalogenation of the more highly-chlorinated biphenyls, which requires
anaerobic conditions [75] is reported to be less amenable to manipulation, by genetic
modification, with fewer potential improvements available [76].
2.69 The aerobic biodegradation of PCBs proceeds in a similar manner to the metabolism
of biphenyl [77], and consists of an upper pathway involving the oxidation of the PCB
congener to the corresponding chlorobenzoate, and a lower pathway through which
the chlorobenzoate undergoes complete mineralisation [78]. However, many PCB
degrading microorganisms are unable to degrade PCBs beyond the formation of the
chlorobenzoate, particularly where the microorganisms are exposed to a number of
different PCB congeners3.
2.70 The initial stages of the aerobic pathway involve the insertion of two atoms of
molecular oxygen at the 2 and 3 positions of one of the biphenyl rings, and are
catalysed by the enzyme 2,3-dioxygenase. The differing ability of microbial
degraders to utilise different PCB congeners may be due to the organisms possessing
single non-specific dioxygenases or multiple dioxygenases. Gibson et al., (1993) [79]
dismissed the idea of multiple dioxygenases and proposed that differences in substrate
specificity of two species of pseudomonad was due to differences in as few as two
amino acids in BphA. Further studies have supported this proposal and have
demonstrated that the BphA1 subunit of the biphenyl dioxygenase (encoded by
bphA1) is responsible for the recognition of the PCB molecule and consequently the
range of congeners that can be degraded by the particular microorganism [80]. This
observation has enabled the bphA1 gene to be modified by site-directed mutagenesis,
to extend the congener specificity of PCB degrading microorganisms. The
3 PCB congeners differ in the number and position of the chlorine atoms on the biphenyl. Because PCBs were
always manufactured as mixtures of different congeners then environments contaminated with PCBs are likely
to contain a range of different PCB congeners.
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modification altered the Thr-376 to Asn-376 in the biphenyl dioxygenase and
extended the congener specificity of the P pseudoalcaligenes KF707 [80, 81].
2.71 The genes that encode the capability to degrade PCBs are organised on the bph
operon, and include bphA1, bphA2, bphA3, bphA4, bphB, bphC and bphD [13]. The
genes bphA1 to bphA4 encode a multicomponent dioxygenase enzyme that catalyses
the degradation of the biphenyl to biphenyl-dihydrodiol. This compound is degraded,
by the bphB gene product, to 2,3-dihydroxybiphenyl. Gene bphC encodes another
dioxygenase which cleaves the 2,3-dihydroxybiphenyl to form a meta cleavage
compound which is then converted to benzoic acid and a pentanoic acid derivative by
the product of the bphD gene [13, 76, 82].
2.72 Overexpression of the bph pathway in Pseudomonas sp LB400 resulted in the GMM
being able to degrade a greater percentage of lightly chlorinated PCBs, compared to
the wild type, and also to degrade the normally recalcitrant 2,4,5,2’,4’,5’-chlorinated
derivative [38]. The genetic modification of the toluene degrading microorganism P.
putida F1, by insertion of the bphD gene cluster, allowed the pseudomonad to grow
on biphenyl [10], and the fusion of the bph and tod (toluene-degrading) operons and
subsequent insertion into a PCB degrading strain of Pseudomonas, enabled the GMM
to degrade both toluene and biphenyl.
2.73 Dowling et al., (1993) [83] reported the development of a transposable genetic
element TnPCB designed to insert the bph operon (from Pseudomonas sp LB440) into
the chromosome of a range of Gram-negative bacteria, including rhizospherecompetent
pseudomonads. Other methods used to insert novel traits into the
chromosome of rhizosphere pseudomonads have required homologous recombination
between chromosomal sequences, which reduces the range of potential recipient
organisms. The introduction of TnPCB into the recipient organism using plasmid
pDDPCB is reported to avoid this problem. Once inserted, TnPCB is reported to
remain stable in the chromosome, with no detectable lateral transfer of the bph genes
to other microorganisms [13].
2.74 The insertion of TnPCB into the rhizosphere-competent pseudomonad P. fluorescens
F113pcb enabled the microorganism to degrade biphenyl as a sole carbon source. The
bph operon was found to be expressed constitutively in the GM pseudomonad, and
importantly had no effect on the rhizosphere fitness (including ability to colonise or
be maintained on plant roots) of the GMM compared to the wild type [13]. The
genetic modification was also stable within the chromosome for at least 25 days in the
absence of positive selection for PCB degrading or biphenyl degrading
microorganisms [13].
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2.75 The introduction of the bph-module into P. putida KT2442 and Pseudomonas sp
B13FR1 expanded the biodegradative ability of these two strains to include biphenyl
and 4-chlorobiphenyl. The survival and function of the GMMs in the environment
was assessed using lake sediment microcosm. The GMMs were able to survive under
simulated natural conditions in the microcosms, and degraded 4-chlorobiphenyl over
a five day incubation period [83].
2.76 The accumulation of chlorobenzoates during the degradation of PCBs by some
microorganisms is a consequence of the inability of PCB degrading microorganisms
to utilise these compounds (particularly chlorobenzoates with a chlorine atom on both
rings4), and the inhibitory effect of the chlorobenzoate metabolites chlorocatechol and
chloromuconate semialdehyde on the enzyme 2,3-dihydroxybiphenyl-1,2-
dioxygenase [85]. Inhibition of this enzyme has a negative feedback effect on the
degradation of the PCB molecule. Several publications have reported the
development of GMMs designed to degrade Cl,Cl’-PCB by pathways that avoid the
formation of inhibitory metabolites [84, 85]. The production of inhibitory
chlorocatechols and the chloromuconate semialdehydes is due to the activity of broadsubstrate
specificity benzoate-1,2-dioxygenases. The elimination of the genes
encoding these enzymes by mutagenesis, and insertion of the cba genes (isolated from
Alcaligenes BR60) carried on the catabolic transposon Tn5271 caused the GM E. coli
and GM Alcaligenes sp to degrade chlorobenzoate to protocatechuate and
chlorodihydroxybenzoate which were not inhibitory to the microorganism [85].
Although the application of these GMMs in the environment was not addressed, the
findings demonstrate the importance of considering the formation of toxic
intermediates when designing bioremediation strategies, as well as the ability of the
microorganism to degrade the parent compound [86].
2.77 An alternative approach to overcoming the accumulation of chlorobenzoates by PCB
co-metabolising microorganisms is the introduction of dehalogenase genes into these
microorganisms. The rational for this, is that the microorganisms able to degrade
PCBs to chlorobenzoate and chloropentadiene are also often able to degrade nonchlorinated
benzoate and pentadiene as part of other existing metabolic pathways
[51]. The introduction of the genes, necessary to enable these microorganisms to
dehalogenate the chlorobenzoates and chloropentadiene, is proposed to allow a more
complete degradation of PCBs by a single population of microorganisms, rather than
by consortia.
4 Designated Cl,Cl’-PCB [84]. McCullar MV, Brenner V, Adams RH and Focht DD (1994). Construction of a
novel polychlorinated biphenyl-degrading bacterium - Utilization of 3,4’-dichlorobiphenyl by Pseudomonas
acidovorans M3GY. Applied and Environmental Microbiology, 60(10): 3833-3839.
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2.78 Hrywna et al., (1999) [51] genetically modified the PCB metabolising Comamonas
testosteroni VP44, so that it was able to grow on, dechlorinate and completely
mineralise ortho- and para-substituted monochlorobiphenyls. The C. testosteroni was
modified by insertion of either plasmid pE43 from Pseudomonas aeruginosa 142
(encoding the oxygenolytic ortho-dechlorination ohb gene), or plasmid pPC3 from
Arthrobacter globiformis KZT1 (encoding the hydrolytic para-dechlorination fcb
gene). The GMM was able grow on and completely dechlorinate high concentrations
(up to 10mM) of 4-CBA (chlorobenzoate), 4-CB (pentadiene), 2-CBA and 2-CB
depending on the modification. The non-GM parent strain was only able to grow on
non-chlorinated benzoate and pentadiene [51].
2.79 Biodegradation of PCBs, particularly in contaminated soils may also be improved
through the application of surfactants. The addition of these compounds to
contaminated sites has a dual purpose in that they improve the bioavailability of the
hydrophobic PCBs, and also provide the PCB degrading microorganisms with a
readily available carbon source. Non-ionic surfactants are reported to be more
suitable than anionic or cationic surfactants, due to their lower toxicity to
microorganisms [82]. Lajoie et al., (1997) [82] genetically modified P. putida and
Ralstonia eutropha (formerly Alcaligenes eutrophus) with the transposon TnPCB
containing the biphenyl/PCB degradative operon (genes bphA1, A2, A3, A4, B, C, K, H,
J, I and D). Both organisms were also capable of utilising surfactants as a source of
carbon and energy. The GMMs were reported to be capable of degrading individual
congeners in Aroclor 1242 over a 20 day period, and may offer a suitable system for
the degradation of PCBs in the environment.
2.80 GMMs designed to utilise a selective substrate such as a surfactant, and express an
inserted gene are described as field application vectors (FAVs) [82]. FAVs offer a
number of advantages over other bioremediation systems designed to degrade
hydrophobic compounds such as PCBs. The addition of surfactants purely to improve
the bioavailability of the pollutant may inhibit the activity of the pollutant degrading
microorganisms [87]. This may be avoided through the use of microorganisms able to
degrade the surfactant and pollutant [88]. Naturally occurring microorganisms are
unlikely to be able to utilise synthetic surfactants such as Ipegal CO-720 [88].
Therefore, the ability of the FAV microorganism to use the surfactant as a source of
carbon and energy provides the organism with a unique substrate, and increases the
chances of survival following addition to the contaminated environment. Also,
because the bph genes inserted into the FAV are controlled by constitutive promoters,
and the microorganism is able to use the surfactant as a growth substrate, then the
growth of the microorganism and the degradation of the PCB are effectively
decoupled from biphenyl [88]. This avoids the addition of biphenyl to the
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contaminated site to induce degradation of the PCBs, and means that in the absence of
biphenyl, there in no selective pressure to discard the bph genes [88].
2.81 The initial reports of the use of FAVs for the biodegradation of PCBs used P
paucimobilis IGP4 (subsequently reclassified as Sphingomonas paucimobilis)
genetically modified to express the PCB degradative genes bphABC. Although this
organism was able to degrade Aroclor 1242 (a commercial mixture of PCB
congeners) in moist agricultural soil, the bph genes, which were plasmid encoded,
were not stably inserted in the GMM. Attempts to insert transposon-encoded genes in
this strain were not successful [88].
2.82 Stable insertion of the recombinant genes into the GMM used in the FAV is important
if the system is to be used to bioremediate PCBs in a field situation. Lajoie et al.,
(1994) [88]compared the two FAV systems for their ability to degrade PCBs, and also
the stability of the inserted bphABCD genes. The P. putida IPL5 was genetically
modified using plasmid encoded (pPCB), or transposon encoded (TnPCB) genes.
Both FAVs were able to degrade some PCB congeners when inoculated into
surfactant amended soil slurries, although the activity in the soil slurries was lower
than in liquid culture, suggesting that the FAV system is affected by bioavailability
constraints in soil. The growth of GMMs in non-sterile soil slurries (105 to 109 cells
ml-1 in two days) indicated that competition with the indigenous microflora was
minimal [88].
2.83 In soil, the GMM modified using the plasmid pPCB showed greater activity against
the PCB congeners, both in terms of numbers of congeners degraded, and the level of
degradation achieved [88]. The plasmid encoded genes were however less stable than
the transposon. The greater activity of IPL5(pPCB) towards the PCB congeners was
reported to be due to a higher level of expression of the bph genes in IPL5(pPCB)
than in IPL5::TnPCB, although this could have been due to the presence of two or
more copies of pPCB. Higher levels of expression of the degradative genes may also
be a consequence of more effective channelling of carbon and energy from the
surfactant to the production of the PCB degrading enzymes [88].
2.84 Further studies with PCB degrading FAVs based on the TnPCB transposon found that
activity towards PCB congeners was inhibited in the presence of biphenyl, and that
the degradation of PCB congeners (Aroclor 1242) was greater using a mixed culture
of P. putida IPL5::TnPCB and Ralstonia eutrophus B30P4::TnPCB than with pure
cultures of either strain [82].
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2.85 Excess overexpression of the nonadaptive pathways, like that used in the FAV may
cause the growth rate of the GMMs to decrease. In a competitive environment this
would increase the selective pressure against the maintenance of the bph genes. The
balance between gene expression and growth is reported to be an important
consideration in the development of FAVs for environmental applications [88]. The
use of FAVs does however represent a potentially successful application of GMMs in
the bioremediation of hydrophobic pollutants such as PCBs or polyaromatic
hydrocarbons (PAHs), either in contaminated soils, or soil washing systems [82].
Hydrocarbons
2.86 Environmental contamination by xenobiotic hydrocarbon compounds is largely a
consequence of the spillage or improper disposal of oil. Such contamination may
consist of a wide range of different compounds, ranging from the low molecular
weight unbranched aliphatic hydrocarbons, which are relatively easily biodegraded by
naturally occurring microorganisms, to the higher molecular weight branched and
aromatic compounds which are more recalcitrant. Bioremediation of hydrocarbons
has focused on two groups of the more recalcitrant compounds. These are the PAHs
and benzene, toluene, ethylbenzene and xylene (BTEX) compounds [89].
2.87 The PAH naphthalene was the target compound in the only field-based
bioremediation application of live GMMs that has been conducted to date [23]. (The
only other field release of a GMM conducted to date for a bioremediation application
used an inoculum of killed E. coli genetically modified to degrade atrazine) [11]. The
microorganism used by Sayler et al., (2000) [23] was Pseudomonas fluorescens strain
HK44 which had been genetically modified to express the naphthalene catabolic
plasmid pUTK21 which had itself been mutagenised by a transposon-inserted lux
gene5. [90]. The parent strains of P. fluorescens (5R and HK9) from which the GM
HK44 had been derived were isolated originally from soil at a manufactured gas plant
facility [91].
2.88 The bioluminescent GM P. fluorescens HK44 was constructed in two stages. The
first involved filter matings between E. coli HB101 (containing the lux transposon
Tn4431 carried on the suicide vector plasmid pUCD623) with P. fluorescens 5R
5 The GMM is reported as the first recombinant microorganism to undergo full USEPA biotechnology risk
assessment review and achieve environmental release status for applications in bioremediation [90] Ripp S,
Nivens DE, Ahn Y, Werner C, Jarrell J, Easter JP, Cox CD, Burlage RS and Sayler GS (2000). Controlled field
release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and
control. Environmental Science & Technology, 34(5): 846-853. Further information on the release of this GMM
and the regulatory process that was undertaken in order to conduct the release is presented in the report of the
workshop at the end of this document (presentation by Prof Sayler).
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(containing the upper and lower pathway operons for naphthalene degradation on
plasmid pKA1). The bioluminescent construct P. fluorescens 5RL (containing
plasmid pUTK21) was selected from the matings illustrated in Figure 2.1 on the basis
of its ability to produce strong inducible light when exposed to naphthalene vapour or
when grown in the presence of salicylate [91].
Figure 2.1 - Construction of bioluminescent reporter plasmid pUTK21) [91]
pUCD613 pKA1
pUTK21
Tn4431 (lux)
upper pathway
lower pathway
Tn4431 (lux)
2.89 The catabolic pathway for naphthalene is encoded by two operons. The upper operon
encodes for the conversion of naphthalene to salicylate, and the lower pathway for the
oxidation of salicylate to acetaldehyde and pyruvate [91].
2.90 The strain selected from the filter matings was designated P. fluorescens 5RL. This
GMM was able to degrade naphthalene to salicylate, which then accumulated,
indicating that the transposon Tn4431 had inserted itself in the lower operon of the
naphthalene pathway (Figure 2.1). The second stage, in the generation of HK44
therefore, involved transferring pUTK21 into another P. fluorescens capable of
oxidising salicylate [91]. P. fluorescens HK9 was able to degrade salicylate but not
naphthalene. Transfer of pUTK21 from P. fluorescens 5RL to HK9 by conjugation
produced P. fluorescens HK44 that had a Nah+Sal+ phenotype and the same light
producing characteristics as strain 5RL [91].
2.91 The construction of the GM HK44 meant that the naphthalene degradation genes and
the lux gene were under the control of the same naphthalene induced promoter.
Exposure of the GMM to naphthalene (or the intermediate metabolites salicylate or 4-
methyl salicylate) resulted in the increased expression of the naphthalene catabolic
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genes and also caused the microorganism to bioluminesce. The bioluminescence was
monitored on-line during the release using fibre optics and photon counting modules.
The advantage of this system was that as the level of bioluminescence was assessed to
be proportional to the rate of naphthalene degradation, then monitoring levels of
bioluminescence allowed the bioremediation rate to be monitored in situ without the
need for chemical analyses of the contaminated soil. Bioluminescence monitoring is
also non-invasive, non-destructive, rapid and population specific [91].
2.92 The GM P. fluorescens HK44 were released into subsurface lysimeters 4 m deep and
2.5 m in diameter containing layers of gravel (31 cm) at the base, then coarse sand (61
cm), uncontaminated soil (92 cm), PAH contaminated soil (92 cm) and a cap of
uncontaminated soil (61 cm) (Figure 2.2). The PAH contaminated soil contained
1000 mg kg-1 naphthalene, 100 mg kg-1 anthracene and 100 mg kg-1 phenanthrene
[90]. The use of lysimeters meant that the release of the GMMs was conducted under
replicated and physically contained conditions. The scale of the test, and the location
of the lysimeters in the field (Figure 2.3), meant that the GMMs were subjected to
environmentally relevant conditions of temperature, water availability and humidity,
and that the study was an accurate approximation of the natural subsurface
environment [23]. The use of non-sterile soil, both for the uncontaminated and
contaminated soil layers meant that the GMMs were exposed to competition from
other soil microorganisms. Although the GMM only had activity against naphthalene,
the additional presence of phenanthrene and anthracene meant that the GMMs were
exposed to a heterogeneous mixture of PAHs which is more representative of a PAH
contaminated environment. The PAHs were added to the soil 260 days prior to
inoculation with microorganisms to allow for PAH sorption and soil weathering [90].
2.93 The population dynamics of the P. fluorescens HK44 were similar in all inoculated
soils (in the presence and absence of PAHs). After an initial inoculation density of
1x106 cfu g-1 soil, numbers of the GMM decreased rapidly during the first 12 days to
a density of approximately 1x105 cfu g-1 soil. This reduction was followed by a more
gradual decline throughout the duration of the trial, with a final population of
approximately 1x103 cfu g-1 soil after 660 days. After 444 days, the GMMs still
showed activity to PAH contaminants, although only in the presence of inorganic
nutrients [90]. The availability of PAHs (used by the GMM as the primary carbon
source and electron donor) and oxygen (used by the GMM as the electron acceptor)
were found to be the significant factors affecting in situ metabolic activity, growth
and aerobic biodegradative activity of the GMM [90].
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Figure 2.2 - Schematic diagram of the lysimeter containing PAH contaminated soil
Gravel
Coarse Sand
Contamination Zone
(GEMs and Hydrocarbon)
Clean Soil Irrigation
Air Distribution System
Manifold
Central Core
Light Sensing
Instrument
Leachate
Ground Water
Supply
O2 Temp Moisture CO2
Fiber 0ptic
Cables PVC Pipes
for Biosensors
Removable Cover
Air
Inlet Liquid
Inlet
Clean Soil
Figure 2.3 - Aerial photograph of lysimeters in the field. Photograph taken prior to
filling the lysimeters and demonstrates the scale of the operation
photograph of lysimeters
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2.94 The results of the lysimeter studies demonstrated that GMMs designed for use in
bioremediation applications can be maintained viably in a field environment [90].
However, the application of GMMs for bioremediation in subsurface environments
may be restricted by inadequate dissemination of the GMM and mass transfer
limitations of electron donors and acceptors [90]. The construction of the lysimeters
used by Ripp et al., (2000) [90], meant that these problems were largely avoided,
although limitations with transfer of electron donors and acceptors were still
encountered.
2.95 The application of GMMs in the bioremediation of hydrocarbons, other than PAHs,
has focused on the genetic modification of microorganisms most suited to the target
environment, rather than just the design of microorganisms able to degrade the
hydrocarbon. The reasons for this may be due to the relative ease by which many
hydrocarbons (other than PAHs) may be degraded by microorganisms, and also the
relatively good level of understanding of the genetic basis of the biodegradation of
many hydrocarbons by microorganisms [29, 92].
2.96 Toluene is reported to be degraded by bacteria by one of five individual pathways
[29]. Although toluene is known to be degraded in contaminated sites by non-GM
bacteria, the current level of understanding of the toluene degradation pathways may
allow the future insertion of the catabolic genes into microorganisms more capable of
surviving in contaminated environments, than the non-GM toluene degrading strains.
2.97 Although the biodegradation of BTEX compounds by microorganisms has been well
characterised and is mediated by the tod and tol pathways, many of the studies
conducted have focused on the biodegradation of just one of the BTEX compounds,
rather than the group of four compounds as a whole [93]. Environmental
contamination by BTEX compounds, which are common components of petroleum, is
likely to involve mixtures of all four compounds, rather than just benzene or toluene
for example [93].
2.98 Lee et al., (1995) [93] reported that biodegradation of benzene, toluene and p-xylene
(BTX) in the environment does not result in the complete mineralisation of the three
hydrocarbon compounds, even by consortia of microorganisms. Biochemical studies
showed that the incomplete biodegradation was due to the 3,6-dimethylcatechol
(formed during the degradation of p-xylene) being a dead-end metabolite in the tod
pathway, and that the enzyme xylene oxygenase in the tol pathway was unable to
degrade benzene. The BTX compounds are degraded by toluene dioxygenase
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dihydrodiol dehydrogenase is present, then this enzyme channels the dihydrodiols into
the tol pathway and allows the complete mineralisation of the BTX mixture. The
genetic modification of Pseudomonas putida, by the insertion of the todC1C2BA
genes (encoding toluene dioxygenase), enabled this microorganism, designated P.
putida TB105, to mineralise BTX without the accumulation of intermediate
metabolites [93]. Because of the presence of BTX compounds in contaminated sites
as a mixture, and the reported failure of indigenous microorganisms to degrade BTX
mixtures completely, then the development of a GMM able to degrade such pollutant
mixes may have significant application in the bioremediation of BTX contaminated
environments.
2.99 Particular attention has focused on the use of GMMs to bioremediate hydrocarbons in
wastewater treatment plants, especially those strains suited to survive and express the
desired bioremediative activities in aquatic environments [53, 94]. Consideration of
the characteristics of the target environment is an important component in the
development of GMMs for bioremediation. A failure to identify this factor may have
contributed to the poor performance of many of the initial attempts to develop GMMs
for bioremediation applications [95]. Whilst indigenous microorganisms may offer
no advantages over non-indigenous microorganisms [96] it is recognised that the nonindigenous
organisms are likely to be more effective if they were isolated from a
similar environment to the target site.
2.100 The use of GMMs has been reported as one approach to improve the biologicallymediated
processes currently applied to the treatment of wastewater [94]. The
limitation with some of the GMMs used in wastewater treatment systems is that
although the GMMs are designed to degrade the pollutants present such as phenol,
and may be inoculated strains directly into the resident microflora, these
microorganisms are often rapidly washed-out of the activated sludge or biofilm fairly
rapidly, and are therefore not present for a sufficient period of time to degrade the
target contaminant. The efficacy of the bioremediation strategy relies on the ability of
the GMM to persist as part of the indigenous microflora for a sufficient length of time
to remove the target pollutant [94].
2.101 Microorganisms that are able to form flocs or to attach to existing flocs are reported to
persist in the wastewater treatment systems for significantly longer periods of time
than cells that do not possess either property. However, whilst the genes encoding for
the degradation of organic pollutants have been well characterised, the ability to form
flocs is more complex and often encoded by multiple genes [94].
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2.102 Soda et al., (1999) [94] therefore genetically modified a floc-forming Sphingomonas
paucimobilus with recombinant plasmid pS10-45 containing phenol catabolic genes
from P. putida BH. The GM S. paucimobilus survived in the wastewater treatment
process longer than a non floc-forming E. coli (also genetically modified with pS10-
45), and conferred better removal of phenol from the wastewater. The plasmid pS10-
45 was, however, reported to be unstable in the GM sphingomonad. Transposonmediated
modification of the microorganism was proposed to provide a more stable
GMM [94].
2.103 Removal of compounds such as phenol from wastewater treatment systems is
important both for ensuring the quality of the plant’s effluent and also to protect the in
situ microflora from the toxic effects posed. The addition of 1 mM methyl- or
chlorophenol to wastewater systems is sufficient to eliminate the populations of
protozoa and metazoa present, and reduce the numbers of culturable bacteria, by three
orders of magnitude [53]. The addition of the GM Pseudomonas sp B13 SN45RE to a
laboratory scale wastewater treatment plant resulted in the degradation of the phenol
compounds present and prevented the toxic effects normally caused by shock loadings
(1 mM) of these pollutants [53]. The use of GMMs in this way demonstrates their use
as an in-direct application of bioremediation.
2.104 Microorganisms such as GM P. putida DOT-T1, that are able to grow in the presence
of high concentrations of solvents, may potentially be used in applications to
bioremediate xenobiotics in sites where levels of organic solvents such as toluene are
sufficient to inhibit the degradative capability of solvent sensitive microorganisms
[97, 98]. P. putida DOT-T1 can grow in the presence of 90 percent toluene (v/v) and
other organic solvents whose octanol/water partition coefficient is >2.36. The
capability of P. putida DOT-T1 to degrade m- and p-xylene and other related
hydrocarbons including toluene was conferred by the insertion of the TOL plasmid
pWWO-Km [97]. Marconi et al., (1997) [98] employed a similar strategy to utilise
the solvent-resistant property of P. putida S12 to degrade naphthalene, toluene and
biphenyl, by insertion of the plasmids encoding the catabolism of these contaminants.
2.105 The use of terrestrial mesophilic microorganisms is also restricted in environments
containing high concentrations of salt. Although non-halotolerant microorganisms
have been used in the bioremediation of crude oil spills in marine environments, the
biodegradation of particular hydrocarbons in oil decreases as the salinity of the water
increases [99]. This can inhibit the overall bioremediation of the oil spill. For
example, the biodegradation of crude oil by a consortium of four pseudomonads was
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inhibited in the presence of 1 percent NaCl (w/v) [100], and growth of the consortium
was inhibited at concentrations of >0.9 M NaCl [101]. Insertion of the proU operon,
from E. coli into each member of the consortium, using a broad-host range plasmid
was found to confer a 25-fold improvement in the salt tolerance of the consortium
[101], and provides a solution to developing microorganisms to degrade pollutants in
hypersaline environments, such as contaminated estuaries and coastal areas.
Nitroaromatic compounds
2.106 One of the most problematic nitroaromatic pollutants is 2,4,6-trinitrotoluene (TNT)
that is often present at sites used to manufacture and handle munitions. Nitroaromatic
compounds can be biodegraded under both aerobic and anaerobic conditions [89]. No
GMMs have been identified in the current literature that have been designed to
degrade nitroaromatic compounds, although if the relevant degradative genes have
been identified, then GMMs could be developed for use in this field7.
2.107 Because munitions waste is likely to be present at contaminated sites alongside other
xenobiotics, particularly organic solvents, then solvent-degrading GMMs might be
employed in the bioremediation of such sites in a bioprotection capacity (similar to
that described for wastewater treatment plants [53].
Use of Bacteria for the Bioremediation of Inorganic Pollutants
2.108 Unlike organic pollutants, inorganic pollutants such as mercury cannot be degraded to
carbon dioxide and water and consequently removed from the contaminated
environment [102]. The objective of strategies to bioremediate inorganic pollutants is
therefore to use the bacteria to:
• transform the pollutant into a non-toxic form (biotransformation); or
• precipitate the metal ions at the surface of the cell (bioprecipitation); or
• sequester the metal ions and bioaccumulate the pollutant within the microbial
cell (biosorption). The strategy used depends on the characteristics of the
pollutant and the level and location of the contamination at the target site.
6 Such compounds are likely to partition within the lipid bilayer of the microorganism’s outer membrane, and
are more toxic than those organic compounds with a partition coefficient of <1 or >5.
7 Further information on the application of GM technology for the bioremediation of nitroaromatic compounds,
particularly munitions waste is presented in the report of the workshop at the end of this document (presentation
by Dr Bruce).
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2.109 The capability of many organisms including microorganisms to biotransform
inorganic pollutants from a toxic to a less toxic state, is often a consequence of the
strategy developed by the organism to survive in environments contaminated by such
pollutants [103, 104]. By transforming the pollutant into a less toxic and/or less
soluble state the organism is no longer exposed to the toxic effect the pollutant may
have. (Reducing the solubility of the pollutant reduces the bioavailability and
therefore the level of exposure of the compound).
2.110 Biosorption and bioprecipitation based strategies are most likely to be applied in ex
situ processes where the microorganism is contained in some form of bioreactor.
Although microorganisms capable of accumulating or precipitating metal compounds
could be added to the contaminated site to localise the pollutant in-situ, the pollutant
will remain in the site, and further measures will still be required to remove the
pollutant/microorganism ‘complex’.
Metallothioneins
2.111 Metallothioneins (MTs) are a group of a range of proteins that are able to sequester
and subsequently accumulate heavy metals [105]. The expression of metallothioneins
in bacteria has been used to enhance the metal-retaining capability of microorganisms,
and has applications in the removal of heavy metals from wastewater and
groundwater if the metallothionein expressing microorganism is immobilised in some
form of permeable matrix [105-107].
2.112 For example, the genetic modification of E. coli to overexpress the outer membrane
protein OmpC, caused the bacterium to display multiple copies of metal-binding polyhistidine
peptides on the surface of its outer membrane. The GM E. coli was able to
adsorb between three and six times more Zn2+, Fe3+ and Ni2+ metallic ions than non-
GM cells expressing the wild type OmpC [108]. The uses of metallothioneins in
bioremediation all operate using this same basic strategy. However, a number of
recent publications have proposed novel variations on this theme, including:
• the synthesis of new metallothionein proteins that are not produced naturally,
and therefore may be able to accumulate different heavy metals;
• the alteration of the system used to express the metallothioneins in the
bacterium, and therefore the location in the cell in which they are expressed.
This is reported to alter the metal-binding capability of the GMM; and,
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• the use of metallothioneins with multiple metal-binding sites.
2.113 The advantages of using metallothioneins with multiple binding sites, are an increased
capacity to remove metals from the solution, a greater stability of the expressed
protein and the potential to use a single strain of GMMs to target a number of
different heavy metals [105]. Mauro and Pazirandeh (2000) [105] genetically
modified E. coli to express the monomeric Mtt1 metallothionein unit from
Neurospora crassa in the cell’s periplasm, as a fusion with the maltose-binding
protein. The E. coli was modified to express between 1 and 12 copies of Mtt1 on a
single maltose-binding protein, and its ability to sequester cadmium measured. In all
cases, the GMM was able to take up approximately 10-fold more Cd2+ than the non-
GM E. coli strain. The relationship between the uptake of Cd2+ and the number of
Mtt1 subunits was linear up to the trimer. Although cells, with more than three copies
of the Mtt1, showed increasing uptake of Cd2+ with the number of Mtt1 units, the
improvements in terms of removal of Cd2+ from the medium were only small.
2.114 The fusion of the Mtt1 metallothionein to the maltose-binding protein meant that the
metal-binding protein was expressed in the periplasm (between the inner and outer
membranes of the cell). The choice of ‘anchor’, for the metallothionein determines
the location in the cell where the protein is expressed, and is reported to affect the
metal-binding capability of the protein [106]. Comparisons between the accumulation
of cadmium by GM E. coli expressing human or mouse metallothionein fused to
LamB, Lpp/OmpA (both membrane associated proteins) and PAL (peptidoglycanassociated
lipoprotein) showed that the LamB-MT fusion was the most stable and
demonstrated the best metal-binding capability. Fusion of the metallothionein to
LamB results in the metal-binding protein being expressed on the outer surface of the
outer cell membrane. E. coli modified with plasmid pLBMT1 (containing the LamBMT
fusion) adsorbed 30 nmol Cd2+ mg-1 biomass, whilst the non-GM E. coli cells
adsorbed only 0.8 nmol Cd2+ mg-1 biomass [106].
2.115 A possible limitation of the metallothionein-based remediation strategies discussed so
far, is that they are not specific to particular metals [106]. Although this non-specific
accumulation of metals may be useful for the general removal of metal contaminants
from wastewater for example, it is not suitable for the extraction of particular
hypertoxic pollutants, such as chromium. Such compounds whilst only comprising a
minor percentage of the total contaminant load in an environment may contribute a
significant proportion of the total toxicity, and should be targeted specifically in order
to reduce the toxic load present at the contaminated site.
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2.116 Specificity of human metallothioneins can be improved by just using the α (Cterminal)
domain of the metallothionein rather than the whole protein, and also by
using shorter synthetic peptides known to have specific metal-binding properties
[106]. E. coli modified to express a fusion of the LamB protein and the α domain of
the human metallothionein showed greater affinity and selectivity for Cd2+ than the
LamB fused to the entire metallothionein [109].
2.117 Kotrba et al., (1999) [110] genetically modified E. coli to express short metal-binding
peptides fused to LamB protein. The two sequences (designated HP8 and CP9),
showed different affinities to Cd2+, Cu2+ and Zn2+, indicating that the use of short
metal-binding peptides may be applied to the removal of specific heavy metal
pollutants from a contaminated environment. Mejare et al., (1998) [111] reported that
the genetic modification of E. coli to express a fusion of the peptide His-Ser-Gln-Lys-
Val-Phe and the outer membrane protein OmpA, conferred resistance to 1.2 mM
cadmium chloride. Such GMMs have applications in the removal of cadmium from
contaminated water, and if used as recipient organisms for further genetic
modification, may also be as degraders of other pollutants present in cadmium
contaminated environments.
2.118 The ability of metallothioneins to accumulate toxic heavy metal pollutants can also be
applied in the bioprotection of other organisms from the toxic effects of the metal.
Such uses are applicable particularly for the protection of agricultural crops. Valls et
al., (2000) [112] genetically modified the metal tolerant Ralstonia eutropha CH34 to
express the gene mtb. This gene encoded a chimeric metallothionein β protein
developed from the fusion of a mouse metallothionein 1 protein with an
autotransporter β-domain of the IgA protease of Neisseria gonorrhoeae. The
metallothionein protein confers the ability to sequester metal ions from the
environment, and the protease from N. gonorrhoeae, ensures expression of the hybrid
protein on the outer membrane of the GM R. eutropha. The GMM (designated R.
eutropha MTB) was reported to have an enhanced ability to immobilise Cd2+ ions
from its surrounding environment, compared to the non-GM strain. The inoculation
of the GMM into soil contaminated with Cd2+ significantly reduced the toxic effects
of cadmium on the growth of tobacco plants (Nicotiana bentamiana), indicating that
the GMM was able to reduce the bioavailability of the cadmium present in the soil, by
sequestration onto the microorganism [112].
8 Gly-His-His-Pro-His-Gly
9 Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly
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Other general strategies for the bioremediation of heavy metals
2.119 Several strategies for the removal of heavy metals from wastewater systems utilise
microbial processes indirectly to either biosorb or bioprecipitate heavy metals from
the wastewater. These include the use of sulphate-reducing bacteria to precipitate
soluble metal species out of solution, as insoluble metals sulphides [113], and the
manipulation of polyphosphate metabolism in bacteria, fungi and protozoa [114].
2.120 Sulphate-reducing bacteria are able to remove heavy metals from solution as a byproduct
of the production of sulphide from inorganic thiosulphate [113]. The gene
(phsABC) responsible codes for thiosulphate reductase, isolated originally from
Salmonella typhimurium. The insertion of pSB74 (containing the phsABC gene)
caused E. coli DH5α to overproduce hydrogen sulphide from thiosulphate [115].
Growth of the GMM in the presence of zinc, lead and cadmium chloride (separately
and in combination) in liquid culture for 24 h at 37 ºC resulted in the precipitation of
the metals as metal sulphides [113]. The GMM was able to remove 99 percent of zinc
(500 µM), 99 percent of lead (200 µM) and 98 percent of cadmium (100 µM) from
the solution. With the lead and cadmium solutions, insoluble PbS and CdS were
visible on the base of the culture flask. At higher concentrations the lead and
cadmium were found to have a toxic effect on the GMM with only 9.3 percent of
cadmium and 31 percent of lead removed from the 500 µM solution [113].
2.121 To assess the potential of the GMM to remove heavy metals from wastewater,
contaminated with a number of heavy metals, the E. coli DH5α harbouring pSB74
was grown in the presence of combinations of zinc, lead and cadmium (each 100 µM).
Although competition between the metals for sulphide delayed their removal in some
combinations, all the heavy metals were removed from the solution within 24 h [113].
2.122 The manipulation of polyphosphate metabolism in microorganisms is another strategy
that may prove useful in the development of GMMs with improved metal tolerance or
metal accumulation capability. Although the modification of polyphosphate
metabolism has yet to prove a realistic option for the bioremediation of heavy metal
contaminated water, it is reported that polyphosphate is involved in the storage of
and/or tolerance to heavy metals [114]. Some microorganisms are reported to use
intracellular polyphosphate to detoxify heavy metals such as lead [116], and others
may use cell-surface associated polyphosphate to chelate cations such as uranium
[117].
2.123 Keasling et al., (1998) [114], genetically modified E. coli CA38 to overexpress the
genes for polyphosphatase (ppk) and phosphate kinase (ppx). Overexpression of just
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ppk (E. coli CA38 pBC29) resulted in the increased production of intracellular
polyphosphate and improved uptake of phosphate, but no greater tolerance to
cadmium than the non-GM E. coli CA38. However, where the GMM was designed to
overexpress both ppk and ppx (E. coli CA38 pBC9), lower intracellular levels of
polyphosphate were observed along with secretion of phosphate from the cells.
Exposure of E. coli CA38 pBC9 to 2 ppm cadmium had no apparent effect on the
growth of the GMM. At a concentration of 4 ppm cadmium, the growth of the E. coli
CA38 pBC9 was reduced, but to a lesser extent than for the non-GM strain and for E.
coli CA38 pBC29. Exposure of 10 ppm cadmium inhibited the growth of all three
strains [114].
2.124 As with the biodegradation of organic pollutants, future applications to bioremediate
inorganic pollutants are likely to utilise the current understanding of the genes
possessed by indigenous microorganisms that are expressed against environmental
contaminants. For example, the soil microorganism Alcaligenes eutrophus CH34 is
able to survive in areas containing high concentrations of heavy metals because it
contains the megaplasmids pMOL28 and pMOL30 which confer resistance to Co2+,
Ni2+, CrO4
2-, Hg2+, Cd2+, Cu2+ and Zn2+ [118]. Two of the operons involved in the
heavy metal resistance, the cnr operon (resistance to cobalt and nickel) on pMOL28
and the czc operon on pMOL30 (resistance to cobalt, cadmium and zinc), have been
linked to the lux reporter system in A. alcaligenes to produce a GMM capable of
monitoring concentrations of metals in wastewater effluent [118].
Mercury
2.125 To date the majority of the work on the use of GMMs for bioremediation has focused
on the treatment of mercury contaminated sites. A limited amount has also been
undertaken on the bioremediation of cadmium, arsenic and nickel, although from the
information available this has focused on the use of GM plants rather than GMMs.
Mercury, cadmium and arsenic have probably been addressed first due to their
relatively high toxicity and the widespread contamination of the environment with
these metals.
2.126 Mercury is reported to be one of the most toxic heavy metals present as an
environmental contaminant. In aqueous environments, mercury present in sediments
in its Hg(II) state, is subject to methylation by both microorganisms and abiotic
processes to the much more toxic form methylmercury (CH3Hg+). This compound
can be bioaccumulated through aquatic food chains, and can give rise to toxic effects
to human health through the consumption of contaminated fish or shellfish [119].
Other organic species of mercury (including alkyl and aromatic derivatives) are also
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capable of bioaccumulating, at potentially toxic levels, in the tissues of higher
organisms [102].
2.127 The bacterial mercury resistance system (Hgr) incorporates a mercuric reductase, a
mercury specific transport system and an organomercurial lyase that is able to cleave
carbon-mercury bonds. Expression of the Hgr system confers resistance to both
organomercurial compounds and mercurial ions [102]. Many of the strategies
reported for the bioremediation of sites contaminated with mercury have used various
parts of the Hgr system, primarily to reduce Hg(II) to the more inert, volatile
elemental form (Hg0).
2.128 The use of bioremediation strategies to remove Hg(II) from polluted sites offers
certain advantages over ‘conventional processes’, such as chemical precipitation,
carbon adsorption and ion exchange. These chemical based processes are often
restricted by variations in pH and the tendency of Hg(II) to form complexes with
various ligands and become associated with suspended solids or sediments.
2.129 The genes conferring resistance to mercury are encoded in a single operon
(merTPABD) regulated primarily by the product of merR. The products of genes
merT and merP encode an integral membrane transport protein and a periplasmic
Hg2+ binding protein respectively, and are therefore involved in the uptake, transport
and accumulation of Hg(II). MerP is reported to sequester extracellular Hg2+ and
transfer it to MerT which then transports it across the cell membrane [102, 120]. The
merA gene and merB gene encode mercuric reductase and organomercurial lyase
respectively, and merD has been linked with a transcriptional coregulatory function
[102].
2.130 Expression of the merA gene enables microorganisms such as E. coli to reduce Hg(II)
to Hg(0) and thereby survive and grow in environments containing mercury, at
concentrations of 50 µM HgCl2 [104]. Survival of the microorganism is important if
it is to fulfil its intended bioremediation function. As mercury concentrations in
contaminated sites rarely exceed 10 µM, then expression of the merA gene offers the
potential to remove Hg(II) from even very contaminated environments.
2.131 However, although the microorganisms are able to express merA and therefore
transform Hg(II) to Hg(0) in their cytoplasm, they may still be restricted by the toxic
effects of Hg(II), in the outer membrane and cell wall regions of the cell [104]. If the
population of merA+ microorganisms in the contaminated environment is large
enough, then sufficient numbers of microorganisms are likely to survive the toxic
effects of the mercury and reduce its concentration to below a threshold level. When
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this point is reached (defined by the rate of cell growth of merA+ microorganisms
overtaking the rate of cell death of the population) Hg(II) should be successfully
removed from the environment [104]. This issue should be considered in deciding the
inoculum size required to bioremediate a contaminated site, where the target
compound is likely to be toxic to the inoculant.
2.132 E. coli cells genetically modified to express merT and merP and also to over express
metallothionein as a glutathione S-transferase fusion protein (GST-MT), were
reported to be capable of accumulating Hg2+ over a concentration range of 0.2 - 4 mg
l-1 [120]. The GMMs were designed so that they would specifically target, sequester
and accumulate Hg2+ in preference to other metal ions and would not be sensitive to
changes in ambient conditions. This was achieved by using the MerP/MerT
membrane transport system to target and sequester Hg2+ specifically, and the
overexpressed metallothionein to accumulate the metal ion intracellularly.
Bioaccumulation within a cell is reported to be more tolerant of changes in
extracellular conditions [121].
2.133 In order to assess the potential for these GM E. coli to be used in a bioremediation
application, for example in the removal of mercury from contaminated water, the
GMMs were immobilised in a hollow fibre bioreactor. Water contaminated with Hg2+
was then circulated through the bioreactor at a rate of 150 ml min-1 (25 ºC). The
system was found to remove Hg2+ from the water, reducing the Hg2+ concentration
from 2 mg l-1 to around 5 µg l-1 [120]. The system was also resistant to changes in pH
(from pH 3-11), ionic strength and the presence of common metal chelators
(ethylenediaminetetraacetic acid (EDTA) and citrate) or complexing agents, and may
therefore be more suitable to remove Hg2+ from the environment than ‘conventional
methods’, particularly where the concentration of Hg2+ is low. The combination of
the mercury transport system and the metallothionein binding proteins was also found
to exhibit good selectivity to mercury in the presence of other metal ions such as
magnesium (200 mM) and cadmium (100 µM) [120, 121]. Although the work was
conducted at laboratory scale, the basic system and approach is intended as an ex situ
bioremediation strategy for the removal of Hg2+ from contaminated water or soil
washings.
2.134 Inorganic pollutants such as mercury are also often present in sites co-contaminated
with some form of radioactive pollutant. In these environments, exposure to ionising
radiation usually inhibits the activities of microorganisms that, in a non-radioactive
environment could be employed to bioremediate the toxic inorganic pollutants
present. Riley et al., (1992) [122] reported the existence of ~1000 sites in the USA
which were contaminated with inorganic and organic pollutants such as mercury and
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toluene, and had radiation levels exceeding 10 mCi l-1. The bioremediation of such
sites requires microorganisms that are able to survive and function under radiation
stress [104].
2.135 The most radiation resistant organism discovered to date is the bacterium
Deinococcus radiodurans [123]. This microorganism is a non-pathogenic, solvent
tolerant soil bacterium capable of growing continuously in the presence of 60 Gy h-1
without an effect on its growth rate or ability to express foreign genes [15, 104].
2.136 Brim et al., (2000) [104] utilised the high tolerance of D. radiodurans to radiation to
develop a GM D. radiodurans designed to transform Hg(II) and degrade toluene in
contaminated sites with high levels of ionising radiation. A wild type strain of D.
radiodurans was genetically modified with the merA locus from E. coli BL308 and
tod genes (encoding the ability to degrade toluene or chlorobenzene) from P. putida.
2.137 Resistance of the GM D. radiodurans to Hg(II) correlated positively with the copy
number of merA genes in the microorganism, with the most mercury resistant strain
(MD737) having 150 copies of plasmid containing the merA gene (plasmid pMD731).
Although the genome of strain MD737 was ~3 Mbp larger than that of the wild type
D. radiodurans, the additional genetic material had no detectable effect on the
survival or growth of the GMM (compared to the wild type). The presence of
multiple copies of the merA gene also had no effect on the ability of the GM strain to
degrade toluene [104]. The report concluded that D. radiodurans offers a unique
system to bioremediate inorganic and organic pollutants concomitantly in the
presence of high concentrations of ionising radiation.
Nickel
2.138 In bacteria resistance to nickel is conferred by the gene ncc-nre. Dong et al., (1998)
[124] reported that the genetic modification of a range of eubacteria with plasmid
pMOL222 (containing ncc-nre isolated from Alcaligenes sp 31A) conferred an
increased resistance to nickel. Where the plasmid was transferred to activated sludge
bacteria in a pilot-scale activated sludge plant, the microflora were able to survive the
shock loading of waste contaminated with nickel at a concentration of 0.25 mM.
Use of Fungi for the Bioremediation of Pollutants
2.139 Although bacteria may be easier and faster to culture and more amenable to genetic
modification, fungi do have several inherent advantages over bacteria that may be
applicable in the bioremediation of pollutants. In addition to being more tolerant of
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environments with low pH, fungi are able to degrade and utilise a wide range of
complex natural substrates such as cellulose, hemicellulose, lignin and pectin [20].
Because the ability to degrade these compounds is conferred by extracellular enzymes
with a relatively low substrate specificity, then fungi are also able to degrade a range
of complex xenobiotic organic pollutants including chlorinated phenols, PCBs,
dichlorodiphenyltrichloroethane (DDT), dioxins, PAHs, alkyl halides and
nitrotoluenes [125-127].
2.140 Fungi also exhibit a number of capabilities that make them suitable for the
bioremediation of inorganic pollutants. These include a tolerance to relatively high
concentrations of metals [128], the ability to bioaccumulate metals by active and
passive processes [129, 130] and the production of extracellular compounds, such as
oxalates and citrates that improve the solubility and therefore the mobility of metals in
the environment [131, 132].
2.141 However, although fungi have been reported to be capable and in some cases uniquely
suitable for the bioremediation of pollutants, particularly heavy metals and high
molecular weight aromatic organic compounds [133-135], no reports of the use or
development of GM fungi have been identified. To date, the only GM fungi that have
been developed that are relevant to bioremediation applications, are those that have
been modified to express particular reporter genes. These include the bacterial gene
uidA (encoding ß-glucuronidase (GUS)) and the gene for green fluorescent protein
(GFP) [136, 137]. Such modifications would enable the GM fungus to be monitored
if released into the environment.
2.142 Other studies of relevance to the application of fungi in bioremediation include work
designed to improve understanding of the degradation of lignocellulose [138], a
complex organic compound whose degradation may be appled to xenobiotic
pollutants with similar structural similarities [139]. Broda et al., (1996) [138] have
reported studies in which individual proteins involved in the degradation of
lignocellulose have been expressed in recombinant systems to determine their
mechanistic use both singly and in combination.
2.143 A number of reasons are proposed for the limited genetic modification of fungi for
environmental applications:
• because of their mycelial structure and consequently non-homogenous growth
in liquid culture, fungi are inherently more difficult to work with than bacteria.
The mycelial growth also makes it difficult to ensure an even inoculation
across a contaminated environment;
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• many fungi are plant pathogens, and therefore their release into the
environment may not be desirable, due to the potentially adverse effects a
release might have on resident flora;
• fungi require a primary growth substrate in order to co-oxidise some aromatic
compounds, and because they are unable to metabolise the products of cooxidation,
then complete mineralisation of the contaminant does not occur
[58];
• the genetic basis for the biodegradation of compounds (including pollutants)
by fungi is relatively complex. Rather than the specific individual genes
present in bacteria, the degradation of compounds by fungi is usually encoded
by multiple gene systems that are induced by a number of different effectors.
This makes the transfer of degradative pathways from one fungus to another
by genetic modification more difficult than a similar transfer in bacteria; and
• the enzymes produced by fungi are largely extracellular and have a low
substrate specificity. This means that the fungi used in bioremediation
strategies are likely to have a degradative activity to a range of compounds in
the environment, in addition to the target pollutant. This lack of focus may
have an adverse effect on other biological processes such as nutrient recycling,
and is therefore less desirable than a strategy which just degrades the target
pollutant and its metabolites.
2.144 Although no reports of the use of GM fungi for bioremediation have been identified,
the use of non-GM fungi in the biodegradation of complex organic pollutants suggests
that GM fungi may have a role in bioremediation in the future. Work with non-GM
fungi has focused on the white-rot fungi such as Phanerochaete chrysosporium, Irpex
lacteus and Coriolus versicolor. Due to their ability to degrade chlorinated phenols,
PCBs, chlorinated pesticides, dioxins, PAHs, alkyl halides and nitrotoluenes, this
group of fungi may therefore be the most likely candidates for genetic modification in
the future.
2.145 In addition to the white-rot fungi, the mycorrhizal fungi have also been reported as
potentially playing an important role in bioremediation applications [140, 141].
Although no work on GM mycorrhizal fungi has been reported, the importance of
these fungi in the rhizosphere, as an interface between plants and soil means that they
may offer particular benefits to phytoremediation applications.
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2.146 Arbuscular mycorrhizal fungi for example are important symbionts of plant root
systems and are able to play both a passive and active role in phytoextraction,
phytodegradation or phytostabilisation. The mechanism of remediation is reported to
involve the fungi’s extracardial mycelium (ERM) which radiates out from the plant’s
root system into the soil, and may involve either the biosorption of the pollutants onto
the ERM or the use of the ERM as a site to harbour specific degradative bacteria
[141].
2.147 The most suitable strategies identified for mycorrhizal fungi in bioremediation are the
development of stress-tolerant indigenous strains rather than a generic strain that may
not be adapted to the target environment [141]. The relatively untapped application of
non-GM mycorrhizal fungi in bioremediation may however mean that work may
focus on the application of non-GM strains before GM mycorrhizal fungi are
developed.
THE USE OF MICROORGANISMS FOR THE MONITORING OF
POLLUTANTS
2.148 Microorganisms can be used to monitor the degradation, presence and toxicity of
contaminants in the environment [12]. Because of their ease of culture, rapid
response to toxins and ability to survive in environments in which the pollutants are
likely to be found, microorganisms are the ideal organisms for pollutant monitoring.
Also, because microorganisms are involved in the degradation of a wide range of
environmental pollutants, they can be used to monitor specific contaminants, as well
as indicating overall metabolic status of the cell, and also the actual level of toxicity in
a contaminated environment [142, 143].
2.149 Genetic modification techniques have enabled reporter genes, such as luc (from the
firefly Photinus sp and Phyrophorus sp.) and lux to be inserted into a range of
microorganisms, and have consequently increased the type of applications for
biomonitoring and biosensing significantly. The lux genes were isolated originally
from the marine bacterium Vibrio fischeri (formerly Photobacterium phosphoreum)
[143], and cause the recombinant microorganism to bioluminesce when they are
expressed.
2.150 Although V. fischeri has been used extensively to assess the toxicity of pure
compounds in liquid media, this microorganism is sensitive to the pH and osmotic
changes and requires a high saline concentration in the analyte under test [142, 143].
Methods such as Microtox and Lumintox, which are based on the response of
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naturally luminescent marine microorganisms such as V. fischeri are therefore
unsuitable for the monitoring of pollutants in non-marine environments [142].
2.151 Other bioluminescent reporter genes that have been inserted into microorganisms
include luc and Rluc from the Click Beetle (Renila reniformis) [144, 145]. The gfp
gene (from Aequorea victoria and Renilla reniformis) encodes for the formation of
GFP (which fluoresces under illumination with blue light) has also been used as a
reporter gene to study the survival and efficacy of GMMs used as inocula for
bioremediation [144].
2.152 The use of bioluminescent reporter genes, such as lux, can be designed for different
applications by the choice of promoter to which the gene is linked. Fusion of the lux
reporter genes, to appropriate heavy metal promoters means that the GMM will emit
light when the heavy metal promoters are induced by the presence of particular heavy
metals. The application of such biosenor technology has been reported for arsenic,
cadmium, chromium, cobalt, copper, mercury, nickel and zinc (cited by) [142], and
organic pollutants including napthalenenaphthalene [23, 91] and PCBs [146].
However, because expression of the genes, encoding the degradation of organic
compounds, is often induced by more than one compound, then biosensing for organic
compounds is usually less compound specific than for heavy metals [142]. For
example, the GM P. fluorescens HK44 used by Sayler and Ripp (2000) [23] to
monitor for the degradation of naphthalene emitted light in the presence of
naphthalene, 4-methyl salicylate and salicylate, although the lux genes were under the
control of the promoter for just the naphthalene catabolic genes.
2.153 If the lux gene is linked to a constitutive promoter, then the system can be employed
to report on the overall metabolic status of the cell, and consequently the level of
environmental stress experienced by that cell or population. The presence of toxic
compounds in the environment will increase the level of environmental stress and
reduce the amount of light emitted by the lux-modified microorganism [147].
2.154 The significant advantage of biosensors compared to conventional chemical
assessment techniques, is that because the control of the sensor is biological it is only
able to respond to the fraction of pollutant that is bioavailable [142]. Determination
of the concentrations of pollutants that are in a bioavailable state is a more
environmentally relevant measurement of the actual toxicity at a contaminated site,
than measurements based on the total amount of pollutant present [23, 147].
Chemical analyses can only determine the total amount of a particular pollutant(s)
present, and are also unable to provide an indication of the level of toxicity where the
pollutants interact to produce a cumulative toxic effect [147]. Cumulative effects are
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likely to be greater than those determined by summing the toxicities of the individual
pollutants present.
2.155 Although, bioluminescence based monitoring for the presence of toxic pollutants is
significantly cheaper than existing chemical based analyses [23], and has a number of
advantages over chemical techniques; the most accurate picture of the toxicity
characteristics of a particular contaminated site is likely to be provided by a
combination of biological, chemical and physical based analyses.
2.156 The use of a lux-modified microorganism in the preliminary assessment of a
contaminated site can identify the location, concentration and type of toxic
pollutant(s) present at the site, enabling subsequent bioremediation or chemical
treatment processes to be targeted at the required areas. This of course improves the
efficiency and reduces the costs of the remediation process.
2.157 Sousa et al., (1998) [147] used Pseudomonas fluorescens 10586s pUCD607
(genetically modified with the lux CDABE genes on a multicopy plasmid), in
combination with chemical treatment methods, to determine the environmental
constraints affecting the remediation of a BTEX contaminated site10. Sediment
supernatant and groundwater samples were taken from the BTEX contaminated site
and inoculated with a cell suspension of the GMM. The level of bioluminescence
recorded from these untreated samples reflected the overall toxicity of the pollutants
in the sample to the GMM. As pseudomonads are ubiquitous to terrestrial
environments, then the results obtained for the GMM were assessed to be applicable
to the indigenous microflora present in the contaminated site.
2.158 Chemical analysis of the BTEX contaminated site identified the presence of heavy
metals, PAHs and chlorinated alkanes at the site in addition to BTEX. To determine
the contribution of each of these groups of compounds to the overall toxicity, samples
were treated chemically and then tested again with the GMM. For example, to
determine the influence of the volatile organic compounds (VOCs) on overall toxicity,
the bioluminescence of the untreated samples was compared with samples that had
been air-sparged to remove the VOCs present. Muffle furnacing can be used to
remove the non-volatile organic compounds such as the PAHs [147]. If the toxicity
due to non-volatile organics and heavy metals is high, then the inoculation of the site
with BTEX degrading microorganisms will not be successful in reducing the
ecotoxicity of the site significantly. If the BTEX degraders are sensitive to heavy
10 Further information on the application of GM biosensor technology for the detection and assessment of
contaminants in a particular environment is presented in the report of the workshop at the end of this document
(presentation by Prof Killham).
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metals for example, then bioremediation of the BTEX pollutants present may also be
unsuccessful [147].
2.159 Although lux-modified GMMs can provide a very sensitive method for the detection
of specific compounds in the environment, any application of this technology needs to
ensure that the insertion of the reporter gene has no effect on both the interaction
between the GMM and the target pollutant, and its overall ecological fitness [142].
The GMM must also be representative of the types of microorganisms present in the
indigenous microbial community [147]. If the GMM behaves differently in the
environment, compared to the wild type strain (with the exception of being able to
bioluminesce), then the activity of the GMM will not be representative of the
indigenous microflora and may provide inaccurate information on the levels and/or
toxicity of the pollutants in the target environment. Sousa et al., (1998) [147] noted
that at some contaminated sites, some taxa within the indigenous microflora may have
adapted to the unique conditions present at the site. Therefore, in order to ensure that
the lux-modified GMM is representative of the indigenous microbial community, it
should ideally be isolated from that specific site, and then be genetically modified.
2.160 Layton et al., (1999) [143] reported a potential limitation of some lux-modified
microorganisms used to monitor the presence of hydrophobic contaminants such as
PAHs and PCBs in terrestrial environments. Because of their low water solubility and
strong lipophilicity, compounds such as PAHs and PCBs tend to absorb to the
particulate matter in soils and sediments. To improve the aqueous solubility and
consequently the biodegradation of these compounds, surfactants are often added to
the contaminated environment. However, if the surfactant is toxic to the lux-modified
GMM, then the amount of bioluminescence recorded will be due to the toxic effects
of both the target pollutant (e.g naphthalene) and the surfactant. Therefore, surfactant
resistant microorganisms such as Stenotrophomonas sp 3664 and Alcaligenes
eutrophus 2050 should be used for toxicological evaluation of pollutants in the
presence of surfactants [143].
2.161 The applications involving the use of lux-modified microorganisms, described above,
have not involved the release of the GMM into contaminated sites. In most of the
applications described, samples of contaminated material are added too a solution
containing the lux-modified microorganisms, and the bioluminescence recorded.
Because the GMMs are not released into the environment, the potential risks of the
use of this type of GM technology to the environment and human health may be
described as essentially zero.
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2.162 Because the lux-modified microorganisms are usually strains that are capable of
surviving in the environment, they could potentially be released directly into the
contaminated site. The detection of bioluminescence in contaminated sites in situ
does require additional electronic equipment and is probably therefore more
applicable to long-term monitoring strategies, for example, the two year study
reported by Ripp et al., (2000) [90]. In this study, the lux-modified P. fluorescens
HK44 enabled the biodegradation and bioavailability of the naphthalene, and the
optimal degradation conditions to be determined in situ. The lux-modified GMMs
were added to the contaminated soil as a direct inoculum and also in fibre optic
biosensors where they were immobilised in alginate11 [90].
2.163 The immobilised cells were used to monitor the presence of naphthalene in the soil
vapour phase with the bioluminescence relayed by a fibre optic cable to a
photomultiplier tube (PMT). The immobilised GMMs survived for approximately
one week, although they could be easily replaced when necessary. Light emitted by
the inoculated GMMs was detected by either PMTs or fibre optic cables, buried
throughout the contaminated soil. The fibre optic cables were however found to be
ineffective at detecting bioluminescence from the GMMs [90]. However, the
biomonitoring system employed to study P. fluorescens HK55 was reported to
provide a suitable online system able to quantify growth and activity of the GMMs in
the soil. Improvements in the encapsulation materials used to immobilise the
microorganisms in the biosensors may increase the survival of the microorganisms in
the biosensors and thereby improve the described system [90].
THE USE OF PLANTS FOR THE BIOREMEDIATION OF POLLUTANTS
2.164 The use of plants for the bioremediation of pollutants is described collectively as
phytoremediation, and has applications both for the removal of contaminants from the
environment and the conversion of pollutants into a less toxic state [148-150]. Recent
advances in molecular biology and biotechnology, coupled with a need for sustainable
technologies, have allowed the further development of phytoremediation as a
potentially cost-effective and environmentally friendly technology [150-154].
Developments in these fields have also provided a faster and more targeted alternative
to traditional plant breeding, and have extended the types of traits and properties that
can be introduced into individual plant varieties [155, 156].
2.165 The use of plants for the bioremediation of pollutants has been presented in this report
as two sections covering the phytoremediation of metals and the treatment of organic
11 Further information on the online monitoring system developed to measure bioluminescence in situ is
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contaminants. However, from the information identified in the scientific literature,
the majority of the work into the use of plants for bioremediation has focused on the
phytoremediation of metal pollutants [151]. This is in contrast to the application of
microorganisms in bioremediation and reflects the unique physiological and
biochemical characteristics of plants.
2.166 Because of the different biological processes involved in the phytoremediation of
metals and organic compounds, it is likely that applications to use plants for
bioremediation purposes will be targeted towards either metal or organic pollutants,
but not both types of contaminant. The biological mechanisms involved in
phytoremediation are addressed in this report as a background to the application of
GM technology in this field. Although applications are likely to be targeted towards
the phytoremediation of either organic or metal pollutants, there are a number of
approaches and advantages that are common to all phytoremediation-based processes.
Approaches and Advantages to Phytoremediation
2.167 As described already in this report, the application of bioremediation-based strategies
is determined by the properties of the organisms employed. Plant-based strategies are
therefore most suitable for the bioremediation of pollutants where:
• the pollutants are relatively close to the surface (within reach of the plant’s
root system), probably within the top 1 m of the soil profile. This is the case
for many, and probably the majority, of anthropogenically derived pollutants,
which tend to be deposited from above (e.g atmospheric emissions, application
of contaminated sludge, etc.). Moreover, metal pollutants normally bind
tightly to clay particles in the soil and so frequently remain in the upper part of
the soil, in principle accessible to plant root systems;
• the pollutants are relatively non-leachable and likely to pose a low risk to the
environment or human health. The comparatively slow growth of plants
compared to microorganisms means that phytoremediation strategies are only
suitable for the treatment of contaminated sites where the pollutant is likely to
remain on-site until the plant has grown [156, 157]. Phytoremediation
strategies may, however, be applicable to the treatment of more mobile
pollutants if the plants are grown and in place prior to the site being
contaminated, for example as a biobarrier ‘downstream’ of a shallow
contaminated aquifer; and
presented in the report of the workshop at the end of this document (presentation by Prof Sayler).
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• the pollutants are present in a contaminated site at low levels or across a wide
surface area [18, 158]. The relatively low cost of phytoremediation strategies
means that compared to chemical- and/or physical-based techniques, they can
be applied for the treatment of large sites, and on sites where the pollutant
concentration is very low.
2.168 The significant advantage of phytoremediation-based strategies is one of cost.
Although reports on the potential savings of phytoremediation applications compared
to traditional methods for the remediation of contaminated land such as ‘dig and
dump’ (and replacement of soils lost), incineration and soil-washing vary,
phytoremediation is proposed as between 10 and 10,000 times cheaper than existing
‘conventional techniques’ [156, 159, 160]. For example, growing a crop plant
capable of phytoremediation on an acre of land can be achieved at a cost of between
two and four orders of magnitude less than the current cost incurred by the physical
excavation and re-burial of the contaminated material [160], and even where the
phytoremediation strategy requires several sequential crops to be grown, overall costs
are still reported to be up to an order a magnitude cheaper than ‘dig and dump’
methods [159].
2.169 Where sites are contaminated with toxic metals, phytoremediation strategies offer the
potential to remove the metals from the site physically. This approach is likely to be
significantly cheaper than the mechanical bulk excavation of the contaminated soil
and re-burial at a designated landfill site, as it involves the removal of significantly
less material from the site [148, 161]. Phytoremediation-based strategies may offer
the only effective and/or financially viable solutions for the remediation of very
extensive metal-contaminated sites (cf. Chernobyl exclusion zone).
2.170 The phytoremediation of some organic pollutants such as TNT may also be more cost
effective than the ‘conventional’ excavation and thermal processing technique most
commonly used to date [162]. In some cases plants may also prove cheaper for the
remediation of organic pollutants than microorganisms [150], particularly where the
microbial-based approach requires the addition of supplementary carbon sources to
stimulate growth and biodegradative activity (although the use of plant-microbe
combined strategies offer a number of alternatives).
2.171 Growing plants is also significantly cheaper than the culture of an equivalent weight
of microbial biomass. The large amounts of biomass that can be produced by plants is
one of the principal advantages of phytoremediation strategies over microbial-based
approaches [163]. Plants do not require the sterile and specific growth conditions, or
the specific organic nutrients needed to culture large volumes of microorganisms, and
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are also generally easier to propagate and to harvest [148, 158]. Bioremediation of
explosives using microorganisms for example, requires the excavation of the
contaminated soil and treatment in bioreactors. Where these methods are as
expensive as more ‘conventional’ soil incineration processes, plants may offer a
cheaper approach, particularly since contaminants from munitions production are
often spread over the large areas near to the soil surface [164].
2.172 Although the use of plants for the remediation of contaminated environments may
offer a number of financial benefits, all phytoremediation strategies are limited by the
properties of the plants involved. As with microorganisms, different plants are
susceptible to different types and concentrations of contaminants, although plants are
reported to be able to survive higher concentrations of certain hazardous compounds
than most microorganisms used for bioremediation [157]. Plant-based strategies are
also depth-limited, as pollutants can only be phytoremediated if they are within reach
of the plant’s root system. Different plants are also restricted to particular growth
climates, with length of the growing season and soil characteristics also having a
bearing on the effectiveness of phytoremediation strategies [165, 166]. Despite these
limitations, in areas where contamination is spread over a large surface area,
phytoremediation may offer a novel, effective solution [18].
Types of Phytoremediation
2.173 Phytoremediation is a process that uses plants to remove, transfer, stabilise or degrade
contaminants present in the growing medium. Different strategies are available for
the phytoremediation of metals and organic compounds in the environment. The
choice of strategy depends on the identity and characteristics of the target compound,
the type of environment to be remediated and the overall objective of the
phytoremediation programme. The general strategies of types of phytoremediation
available are:
• for metal pollutants - phytoextraction (also often referred to as
phytoaccumulation); phytostabilisation; rhizofiltration and
phytovolatilisation;; [151, 152, 155, 156, 167]; and
• for organic contaminants - phytodegradation and phytostabilisation [151, 156,
157].
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Phytoextraction
2.174 Phytoextraction (or phytoaccumulation) refers to the use of plants capable of
removing metals from the soil and accumulating them in the above-ground parts of
the plant, that can then be harvested [151, 152, 154, 168].
Rhizofiltration
2.175 Rhizofiltration is a similar process to phytoextraction except that plant roots are
grown hydroponically to adsorb and, to some extent, absorb metals directly from
polluted aqueous environments [151, 154, 155]. Although both phytoextraction and
rhizofiltration can also be used for the phytoremediation of organic pollutants [168],
to date this is only a minor application of these processes, and this report will
therefore focus on their use for the treatment of metal pollutants by GM plants.
Phytostabilisation
2.176 Phytostabilisation is a process which uses plants to either reduce the bioavailability of
the metal pollutants in the environment, or to transform them into a less toxic form.
Unlike phytoextraction, phytostabilisation does not result in the removal of the
pollutant(s) from the soil [151, 152, 155]. However, this process can be very
important in preventing dispersal of the pollutant(s) from the contaminated site by
wind erosion or leaching [156].
2.177 Phytostabilisation can also be applied to the remediation of organic pollutants. This
area of phytoremediation research has however been less well studied than
phytoextraction, rhizofiltration or phytovolatilization for the treatment of metal
pollutants [152].
Phytovolatilisation
2.178 Phytovolatilisation uses the ability of some plants to convert specific pollutants into
volatile and usually less toxic forms, and is particularly relevant for the removal of
mercury and selenium from the soil [151, 155, 156].
Phytodegradation
2.179 Phytodegradation is the process where plants are used to degrade organic pollutants,
either through the uptake of the pollutants and their subsequent break-down within the
plant, or by the secretion of degradative enzymes from the plant into the environment
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[151]. The exact role of the plant in the phytodegradation of an organic pollutant is
still somewhat controversial [169]. To date, studies have been unable to demonstrate
whether the plant is able to degrade the pollutant in the absence of rhizosphere
microorganisms, or if the plant is required to stimulate or assist in the degradation of
the pollutants by the microorganisms. Where microorganisms are not involved
directly in the biodegradation process, some synergistic process may still be involved
[169]. GM plants are reported to have applications in both types of phytodegradation
strategy.
Phytoremediation of Metals
2.180 The phytoremediation of metals is restricted to the sorption of the metal onto a solid
matrix or the conversion of the metal into a less toxic or non-toxic state [170]. The
advantage of phytoremediation-based processes is that, unlike other in situ
remediation strategies (with the exception of vitrification- or concretisation-type
approaches), the phytoremediation process does not always require the conversion of
the metal pollutant into a more mobile form. Increased mobility of the pollutant is
likely to represent a greater hazard to the environment [171].
2.181 As discussed in the microbial section of this report, the application of microorganismbased
bioremediation strategies to the in situ treatment of metal-contaminated sites, is
limited to the immobilisation of the metal through precipitation, or its reduction into a
less toxic or non-toxic state [159]. Plants are able to bioremediate metals in this way
but, most importantly, can also accumulate the metal into above-ground parts of the
plant allowing the metal to be removed physically from the site following harvesting.
2.182 The application of phytoremediation-based strategies for the treatment of
environments contaminated with metals is based on the ability of naturally occurring
(non-GM) plants to extract and concentrate elements and compounds from their
environment. In particular, plants require a number of metals for use as electrolytes,
solutes, cofactors and essential components of proteins, and possess specialised
strategies to obtain these compounds from the environment. These include calcium,
copper, iron, potassium, magnesium, manganese, molybdenum, nickel and zinc. To
protect themselves from any potentially harmful effects of accumulating these
compounds, plants have developed mechanisms that inactivate or chelate the metal
ion upon its entry into the plant cytosol [172]. This system prevents the metal from
inactivating active or structural proteins, whilst at the same time allowing elements
essential for the plant’s metabolic function to be taken up and transformed into forms
that are tolerable to the plant [103]. However, this system is proposed only to protect
the plants up to a certain level, as soils that contain high concentrations of metals such
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as potassium, magnesium, manganese and molybdenum are usually toxic to plants
(Thurman and Hardwick cited by) [173].
2.183 The genetic basis of the ability of plants to tolerate metals is not yet well understood
compared to other species such as microorganisms [161, 163, 174]. However, the
introduction into plants of genes that confer resistance (or greater tolerance) to
pollutants in other species, have been reported [152]. The biological mechanisms and
genes involved and their potential application in phytoremediation of metals by GM
plants, are discussed below12.
Phytoextraction
2.184 The ideal plant for use in a phytoextraction application would have a large biomass,
be able to grow rapidly and accumulate the target metal pollutant to a higher
concentration than that found in the soil. Unfortunately, naturally occurring plants
with all three of these desired characteristics do not exist [168]. Some plants which
occur naturally on metalliferous soils are able to accumulate very high concentrations
of metals such as nickel, cobalt, copper, zinc and lead [148], with metal
concentrations in above-ground plant structures reaching between 0.1 and 3 percent of
their shoot dry biomass [168]. Several Thlaspi species have been found to accumulate
nickel and zinc to between 1 and 5 percent of their dry biomass [155].
2.185 However, although these plants are capable of hyperaccumulating metals, their low
biomass and relatively slow growth rate means that they are not suitable for
phytoextraction applications [17, 159]. Naturally occurring hyperaccumulators are in
general further limited by their ability to only accumulate a specific metal (with the
exception of Thlaspi sp.) and not the range of metals likely to be present on a single
contaminated site. The metals that are accumulated primarily by these plants are
nickel, zinc, manganese and cobalt, which are not among the more hazardous
environmental pollutants [154], although there are occasional reports of
phytoaccumulation of lead, copper and arsenic [175, 176]. Natural
hyperaccumulating plants are also often rare, often of limited population sizes and
growing in remote regions that may be threatened by mining, development and other
activities. Little is known, therefore, about the ability to cultivate these plants for
phytoextraction purposes. However, due to their patchy natural growth habits, it is
unlikely that they would be suitable for monoculture [18, 154].
12 Further information on the natural mechanisms employed by plants to accumulate metals, and the application
of GM technology to improve or modify these processes is presented in the report of the workshop at the end of
this document (presentations by Prof Smith and Prof Meagher).
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2.186 The transfer of the genes responsible for metal hyperaccumulation from natural metal
hyperaccumulating plants to faster-growing, high-biomass plants, without a
consequent reduction in plant yield or metal accumulating ability is therefore one
approach to the use of GM plants in phytoextraction strategies.
2.187 The most likely mechanism by which hyperaccumulating plants are able to tolerate
the relatively high concentrations of metals that they accumulate is reported to be the
production of a compound(s) that binds to (coordinates) the metal in the plant’s
cytoplasm [172]. Once bound, further interference between the metal and the plant’s
normal metabolic processes is prevented, and the metal complex may stay within the
cytoplasm or move to another part of the cell, such as the plant vacuole [173].
Current applications of this approach are however restricted to those compounds
whose genetic and biochemical pathways have been elucidated. Effective genetic
engineering to develop a range of plants for use in phytoextraction applications will
depend on an understanding of the mechanisms of metal uptake, transport and
tolerance, and the rate limiting steps involved in these processes [17-19, 164, 168,
177, 178].
2.188 Although not related directly to the bioremediation of contaminated sites, plants
capable of extracting metals from their environment also have applications in the
recovery of ‘precious metals’ from the environment. The use of plants in this way is
particularly relevant for the removal of metals from spoil waste, where the target
compounds are often present at relatively low concentrations across a wide surface
area [179].
♦ Metal-binding compounds in plants - Phytochelatins
2.189 Most of the work reported in the scientific literature that has addressed the
development of GM plants for phytoextraction applications has focused on the use of
phytochelatins (PCs) by plants to remove metals from their environment.
Phytochelatins are a family of heavy metal-inducible peptides that have been
identified as important in the detoxification of heavy metals in plants and some
microorganisms [180]. They are produced by a wide range of plants in response to
exposure to metal pollutants, and are proposed to form ligand complexes with the
metal thereby aiding its transport into the cell vacuole, where the metal is sequestered
and the phytochelatin degraded (Figures 2.4 and 2.5) [19, 103, 181].
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Figure 2.4 - Cadmium ions entering the cell activate PC synthase that catalyses the
transformation of GSH to PC [103]
Cadmium ions entering the cell activate PC synthase that catalyses the transformation of GSH to PC. The Cd2+-
PC complex is actively taken up into the vacuole. Within the vacuole the Cd2+-PC complex eventually
dissociates. The metal is stored there while the PC peptide is degraded.
Figure 2.5 - Possible structure of a phytochelatin (n=3, X=Gly) binding
three Cd(II) ions [19]
Phytochelatins, in this case a trimeric PC3, form tetrahedral complexes with thiol-reactive metals like cadmium
(Cd2+) enhancing tolerance. These structures should aid in the transport, and sequestration of, metals into
vacuoles via the glutathione S-conjugate pump (GCP).
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2.190 Cadmium ions have been reported to be the strongest inducers of phytochelatin
formation in vivo, with differentiated plants and suspension cultures of mosses, ferns
and angiosperms all found to detoxify Cd2+ ions through the production of
phytochelatins of varying chain length [103].
2.191 In mesophyll protoplasts derived from tobacco plants that had been exposed to
cadmium, almost all of the accumulated cadmium and phytochelatins were identified
as being confined to the vacuole, thereby confirming the sequestration of the toxic
metal within the plant. The identification of an ATP-dependent, proton- gradientindependent
active mechanism, capable of transporting both phytochelatins and PCCd
complexes into tonoplast vesicles derived from oat roots has also been reported
[178].
- Structure and biosynthesis of phytochelatins
2.192 Phytochelatins have the structure (γ-Glu Cys)nX where n is between 2 and 11 (but is
generally in the range 2-5), and X is commonly Gly (but can be β-Ala, Ser or Glu in
some plant species) [19, 178, 182]. Phytochelatins are just one family of the γ-Glu
Cys peptides (also known as class III metallothioneins) [182]. The direct precursor of
phytochelatin is the reduced form of glutathione (γ-Glu-Cys-Gly, GSH). Glutathione
(GSH) is synthesised from its constituent amino acids in two sequential, ATPdependent
enzymatic reactions catalysed by γ-glutamylcysteine synthetase (γ-ECS)
and glutathione synthetase (GS), respectively. PC synthase subsequently catalyses the
elongation of the (γ-Glu-Cys)n by transferring a γ-Glu-Cys group to glutathione or to
phytochelatins (Figure 2.6). The proposal that phytochelatins arise from glutathione
is based on:
• the structural resemblance of phytochelatins and glutathione;
• the appearance of phytochelatins at the same time as the disappearance of
glutathione; and
• the inhibition of PC synthesis by buthione sulfoximine, an inhibitor of γ-ECS
which can be reversed by the addition of glutathione to the growth medium
[178, 182].
2.193 The final stage of the synthesis of a phytochelatin is strictly dependent on the
presence of metal ions, since the PC synthase ion is only activated by metal ions. The
enzyme is also self-regulating since the reaction product, phytochelatin, chelates the
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metal responsible for activating the enzyme [103, 182]. Evidence for this mechanism
comes from the fact that the synthesis of PC is halted abruptly through the
introduction of another metal-chelating agent, such as EDTA [103].
2.194 Because PC synthase is only activated in the presence of metals, it is unlikely that this
enzyme is the rate-limiting step in phytochelatin synthesis [17]. It is thought that the
rate-limiting step in this process is a combination of the reactions catalysed by γ-ECS
and GS, depending on whether the plant is under metal stress or not [17].
Figure 2.6 - Regulation of GSH/PC biosynthesis in plants [17]
Regulation of GSH/PC biosynthesis in plants: cadmium enhances the transcription of ECS and activates the PC
synthase enzyme, leading to the production of PCs and the depletion of GSH. γ-ECS is also subject to feedback
inhibition by GSH.
- Evidence for importance and function of phytochelatins
2.195 Correlatory evidence for the importance of phytochelatins came from work by Speiser
et al., (1992) [183], which showed that selenium-tolerant Brassica juncea produced
two types of PC-Cd complex on exposure to cadmium. These included a more stable
high molecular weight PC-Cd-sulphide form, which could contribute to higher metal
tolerance by more effective metal sequestration. Increased cadmium tolerance in
tomato cell lines was found to be accompanied by increased production of Cd-binding
phytochelatins, most of which were higher molecular weight compounds. At least 90
percent of the cadmium in most tolerant cells was found to be associated with Cd-PC
complexes [184].
2.196 The importance of phytochelatins for tolerance to cadmium has been demonstrated by
the isolation of a Cd-sensitive mutant (designated cad1) of Arabidopsis thaliana that
appeared to be unable to accumulate or sequester cadmium [185]. Further
experiments revealed an allelic series of cad1 mutants that were all deficient both in
their ability to accumulate phytochelatins when exposed to cadmium and in PC
synthase activity compared to the wild type [186]. The level of phytochelatins
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observed correlated with the level of sensitivity of the mutant. The mutant strains had
wild type levels of glutathione, suggesting that cad1 mutants were defective in the
gene for PC synthase. Further experiments found that all four, independent cad1
mutants had base-pair substitutions on the same cad1 gene [180]. This result was
confirmed by extracts of E. coli cells expressing the cad1 gene product being able to
catalyse glutathione-dependent, metal-activated synthesis of phytochelatins [180].
2.197 A second cadmium-sensitive Arabidopsis mutant (designated cad2) was affected at a
different locus to the cad1 mutants [187]. The cad2 mutant was also deficient in its
ability to sequester cadmium compared to the wild type. The accumulation of
phytochelatins was also found to be only about 10 percent of that in the wild type and
glutathione levels were also lower. The deficiency in phytochelatin synthesis was
proposed to be due to a deficiency in glutathione [187], and was supported by the
observation that phytochelatins are synthesised from glutathione. Further experiments
on the cad2 mutant found that this mutant was actually deficient in the first enzyme in
the pathway of glutathione biosynthesis, γ-ECS. Enzyme assays showed that the cad2
mutant had only 40 percent of γ-ECS activity compared to the wild type, and that the
activity of the second enzyme in the pathway (GSH synthetase) was unchanged
compared to the wild type. In particular, the cad2-1 mutant was partially deficient in
glutathione and γ-ECS activity (the first of the two glutathione biosynthetic enzymes).
The cad2-1 mutation was found to be a six base pair deletion within an exon of the γ-
ECS gene, which affected residues in the vicinity of the presumed active site of the
enzyme [188].
2.198 These findings were reported to demonstrate the importance and function of
phytochelatins in protecting plants from toxic metals, particularly cadmium. The
sensitivity of the cad1 mutants to specific heavy metals gives some indication of the
importance of phytochelatins for the detoxification of metals in vivo (for Arabidopsis
at least) [180].
- Application of phytochelatins in the development of GM plants for
bioremediation
2.199 The manipulation of glutathione and PC concentrations therefore appears to hold
significant potential for increasing the accumulation of toxic metals by plants [19].
None of the γ-Glu-Cys peptides are synthesised on ribosomes, and all are formed
through enzymatic reactions [182]. Because of their identification as possible ratelimiting
steps in the synthesis of phytochelatins, the over-expression of GS or γ-ECS
may have the potential to improve metal accumulation in plants [189]. The
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overexpression of the bacterial γ-ECS gene in hybrid poplar for example resulted in
increased levels of foliar glutathione [190]. Unlike other genetic modifications where
the modified gene is designed to have a direct phytoremediation application, for
example the insertion of the mer genes, the manipulation of the expression of GS or γ-
ECS is intended to have a more indirect effect. The levels of phytochelatins in plants
are not increased directly as a result of overexpression of GS or γ-ECS, but are a
secondary result of the modification.
2.200 Alteration of the phytochelatin biosynthetic pathway of Indian mustard (Brassica
juncea) by genetic modification was reported to demonstrate the possibility of
producing GM plants with superior phytoextraction capability [17, 191]. This plant
was modified to overexpress the gshII gene (encoding for GS and isolated from E.
coli) in its cytosol. B. juncea was selected for genetic modification because the wild
type has a rapid biomass production and a high trace element accumulation capacity
[17]. B. juncea has also been shown to produce a high molecular weight PC-Cdsulphide
complex. Such complexes have been found to have greater stability
compared to low molecular weight complexes and could therefore contribute to
higher metal tolerance due to more effective sequestration [183].
2.201 Overexpression of the gshII gene in the GM B. juncea resulted in enhanced
production of glutathione and phytochelatins and improved accumulation of, and
tolerance to cadmium [17]. Increased levels of both compounds by the plant
correlated positively with the gshII expression levels [17]. The corresponding
increase in phytochelatin levels and improved accumulation of cadmium in the GM
plant was expected as greater levels of phytochelatins mean an improved ability to
bind and sequester the metal pollutant in the vacuole. Further complexation of the
cadmium with sulphide is reported to occur in the vacuole [17, 103].
2.202 In unstressed GM plants expressing gshII, GS was found not to be rate limiting for the
synthesis of glutathione, as the glutathione levels were not significantly different in
the unstressed GM plants compared to the wild type. There was also no detectable
phytochelatin in either the unstressed GM plants or the wild type variety [17]. Under
situations of cadmium stress however, the GS enzyme appeared to become rate
limiting for the biosynthesis of glutathione and consequently phytochelatins. In the
roots of wild type plants exposed to cadmium, glutathione levels were three-fold
lower than in similar plants in the absence of cadmium. This was reported to be due
to depletion of glutathione following the synthesis of phytochelatins by the stressed
plant. In the GM plants the modification meant that the rate limitation was removed
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and the glutathione levels were found to be the same irrespective of the presence of
cadmium [17].
2.203 Similar experiments have also been reported using B. juncea genetically modified to
overexpress gshI (encoding for γ-ECS) in the plant’s chloroplasts [191].
Overexpression of the gshI gene (isolated from E. coli) in the GM plant resulted in
increased γ-ECS activity compared to the wild type. In plants treated with cadmium,
overexpression of the γ-ECS gene increased the formation of γ-EC (its direct product),
and also glutathione and phytochelatins further down the biosynthetic pathway [191].
2.204 In unstressed transgenic plants (those not treated with cadmium) there was increased
production of γ-ECS and glutathione, compared to the wild type, although production
of phytochelatins remained unchanged [191]. The findings from this work were
reported to suggest that γ-ECS was limiting for glutathione and therefore production
of phytochelatins. The enzymes γ-ECS and GS were only reported to co-limit
glutathione production under conditions of cadmium stress [191].
2.205 The work reported with B. juncea demonstrates the potential of genetic modification
for the development of cadmium accumulating plants to treat contaminated land [17,
191]. The application of this type of genetic modification to other metal pollutants
depends on the ability of plant ligands to be induced and sequester other metals.
Although various other metals, for example copper, nickel and zinc, have been found
to induce the formation of ligands in plants, their function in sequestering the metal in
vivo has only been demonstrated extensively for cadmium, with comparatively little
work reported for other metals [182, 192]. Therefore, the determination of the
functional importance of plant ligands in cellular metal sequestration and the
elucidation of the genes involved in metal tolerance should improve the applications
of phytoremediation in the treatment of contaminated environments [182].
2.206 Studies on the cad1 mutants of Arabidopsis have shown that PC synthase is activated
in the wild type in vivo and in vitro by a number of metal ions to which the cad1
mutants are not hypersensitive. For example, in the wild type, PC synthesis in vivo is
activated effectively by copper. However, the cad1 mutant is only slightly more
sensitive to copper than the wild type [180].
2.207 Therefore, although synthesis of phytochelatins may be activated by a number of
different metal ions, this may not necessarily mean that phytochelatins play a major
role in their detoxification. From the information available, it appears that
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phytochelatins are a major component of certain heavy metal detoxification, but that
the increased tolerance of plants to metals other than cadmium may involve other as
yet undetermined aspects of phytochelatin function, or may involve the operation of
other more effective biological pathways for the detoxification of these metals [180,
186]. For metals other than cadmium there are few studies demonstrating the
formation of phytochelatin-metal complexes either in vitro or in vivo [178].
2.208 Studies of both naturally occurring metal-tolerant plants and laboratory-selected metal
tolerant plant cell lines do not show a clear correlation between increased resistance
and increased production of phytochelatins [186]. Although phytochelatins have been
shown to be able to bind more than one metal, most of the naturally occurring plant
species that are hyperaccumulators are only able to tolerate high levels of just one
metal [173], suggesting that hyperaccumulation is a consequence of more than just the
production of phytochelatins. Chaney et al., (1997) [155] proposed that the properties
identified in plants as cadmium tolerance mechanisms are in fact incidental
biochemical phenomena. An improved understanding of the genetic basis of natural
hyperaccumulating mechanisms is required to enable their manipulation in a wider
variety of plant species [19].
♦ Metal binding compounds in plants - Metallothioneins
2.209 Metallothioneins (MTs) have been proposed as the mammalian equivalent of
phytochelatins [193]. However, further studies of metal-binding complexes in plants
have revealed that some plants have metallothionein-like genes and proteins in
addition to phytochelatins, suggesting that these two groups of compounds may have
different roles [178, 186]. Kawashima et al., (1991) [194] identified a
metallothionein-like protein from soybean using a synthetic oligonucleotide probe
that corresponded to part of the nucleotide sequence of the mammalian
metallothionein.
2.210 A number of reports have proposed that metallothioneins and phytochelatins may
have relatively independent or overlapping functions in metal detoxification and/or
metabolism, although it has not been determined to what extent their individual roles
are complementary or redundant in plants [19, 178, 180, 186]. In animal cells and in
some fungi, metallothioneins appear to play a major role in heavy metal detoxification
[186]. The expression of the mouse metallothionein 1 gene (mt1) in tobacco plants,
found that the cadmium concentration in the transformed plants was 24 percent less in
the shoots, and approximately 5 percent more in the roots compared to non-GM
tobacco seedlings [195]. Although the results demonstrated that insertion and
expression of the mouse metallothionein did affect the sequestration of cadmium in
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the roots and shoots of the plant, the relatively low increase in cadmium accumulation
meant that this modification is not applicable to phytoextraction applications.
However, the modification may be useful in the development of a crop plant that has
lower concentrations of the accumulated metal pollutant in the consumable parts of
the plant.
2.211 In a similar study a gene construct encoding the α-domain of the human
metallothionein gene IA (MT-IA) was introduced into tobacco cells on a disarmed Ti
plasmid of Agrobacterium tumefaciens. The transgenic plants were tolerant to levels
of cadmium that were toxic to the non-GM control plants, and suggested that the
transgene was involved in metal detoxification and/or sequestration in the tobacco
plant [196]
♦ Other strategies for the development of GM plants for phytoextraction
2.212 Harmens et al., (1993) [197] found that increased tolerance to zinc in the plant Silene
vulgaris, which is naturally either sensitive or tolerant to zinc, was not related to an
increased accumulation of phytochelatins. The relatively low affinity of
phytochelatins for zinc compared to cadmium meant that the findings were not
unexpected. The results were reported to support the hypothesis that zinc
detoxification in the roots of plants involves other ligands such as organic acids, for
example citrate or malate, that may facilitate the transport of zinc through the xylem
and its ultimate storage in the vacuoles of cells in the shoot.
2.213 In the nickel hyperaccumulator Alyssum lesbiacum, histidine has also been identified
complexed with a proportion of the nickel present in roots, shoots and xylem [168,
178]. Moreover, the free amino acid histidine is produced in the roots of A. lebiacum
as a direct and proportional response to nickel exposure [198]. However, it is not yet
known at what level (e.g transcriptional, post-translational), the regulation of histidine
biosynthesis occurs, at least in Thlaspi goesingense [168].
2.214 Since the genetic basis for the above observations is unknown, it is difficult to use this
knowledge to devise genetic engineering strategies to improve the suitability of.
plants for the phytoextraction of nickel and/or zinc from contaminated sites. No work
has been reported in the scientific literature on the over-expression of genes encoding
the enzymes involved in histidine biosynthesis, although this field would be expected
to hold considerable promise for future work on phytoextraction of zinc and nickel by
certain plant species.
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2.215 The transfer of the gene(s) encoding the ‘hyperaccumulating’ phenotype into higher
biomass plants has been restricted up to now by a lack of fundamental knowledge
regarding the molecular, physiological and biochemical basis of hyperaccumulation
[169]. However, recent research into the genes encoding for proteins involved in the
transport of metals has improved the knowledge base in this area and is reported to
show some promise for its application to phytoextraction [177].
2.216 Radiotracer studies with Thlaspi caerulescens and a closely related nonhyperaccumulating
species, T. arvense, have suggested that the uptake of zinc is
controlled by the number (area density) of active transporters located in the
membranes of the root cells. Once in the plant’s root, zinc is proposed to be
transported into the xylem and taken up by the leaf cells, thus preventing the build-up
of toxic levels of the metal in the cytoplasm [199-201]. These observations are
proposed to indicate that zinc transporter systems exist within plants and operate to
transport the metal between cells and into subcellular compartments within the plant.
2.217 The first plant transporter genes for zinc were successfully isolated and characterised
from Arabidopsis thaliana [202]. Four genes encoding a zinc transporter were
identified (designated zip1, zip2, zip3 and zip4). The genes zip1, zip2 and zip3 were
found to encode for transporters that showed unique sensitivities to metal ions other
than zinc (Mn, Fe, Co, Cd and Cu were tested). This was proposed to be due to
differences in their substrate specificity [202]. Multiple zinc transporters are likely
since after the metal ion has entered the plant it must cross cell and organelle
membranes as it is distributed in the plant. Each of the transporters identified may
perform different roles in different parts of the plant. For example zip1 and zip3 were
found to be most strongly expressed in the roots of zinc-deficient plants. Little or no
mRNA of these genes was detected in the roots of zinc-sufficient plants or in the
shoots of zinc-deficient or zinc-sufficient plants [202]. The mRNA from zip4
however, also responds to zinc deficiency like zip1 and zip3, but is induced in the
shoots as well as the roots. These results were consistent with the theory that zip1 and
zip3 are involved in the uptake of zinc from the rhizosphere and zip4 is involved in
the transport of zinc in the plastids [202]. Histidine residues were identified in nearly
all the variable regions of these ZIPs, as well as within the transmembrane regions of
the protein, suggesting that these residues play a role in metal recognition and/or in
the transport of zinc through the membrane [202].
2.218 A gene encoding a zinc transporter has also been identified in Arabidopsis thaliana
(designated zat). Compared to the wild type transgenic Arabidopsis plants overexpressing
zat were found to have enhanced resistance to zinc and the ability to
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accumulate higher concentrations of zinc in the roots when exposed to high
concentrations of zinc [203].
2.219 A set of mammalian genes closely related to zat that are also involved in the uptake of
zinc have been found to encode proteins that are involved in the facilitation of the
vesicular sequestration of zinc. Expression of these proteins was found to result in an
increased resistance to otherwise toxic levels of extracellular zinc. The zat gene may
also encode for a protein that has a similar function, possibly transporting zinc into
the central vacuole of the plant cell, thereby contributing to the resistance of zinc in
plants [203].
2.220 A set of genes coding for a reported calcium transporter system have also been
identified in Arabidopsis [204]. These genes (designated cax1 and cax2) were
identified by their ability to suppress mutants of a yeast defective in vacuolar calcium
ion transport [205]. The expression of cax1 in tobacco plants resulted in the
disturbance of normal vigour, including necrotic lesions, chlorosis and a reduction in
root mass, compared to the non-GM plants. All of the symptoms identified are
characteristic of calcium ion deficiency. The GM plants were also more sensitive
than the non-GM plants on exposure to other ions and to cold-shock. When the GM
tobacco plants were grown in a media containing supplements of calcium ions, the
number of transformed plants showing abnormalities decreased dramatically [204].
The altered phenotype and increased stress sensitivities emphasised the importance of
cax1 for normal growth and several biological responses [204].
2.221 Similar altered phenotypes were observed in tobacco plants genetically modified to
express cax2, although the majority of the GM plants were as vigorous as the non-GM
controls. T2 plants from the healthy cax2 modified plants had a slight reduction in
root mass but the majority of plants appeared normal [206]. Studies showed that the
transporter encoded by cax2 was localised in the plant’s vacuolar membrane and was
responsible for transporting divalent cations, including Ca2+, Cd2+ and Mn2+ into the
vacuole [206]. The expression of cax2 was also found to result in increased
accumulation of calcium, cadmium and manganese ions in the roots and shoots of the
GM plants compared to the control. The transgenic plants were also slightly more
tolerant to manganese ion stress than the non-GM controls [206].
2.222 Studies with the cax2 modified tobacco plants [206] suggested that the expression of
cax2 in transgenic crops could alleviate Mn2+ toxicity problems and aid the
phytoremediation of Cd2+ in contaminated soils through the accumulation of the metal
ions within the plant vacuoles. Since various plant transporters appear to have a
broad selectivity in ion transport, cax2 may also be capable of conferring increased
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resistance/accumulation of other metal ions [206]. The cax2 modified plants however
showed no enhanced Cd2+ tolerance, and only limited increased tolerance to Mn2+
[206].
2.223 However, in addition to the genetic modification of the plant to sequester the required
metal pollutants, modifications may also be required in the control of metal uptake in
the roots, long distance transport of the metal within the plant and additional tolerance
factors to accommodate high concentrations of these metal ions.
2.224 An important trait of natural hyperaccumulating species is enhanced translocation of
the absorbed metal to the shoot [168, 175]. It is proposed that continued research in
the elucidation of the molecular basis for heavy metal transport in natural
hyperaccumulators, the tools to allow genetic modification of high biomass metal
accumulating plants for phytoextraction will become apparent. For example, a recent
study on the natural hyperaccumulator Thlaspi caerulescens identified another zinc
transporter, encoded by the gene znt1. This transporter is expressed at very high
levels in the roots and shoots of the plant and was shown to mediate high-affinity Zn2+
uptake as well as low-affinity Cd2+ uptake [201].
Phytostabilisation
2.225 The main distinction between phytostabilisation and other types of phytoremediation
is that phytostabilisation does not actually reduce the amount of pollutant present at a
site. Phytostabilisation uses methods such as the secretion of compounds into the soil
to alter the soil chemistry, formation of humic matter and accumulation of other
organic phases to reduce the bioavailability of the pollutant [18].
2.226 The ideal plants for phytostabilisation applications are therefore those capable of
tolerating high levels of heavy metals and having the capability to immobilise those
metals in the soil [159]. In biologically active soils, organic and inorganic
contaminants can form chemical and biological associations of varying intensity.
These associations can decrease the bioavailability of a contaminant, therefore
effectively reducing its risk of causing a toxic effect [18].
2.227 Plants are particularly well suited to the sequestration and/or immobilisation of
pollutants as they produce dense root systems that infiltrate large volumes of soil and
help to stabilise disturbed ecosystems [174]. Root densities equivalent to 4.8 x 108
km of roots per hectare have been reported [149]. Because of their large spreading
root system, trees are particularly suited for phytostabilisation purposes [149, 158].
Because trees are perennial and relatively slow growing, plant establishment will
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occur in parallel with soil stabilisation for a period of years with little maintenance
required [158].
2.228 No work has been identified in the scientific literature into the use of GM plants in
phytostabilisation. However, studies have been conducted that have demonstrated the
potential for phytostabilisation as a treatment for contaminated sites. The infection of
trees with the bacterium Agrobacterium rhizogenes results in the formation of larger
than normal root masses. Preliminary results showed that these non-GM trees had
higher growth rates with greater potential to establish on and stabilise the soil of a
contaminated site more quickly [158].
2.229 Deep-rooted plants have been shown to reduce the highly toxic form of chromium.
(Cr6+) to the more insoluble and significantly less toxic Cr3+ form [155]. The roots of
Agrostis capillaris have been found to cause the formation of pyromorphite, an
insoluble and bio-unavailable form of lead, in soil containing concentrations of lead
and phosphate. However, the mechanism involved remains unknown [155].
2.230 Investigation into the various mechanisms that plants use to render metal species
unavailable may provide useful insights into using molecular modification techniques
for those metals that cannot be remediated in other ways. Alternatively, stabilisation
of the soil surface could be achieved using metal-tolerant GM, or non-GM, species in
order to at least achieve stabilisation of a contaminated area to prevent erosion and/or
leaching of the pollutant and spreading of the pollutant into the surrounding area.
Rhizofiltration
2.231 Rhizofiltration has applications in the removal of pollutants from aqueous
environments through the adsorption of the metal compounds onto the plant’s roots.
Hydroponic plants therefore offer the greatest potential for rhizofiltration applications.
The ideal plant for rhizofiltration should be able to produce large amounts of fine root
biomass rapidly, and be able to remove toxic metals from solution over an extended
period of time [152, 159, 168]. Rhizofiltration applications include the treatment of
surface waters and groundwater aquifers, industrial and residential effluents, storm
waters, acid-mine drainage, agricultural run-off, diluted sludges and radionuclidecontaminated
solutions [207].
2.232 Research on the use of plants for rhizofiltration has been limited predominantly to
non-GM plants. The only application of GM plants in this field is the use of GM
plants modified to express a bacterial enzyme capable of detoxifying and removing
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mercury from experimental solutions. This is addressed further in the following
section of this report.
2.233 Young seed-lines of certain species grown in aerated water have been shown to be
effective at removing metals from water. Indian mustard seeds grown in this way
rapidly generate a large seedling biomass, which is capable of accumulating various
metals including Cd, Pb, Sr, Ni and Cr [159]. In order to improve the performance of
such systems, the affinity of the plant roots for metals will need to be increased. This
is reported to be possible using molecular techniques to produce GM plants with the
capacity to express high-affinity metal binding peptides on their roots [148].
Continued studies of the mechanisms of heavy-metal uptake by root tissue should also
provide important insights into increasing the efficiency and applications of
rhizofiltration [207].
Phytovolatilisation
2.234 Phytovolatilisation strategies are applicable for the treatment of volatile metal
pollutants, such as mercury and selenium. Plants used in phytovolatilisation
applications are able to sequester the metal, convert it into a less toxic form and then
volatilise the metal into the atmosphere, thereby removing the pollutant from the
contaminated site13.
♦ Phytovolatilisation of mercury
2.235 In the environment mercury exists mainly as a divalent cation (Hg2+), but may
bioaccumulate as methylmercury. Conversion of ionic mercury to its elemental state
reduces the risk posed to the environment due to the lower toxicity, aqueous solubility
and reactivity of elemental mercury compared to the other forms [171, 208].
Elemental mercury and Hg2+ are released into the environment as a result of gold
mining, various industrial processes, burning of fossil fuels and the disposal of
medical waste. On entering the lower trophic levels of the food chain, Hg2+ can be
converted to the even more toxic compound methylmercury by microorganisms.
Methylmercury is also bioaccumulated through the food chain [149, 156, 174].
Bioaccumulation can lead to mercury poisoning in organisms in upper trophic levels
[19]. Although releases of mercury to the environment have decreased since the
1960s, there are still large areas contaminated with mercury which are likely to
remain hazardous to the environment for many years, unless remediated [208].
13 Further information on the application of GM technology to phytovolatilise pollutants from contaminated
sites, particularly mercury is presented in the report of the workshop at the end of this document (presentation
by Prof Meagher).
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2.236 Mercury contaminated sites can be remediated ‘conventionally’ through ‘dig and
dump’ processes, although this simply transfers the contamination to a land-fill site
and also is severely disruptive to the contaminated area. With respect to the potential
for the phytoremediation of mercury, plants cannot detoxify mercury naturally, and
their tolerance to mercury is generally low. Therefore the potential to use naturally
occurring plant species for the phytoremediation of mercury contaminated sites is
limited [156]. Microorganisms have been isolated from mercury contaminated
environments, and have been found to have some natural resistance to mercury.
Resistance of microorganisms to mercury is encoded by the mer operon14. The two
mer genes that have been used in phytovolatilisation systems are the merA and merB
genes [208].
2.237 The merA gene codes for an NADPH-dependent mercuric ion reductase that converts
ionic mercury (Hg2+) to elemental mercury (Hg(0)), which is then volatilised into the
atmosphere. The merB gene encodes an organomercurial lyase that degrades
methylmercury to methane and Hg2+ (Figure 2.7).
Figure 2.7 - The bacterial enzymes MerA and MerB catalyse the detoxification of
methyl and ionic mercury respectively to produce volatile Hg(0) [19]
2.238 Although microorganisms have been proposed for use in the bioremediation of
mercury contaminated sites, the limited sphere of influence of individual cells means
that the treatment of large-scale sites with microorganisms requires a large inoculum
density and would therefore be unlikely to be financially viable [149, 208]. Since
plants are autotrophic and have large root systems, they should be able to increase the
rate at which mercury is eliminated from the soil by orders of magnitude over and
above the rate of mercury remediation by bacteria expressing the mer operon [19]. In
theory, plants genetically modified to express the mer genes should be able to extract
organomercuric compounds from the environment and convert them using the same
system conducted by mer+ microorganisms. The modified plants should then be able
to transpire Hg (0) from their leaves [174].
14 For further information on the bioremediation of mercury by microorganisms, see section on the use of
bacteria for the bioremediation of inorganic pollutants - mercury.
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2.239 Initial work on the genetic modification of plants to express the mer genes was
conducted with Arabidopsis thaliana [208]. Initial attempts to express the merA gene
in the Arabidopsis plant were unsuccessful. The coding sequence of the bacterial
merA is guanine and cytosine rich, contains 218CpG dinucleotides and is skewed
toward GpC-rich codons, which are uncommon in plants. In order to express the
merA gene, a modified version of the gene was constructed so that nine percent of the
original coding region was replaced with nucleotide combinations and codons that
were more similar to those in highly-expressed plant genes. In addition, 15 bp of the
5′ region immediately upstream from the initiator codon of the merA gene was
replaced with a plant translation signal code [171]. Plants expressing the modified
merA gene were reported to grow in an environment containing concentrations of
mercury normally toxic to most plant species, including the wild type Arabidopsis.
The GM plants also reduced Hg2+ to Hg (0) several times more efficiently than the
non-GM control plants [148, 171, 208].
2.240 Similar studies were conducted with the merB gene using A. thaliana. As with merA,
the bacterial merB gene had to be modified in order for it to be expressed efficiently
in the plant. Flanking regions were added containing plant and bacterial translation
signals. The transgenic plants were able to germinate and grow well in environments
containing concentrations of methylmercury that caused adverse effects and death to
non-GM plants [209].
2.241 Although the GM Arabidopsis was found to be capable of removing mercury from a
contaminated environment, the small biomass of this plant and its unsuitability for
large-scale cultivation means that it would not be effective for the phytovolatilisation
of mercury in a field situation [148]. Rugh et al., (1998) [149] selected yellow poplar
(Liriodendron tulipifera) for genetic modification using merA gene constructs. The
transgenic poplars expressing merA were found to be resistant to levels of Hg2+ that
were toxic to the wild type. High levels of Hg(0) were found to be released from GM
poplar plantlets rooted in a medium containing Hg2+. However, these transgenic
plants were not tested in model phytoremediation experiments using mercury
contaminated soils [149].
2.242 Plants expressing the merA construct are therefore reported to have some potential for
the remediation of Hg2+ from soil. Reduction in concentrations of Hg2+ also has the
great benefit of slowing the formation and accumulation of methylmercury, although
the use of merB+ plants would be able to reduce the levels of methylmercury present.
Plants expressing both merA and merB should therefore be capable of removing both
methylmercury and Hg2+ from mercury contaminated environments [208].
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2.243 Arabidopsis genetically modified to express the modified merA and merB genes had a
higher level of resistance to methylmercury concentrations than either merB plants or
the wild type. The GM plants were also able to convert methylmercury to Hg(0),
which was subsequently volatilised from the plant [209]. Further experiments found
that the rate of evolution of Hg(0) from individual GM plants correlated positively
with MerB concentrations. Regression analysis confirmed this relationship [174].
These experiments were carried on culture media containing methylmercury; a real
test of this technology would be to grow these plants on methylmercury contaminated
soils from the field.
2.244 Because methylmercury poses a greater environmental hazard in aquatic and marine
sediments (due to its higher toxicity to aquatic organisms), it has been proposed that
the modification of aquatic and saltmarsh plants would provide the most effective
solution to bioremediate contaminated aquatic habitats. Target species for the
insertion of the mer genes are wetland species and water-tolerant trees, such as
cordgrass (Spartina), cat-tail (Typha), bulrush (Scirpus), poplar (Populus) and willow
(Salix). These species could be planted in aquatic and/or wetland environments where
methylmercury pollution is most prevalent [156, 210].
♦ Phytovolatilisation of selenium
2.245 Another element that can be volatilised by plants is selenium. This metal is a
common contaminant in oil-refinery wastewater and can cause death and deformities
in wildlife [211]. Microorganisms are known to be involved in the volatilisation of
selenium from soils, and the ability of a naturally occurring plant to volatilise this
compound has only recently been identified in Brassica juncea. This species has high
rates of selenium accumulation and volatilisation, which combined with the fast
growth rate and high productivity make B. juncea a very suitable species for the
bioremediation of selenium by phytovolatilisation [211].
2.246 Volatilisation of selenium in the form of methyl selenate has been proposed as a
mechanism of selenium removal by plants. Volatile forms of selenium have been
reported to be 500 to 600 times less toxic than the inorganic forms [211]. Some
plants can also remove selenium from the soil by accumulating non-volatile selenium
compounds in their foliage. Naturally occurring plants such as Astralagus sp
hyperaccumulate high concentrations of selenium, although the mechanism for how
these plants are able to cope with toxic levels of selenium is unknown [19, 152].
2.247 Selenium has very similar chemical properties to sulphur. Both of these compounds
are taken up and assimilated by plants through a common pathway activated by ATP
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sulphurylase [19]. The reduction of selenate, mediated by ATP sulphurylase, has
been proposed as the rate-limiting step for the assimilation of selenate [211]. This
proposal was tested by modifying B. juncea to overexpress ATP-sulphurylase, and
resulted in the increased reduction of supplied selenate, compared to the wild type.
The findings supported the theory that the enzyme ATP-sulphurylase mediates
selenate reduction in vivo and that the enzyme is rate-limiting for the uptake and
assimilation of selenate to organic selenium [211].
2.248 Earlier studies found that, in a second detoxification mechanism, selenate can be
converted to dimethylselenide, which is 100 times less toxic than selenate, and can be
volatilised from the leaves and the roots (predominantly the roots) (Figure 2.8) [19,
211]. It was proposed that in the transgenic plants expressing the gene for ATP
sulphurylase, the rate-limiting step for selenium-volatilisation shifts from the
reduction of selenate to the volatilisation of organic selenium [211]. The
development of a GM plant capable of volatilising selenium from the soil into the
atmosphere was, however, not achieved (Terry, cited by Black, 1995) [166].
Phytoremediation of Organic Pollutants
2.249 The application of plants for the bioremediation of organic pollutants can be divided
into phytodegradation and phytostabilisation strategies. The concept of using plants
to bioremediate soils contaminated with organic compounds stems from the
observations that organic chemicals disappear faster from vegetated soils compared to
non-vegetated ones [151]. As with microbial-based strategies, the phytodegradation
of organic pollutants is intended to result in the breakdown of the pollutant to its
relatively non-toxic constituents [19]. Organic compounds that are potential targets
for phytoremediation include persistent organic pollutants that are also known to be
toxic, teratogenic and carcinogenic [19, 150]. Suggested appropriate organic targets
for phytoremediation include:
• petroleum products and by-products. These compounds probably represent the
largest volume of organic pollutants requiring remediation;
• industry-specific chlorinated organics (PCBs, dichlorobenzenes and TCE);
• industry-specific nitroaromatic compounds (TNT and dinitrotoluene (DNT));
and
• pesticide residues that are historic “off-label” or accidental spills [212]
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Figure 2.8 - A proposed model for the selenium flow in Indian mustard plants [211]
The compounds shown in boxes are the Se forms that accumulate in selenate-supplied plants. Selenate is
translocated rapidly from root to shoot, and is accumulated in shoots and roots of wild type plants because ATPsulphurylase
activity is limiting. When ATP sulphurylase is overexpressed (in APS plants), an organic form of
Se (possibly SeMet) is accumulated in shoots and roots. Because detopped roots of APS plants do not
accumulate organic Se, selenate assimilation appears to be a predominantly shoot-specific process and there
must be a flow of organic Se from shoot to root. (SP, sulphate permease; ATP-S, ATP sulphurylase; OrgSe,
organic selenium).
2.250 With the exception of some PAHs that are acquired by plants from the atmosphere,
organic compounds are taken up by naturally occurring plants in the liquid phase
[151, 213]. Although plants can transform and mineralise a wide variety of complex
organic compounds, only a few of these chemicals appear to be completely
mineralised by naturally occurring plants to water and carbon dioxide [19]. This puts
plants at a disadvantage compared with some bacteria in providing an effective
mechanism for the degradation of organic pollutants.
2.251 The modification of plants with microbial genes that confer the ability to remediate
pollutants may, however, provide a more effective method for the phytoremediation
of organic pollutants in the environment [163]. An important consideration in the
bioremediation of all organic pollutants by both plants and microorganisms is the
toxicity of the compounds produced as intermediate end products of metabolism
[151].
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Phytodegradation
2.252 The ideal plant for phytodegradation would have a high growth rate and be able to
mineralise the target organic compound without accumulating or releasing toxic
metabolites. Only a limited number of studies have been reported that have used GM
plants for the phytodegradation of organic compounds.
2.253 French et al., (1999) [163] genetically modified tobacco plants to express
pentaerythritol tetranitrate (PETN) reductase, a microbial enzyme responsible for the
denitration of TNT and GTN. The bacterial gene encoding for the reductase was
modified to include a plant consensus start sequence [163, 164, 214].
2.254 Although certain naturally occurring plants are able to degrade TNT, their application
for the treatment of contaminated sites is limited as the principal products of the
degradation are aminodinitrotoulenes, which are potentially more toxic than the
parent compound [163]. Transgenic plants producing PETN reductase were able to
germinate and grow in media containing higher levels of glycerol trinitrate (GTN) or
TNT compared to the wild type seeds [163]. The transgenic tobacco seedlings were
able to denitrify GTN. The ultimate products of TNT reduction by PETN reductase
have not been identified, but the results indicate that the products are less inhibitory to
plant growth than aminodinitrotoulenes [163].
2.255 The degradation of TNT is undergoing further investigation since this compound is a
more significant environmental pollutant that GTN, due predominantly to its wider
use and greater recalcitrance and toxicity. Much of the land polluted by TNT and
other types of munitions contamination is spread over large areas in near-surface
soils; conditions ideally suited for phytodegradation-based treatments [164].
However, numerous sites exist where TNT and GTN are just two components of a
diverse mixture of explosive contaminants. For phytodegradation to provide an
effective remediation solution at these sites, plants must be able to take up and
degrade effectively a number of compounds [164]. Further study is also required to
see if these transgenic plants are able to degrade explosive residues in soils in field
conditions, rather than in plant growth media [163].
2.256 Work on the phytodegradation of TNT and GTN demonstrates the potential for the
introduction of transgenes into a plant genome, to enhance the natural capacity of
plants to break down organic compounds. Similar applications of other bacterial
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degradative pathways in plants, give an indication of the potential for
phytodegradation applications15 [163].
2.257 Studies have also been reported for the use of GM plants for the treatment of TCE
contamination. Non-GM yellow poplar plants were found to be able to take up and
degrade TCE to several known metabolic products [215]. The introduction of a
transgene such as the mammalian cytochrome P450 2E1, into the plant may however
improve the rate and or extent of the biodegradation of this compound [150]. The
mammalian cytochrome P450 2E1 is a reported to be capable of oxidising a wide
range of compounds including TCE, ethylene dibromide (EDB), carbon tetrachloride,
benzene, styrene, chloroform, 1,2-dichloropropane and vinyl chloride [216],
(Guengerich et al., 1991 cited by) [150].
2.258 After exposure to TCE for five days the GM plants were found to contain
significantly higher concentrations of the TCE metabolite trichloroethanol in their
tissues than the non-GM wild type. The greatest difference in concentrations of
trichloroethanol between the GM and non-GM plants was in the roots, with the
smallest difference in the leaves. The metabolite was further degraded in the plant,
with the fastest removal in the roots. The reason for these differences between the
plant tissues was not determined, but was proposed to be due to transport of the
trichloroethanol from the roots to the leaves, loss of trichloroethanol from the roots
into solution, or because of faster metabolism of the trichloroethanol in the roots and
stem of the plant compared to the leaves [150].
2.259 Tobacco plants were genetically modified with a gene construct containing a plant
promoter and terminator and the P450 2E1 cDNA. The transgenic tobacco plants
expressing human P450 2E1 metabolised TCE and EDB at an enhanced rate
compared to the wild type. However, further work is reported to be required for these
transgenic plants to identify the downstream metabolic products of the reaction and
ensure that no toxic intermediates are released into the environment [150].
Phytostabilisation
2.260 No applications for the phytostabilisation of organic pollutants as a strategy for the
treatment of contaminated land have been reported to date. However, as with metal
contaminants, organic pollutants form chemical and biological associations of varying
intensity within the soil and plants. These associations can decrease the
15 Further information on the application of GM technology to phytoremediate nitroaromatic compounds from
munitions contaminated sites is presented in the report of the workshop at the end of this document (presentation
by Dr Bruce).
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bioavailability of the contaminant and therefore effectively reduce its risk of causing a
toxic effect. For example, organic compounds can be incorporated into lignin,
thereby becoming irreversibly trapped in plant cell wall constituents [18]. One
realistic benefit of the use of plants on contaminated sites is to stabilise the soil to
prevent erosion and/or leaching of the pollutant and further spreading of the pollutant
into the surrounding area [157].
Technical Problems Encountered with Transgene Expression in Plants
2.261 The purpose of this section is to address briefly the technical problems of transgene
expression in plants that are relevant to phytoremediation applications. This section is
not intended as an exhaustive consideration of the issues, which would be best
addressed in a separate report, and is only intended to highlight the potential problems
that could be encountered.
2.262 The genetic basis of contaminant degradation and/or accumulation is not as well
studied in plants as in bacteria. Therefore much of the work conducted to date in this
field has focused on the use of bacterial genes to enhance the bioremediation abilities
of plants. The modification of plants with non-plant genes is not technically
straightforward. It has been shown that in plants, (trans)gene expression is affected to
a large extent by the codon composition [217]. For example, certain bacterial genes
were not expressed in transgenic plants, in spite of their presence in very efficient
plant expression systems (Agrobacterium tumefaciens mediated transformation)
[171]. This was thought to be because the coding sequence of the bacterial gene was
GC-rich, and used codon sequences that are rarely found in highly expressed plant
genes [149]. Although these problems can be resolved by making changes to the
sequence of the bacterial gene, and creating a new gene construct which is more
compatible for plant expression without losing the resistance encoded for in the
original bacterial gene, such changes may be required for all bacterial genes intended
for use in plant systems.
2.263 In another example in which the bacterial gene has required modification, the gene
encoding PETN reductase was modified to introduce a plant consensus start sequence
so that the gene was expressed efficiently within the plant [163]. Similarly, the
mammalian P450 2E1 cDNA was placed between a plant promoter and plant
terminator sequence in order to effect efficient expression within the recipient plant
[150].
2.264 Work with merA and merB demonstrated that a plant’s ability to bioremediate
pollutants can be expanded beyond the plant’s natural capacity, by the incorporation
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of multi-gene pathways from other organisms [174]. Both the mer genes were
modified in order to facilitate their efficient expression in the transgenic plants. A
number of reports have stated that in order for phytoremediation to be a truly
successful technology then it must be able to remediate sites that have a number of
pollutants present, including those with a mixture of organic and or inorganic
pollutants. Such technologies will require the identification, characterisation and
cloning of the coding regions of genes for a number of enzymes from plants and other
organisms involved in pollutant transformation. These will then have to be
introduced into the recipient organism at the same time. The introduction of multiple
genes is likely to be important for the development of phytoremediating plants
capable of remediating sites that are contaminated with more than one type of
pollutant.
2.265 The merA and merB genes were introduced to a single plant by crossing
independently transgenic merA and merB plants. However, Chen et al., (1998) [218]
demonstrated that it was possible to introduce at least 13 different genes into the rice
genome using the co-bombardment method, where the genes carried on separate
plasmids are mixed prior to transfer by particle bombardment.
2.266 Several other factors related to the integration and structure of transgenic DNA may
influence the expression of transgenes within plants, as well as environmental and
developmental factors. These include the transgene copy number, the number of
rearranged and truncated transgene copies, their position in the genome and level of
methylation [149, 219-221]. Desirable new phenotypes created in plants can become
unstable following propagation, leading to the loss of the newly acquired trait (a
phenomenon is known as gene silencing). The way in which plants recognise and
specifically inactivate foreign DNA is unknown, and several different mechanisms are
probably involved [220, 222]. This will also affect the potential for the development
of effective phytoremediative plants.
2.267 Another example of loss of transgene, not related to gene silencing, was seen in the
Arabidopsis thaliana plants that were engineered with merA and merB gene
constructs independently. These were crossed to produce independent merA and
merB alleles within the same genetic background. The F1 plants were heterozygous
for one or more merA and merB insertions, having received one haploid chromosome
set from each parent. The F1 generation plants were then selfed to produce an F2
population containing both homozygous and heterozygous plants. Some of the plants
of the F2 generation were found to lack merA and/or merB, probably due to the loss of
the alleles through segregation (Mendel’s first law) [174].
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COMBINED STRATEGIES FOR THE BIOREMEDIATION OF
POLLUTANTS
2.268 Combined strategies for the bioremediation of pollutants are defined as those
processes that require the use of more than one organism to bioremediate the
contaminant successfully. Not all of the organisms involved are required to be
genetically modified. Combined strategies, particularly those involving plants and
microorganisms offer some particular advantages not available to strategies involving
single GMOs.
Multi-plant Strategies
2.269 Plants are able to infiltrate the soil to provide soil surface stabilisation. Where GM
trees are used in the phytoremediation of a site (e.g transgenic yellow poplars
expressing merA gene constructs), the treatment process may be more efficient if the
trees are grown in combination with smaller herbaceous species [149]. These smaller
species will fill the gaps between the larger trees. This will promote further
stabilisation of the soil surface and, if the herbaceous layer was also genetically
engineered to express the merA construct, increase remediation of the site’s pollution.
Plant-microbial Strategies
2.270 Plant-microbial strategies have been identified involving either GM plants or GMMs.
In the absence of plants, soil is a relatively oligotrophic environment, with the lack of
bioavailable nutrients having a significant negative effect on the activity of
microorganisms present (with respect to the level of microbial activity in the
rhizosphere). The lack of substrates is also likely to have an adverse effect on
bioremediation applications, particularly where the degradation of the target
compound provides the microorganism with little or no source of energy, for example
PCBs. An insufficient supply of nutrients may result in either the population of
inoculated GMMs dropping to below an effective level to degrade the pollutant, or the
GMMs losing the degradative trait (particularly where the recombinant genes are
present on a mobilisable plasmid) [13].
2.271 Plants could participate in the bioremediation of a contaminated site indirectly
through their support of symbiotic, root-associated microorganisms that carry out the
actual bioremediation of the contaminant [158]. In cases where plants promote the
microbial breakdown of the pollutant(s), the process is described as phytostimulation
or enhanced rhizosphere degradation [156-158]. The plant’s role in this type of
process could be through the release of exudates and/or enzymes into the rhizosphere
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that stimulate microbial activity and biochemical transformations. Plants are reported
to secrete 10 to 20 percent of their photosynthate in root exudates [151, 154, 157].
2.272 In the rhizosphere, the exudation of potential microbial growth substrates from plant
roots into the surrounding environment means that microbial activity is significantly
higher than in plant-free soil. Recalcitrant compounds such as TCE and the herbicide
mecoprop are reported to be less persistent in the rhizosphere [223, 224]. The
successful modification of two rhizosphere-competent GM strains of P. fluorescens to
degrade biphenyl (following insertion of bph genes on transposon TnPCB)
demonstrates that these microorganisms can be suitable recipients for genetic
modification to degrade environmental pollutants [13]. Plants also release oxygen
from their roots into the rhizosphere, thereby ensuring good aeration of the soil.
Penetration of plant roots through the soil not only improves the aeration, but also
water availability [149, 157]. Because the macro-movement of many microorganisms
and nutrients through soil is mediated by the flow of water, then root growth is also
likely to promote the spread of microbial degraders through a contaminated site. It
should also be noted, however, that exudation of organic compounds from plant roots
may (at least transiently) retard the degradation of a xenobiotic if the plant products
are preferred substrates for microbial growth.
2.273 The success of the GMMs reported by Brazil et al., (1995) [13] was due to the ability
of the microorganisms to use the root exudates as a general source of carbon and
energy. However, the discovery that some plant exudates may be able to induce the
degradation of pollutants such as PCBs means that a more targeted use of
rhizosphere-competent microorganisms may be employed. Terpenes are compounds
exuded by some plants into the rhizosphere, and are reported to induce co-metabolism
of PCBs by P. putida LB400, A. eutrophus H850 and Rhodococcus globerulus P6 to
the same degree as when the microorganisms were grown in the presence of biphenyl
[26]. Although these three strains were not genetically modified, recombinant
versions of these microorganisms have been designed for the enhanced degradation of
PCBs. The combination of terpene producing plant species and PCB degrading
GMMs at PCB-contaminated sites may avoid the addition of biphenyl supplements to
the site to promote the degradation of the PCBs.
2.274 The use of GM plants in plant-microbial strategies is likely to be based on the
development of plants capable of producing particular compounds required by
microorganisms to degrade or chelate specific pollutants. Although no such GM
plants have been produced to date, future applications may include the plants able to
secrete phytochelatins to concentrate metal pollutants in the rhizosphere for
subsequent bioremediation by GM or non-GM microorganisms. GM plants may also
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be designed to reduce the pH of the environment, which release metals from fixed
cation-exchange sites and will improve the bioavailability of metal pollutants for
microorganisms. It is unknown whether the action of these phytochelatins or pHreducing
compounds would be restricted to the rhizosphere, or may potentially have a
more widespread action.
2.275 Improvements in phytoremediation by GM plants e.g through rhizofiltration or
phytoextraction, may also be achieved through a better understanding of the role of
rhizosphere microorganisms in metal uptake and precipitation by plant roots [207].
Roots may be able to employ rhizospheric organisms (mycorrhizal fungi or rootcolonising
bacteria) to increase the bioavailability of metals. Mycorrhizal fungal
associations with plant roots have been found to affect plant tolerance to heavy
metals, metal uptake and translocation and plant growth parameters (e.g root: shoot
ratio) in a number of plant species [152, 158]. For example, the volatilisation of
selenium by plants is enhanced by the inoculation of the plants with rhizospheric
bacteria, as the presence of the bacteria affected the root surface area of the plant and
the rate of selenium uptake [225]. It has also been shown that natural microbial
populations can be manipulated to improve the metal accumulation abilities of plant
roots [168]. The ability of plant-leaf microflora and endophytic organisms are also
being investigated to determine their effect on phytoremediation. However, the
significance of microorganisms for the phytoremediation of pollutants remains mostly
unknown [18, 212].
2.276 Strategies involving both GM plants and GMMs may include the use of GMMs to
improve the mobility of xenobiotic compounds, particularly heavy metal
contaminants by changing their state. This could increase the uptake and
bioremediation of the contaminant by the GM plant. GM plants could be engineered
to exude specific molecules required to induce GMM bacteria to degrade pollutants
[160]. Rhizosphere-competent microorganisms can of course be added to the existing
rhizosphere at a contaminated site by inoculation directly into the soil. However, if
the site does not contain sufficient plant cover, then both the plant and GMM must be
added. The inoculation of the microorganism onto the outside of the seeds used to
sow the site reduces the amount of labour required and ensures a close interaction
between the GMM and the plant. Wheat seeds coated with a GM pseudomonad were
used as a plant-microbial strategy to remove TCE from contaminated soil [226]. The
GM P. fluorescens expressed the tomA(+) genes (encoding toluene monooxygenase)
isolated from Burkolderia cepacia PR1(23)(TOM23C). The efficacy of the system
was tested by adding TCE to soil microcosms containing wheat plants and the
rhizosphere associated GMM. The level of TCE in the soil was reduced by 63 percent
in 4 d (20.6 nmol TCE d-1 plant-1) in the soil containing the GMM, compared to only a
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9 percent reduction in soil microcosms containing wheat plants and the non-GM wild
type P. fluorescens 2-79 [226].
Multi-microbial Strategies
2.277 The application of consortia of microorganisms is a common strategy for the
biodegradation of pollutants by non-GM microorganisms. However, as described in
the section on ‘General strategies for the optimisation of bioremediation’, the use of
consortia may not be the most efficient method of biodegrading pollutants in the
environment, if genetic modification techniques can be applied to enable single taxa
of microorganisms to degrade a pollutant. No reports have been identified during the
compilation of this review that have used GMMs in consortia with other
microorganisms (GM or non-GM) to bioremediate pollutants. Indeed, many of the
GMMs reported have been designed to replace consortia [51, 53, 93].
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3. ASSESSMENT OF THE RISKS OF THE USE OF GMOs FOR
THE BIOREMEDIATION TO THE ENVIRONMENT AND
HUMAN HEALTH
3.1 Assessment of the risks posed by the use of GMOs in bioremediation strategies to the
environment and human health is a key component of the regulatory process that must
be completed before any release of a GMO can take place (in either the field or
contained facilities).
3.2 For the purposes of this report, the risk assessment has been divided into two parts to
address the risks posed by GMMs and GM plants separately. This has been done due
to the inherent differences between the two groups of organisms, the bioremediation
applications in which they are employed and consequently the risks posed to the
environment and/or human health.
3.3 For each group of organisms, the risk assessment has been conducted in two stages;
the identification of the characteristics or properties of the GMO which may cause an
adverse effect(s) to the environment and/or human health (the hazard identification
stage), and the assessment of the likelihood of each of the adverse effects identified
being realised (the risk assessment stage). A hazard or adverse effect of a GMO is
defined as an intrinsic property or characteristic of the GMO that could result in harm
to the environment and/or human health.
3.4 Although the hazards posed by a particular GMO are likely to vary depending on the
characteristics of the GMO and the environment in which it is intended to be used,
there are a number of key hazards common to GMOs involved in bioremediation
applications:
• transfer of the inserted genetic material from the GMO to other organisms in
the environment;
• accumulation of above ambient concentrations of toxic compounds such as
heavy metals by the GMO;
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• production of toxic metabolites or by-products by the GMO during the
degradation of the target pollutant; and
• disruption of indigenous organisms by the GMO, and the potential knock-on
effects on food-chains and biological processes such as decomposition and
nitrification. It should be noted that the diversity of organisms present in a
polluted environment is likely to be a consequence of the contamination
present, and is unlikely to be representative of an equivalent non-contaminated
environment. This may need to be addressed in assessing the level of
disruption caused by the introduction of the GMO.
3.5 The risk posed by the GMO to the environment and/or human health is determined by
the likelihood of the particular hazard being realised, and the magnitude of the effects
if they occur. Therefore the level of risk will vary between GMOs depending on
whether they possess a specific hazardous property and are able to express it in the
environment.
3.6 It should be noted that not all of the hazards described above apply to all GMOs used
in bioremediation applications. For example, GMMs modified by insertion of the lux
gene will not accumulate toxic compounds or produce toxic metabolites, whereas all
of the hazards described may be realised with GMMs designed to degrade organic
pollutants and bioaccumulate heavy metals. Where the GMO is used in such a way so
that it is not released to the environment, then it is likely that none of the hazards
identified will be realised. Examples of such applications include lux-based
biosensors where samples of the contaminated site (soil or water) are added to a
suspension of the biosensor ex situ and the level of bioluminescence quantified.
3.7 The level of risk posed by a particular GMO (defined by the likelihood of the hazard
being realised) is expected to vary between different GMOs, and also potentially for
the same GMOs used in different environments or in different applications.
3.8 Although a number of hazards may potentially be associated with the use of a GMO
for the bioremediation of a pollutant(s), it should be noted that in many cases, the use
of a GMO may provide a number of advantages (or fewer potential hazards)
compared to the other options available, namely:
• the use of physical and/or chemical remediation strategies;
• the use of non-GM based bioremediation strategies; or
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• leaving the contaminant in place.
3.9 As discussed previously in this report, physical and/or chemical remediation strategies
may pose their own inherent hazards to the environment, through for example, the
increased mobilisation of the contaminant(s) or the use of compounds that are
themselves environmental pollutants. A potential limitation with non-GM based
strategies is that the organism involved may not be capable of remediating the more
recalcitrant or toxic pollutants. In situations where the pollutant is relatively
immobile in the contaminated site, or is non-toxic, then it may be less hazardous to
leave the contaminant untreated. However, changes in land-use or climate, for
example, may alter the mobility and level of likely exposure of the compound.
3.10 In addition to the potential direct applications for plants in bioremediation discussed
in the previous chapter, plants also offer a secondary advantage in reducing erosion at
the contaminated site which may otherwise result in the increased exposure of the
surrounding environment to the contaminant(s). The growth of plants with no direct
ability to degrade or sequester pollutants at a contaminated site may be regarded as
some form of phytoremediation strategy [212].
3.11 The other issues that will be considered in selecting the appropriate remediation
strategy include cost and public acceptability. Whilst these subjects are outside the
scope of this technical report, it has been reported that bioremediation-based process
may offer significant financial savings compared to physical/chemical-based
techniques for particular sites [151, 152, 154, 156, 159, 160]. The choice of whether
or not to leave the pollutant on-site is likely to depend on the intended future use of
the area, but is unlikely to be publicly acceptable if the site is intended for domestic or
recreational usage, irrespective of the potential for pollutant exposure16.
THE USE OF MICROORGANISMS
3.12 The use of GMMs in the bioremediation of pollutants is only likely to pose a risk to
the environment and/or human health if the microorganism can survive in the
environment into which it is released. The only exceptions to this statement are:
• where genetic material is released into the environment prior to or during cell
death and remains in the environment in a state where it can be transformed
into other microorganisms; and
16 The issue of public perception of contaminated sites, and the measures available to remediate them was
dicussed during the workshop. The outputs from this discussion are presented in the report of the workshop at
the end of this document (General discussion section).
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• where the GMM is designed to accumulate toxic inorganic pollutants such as
heavy metals. The use of such organisms usually results in the localisation of
comparatively high concentrations of the toxic compound in an environment
where the contamination was originally more dispersed. Because such
compounds are not biodegraded, then these localised accumulations may
remain even after the death of the microorganism.
3.13 Survival of GM microorganisms in the contaminated environment is important if they
are to perform their bioremediative function to the desired level [57, 227]. Early
reports on the use of GMMs in the environment suggested that the microorganisms
were unlikely to survive, grow and compete with the indigenous microflora, due to
the greater energy demands imposed by the foreign genes and the consequent
reduction in the fitness of the GMM [8, 228-230]. However, the findings from
various releases of GMMs in the environment have not supported this proposal, and
have found that GMMs are able to survive for long periods of time (up to 6 years)
[231] in the environment in the presence of natural microbial populations [90, 232-
234].
3.14 Ripp et al., (2000) [90] found that the GM P. fluorescens HK44 introduced into PAH
contaminated soil persisted for up to 660 days in the soil under field conditions.
Assessment of the risks posed by the GMM should assume that the microorganisms
are capable of surviving in the environment, although determining the level and
duration of the survival of GMMs in the environment is not easy to predict [23].
3.15 Roberts (1989) [95] proposed that many of the earlier studies that were conducted to
assess the survival of GMMs in the environment were poorly designed and many
ecological aspects of the release were not taken into account in the design of the
GMM. Consequently, many of the GMMs tested were inherently less fit than the
indigenous microflora and therefore unlikely to persist in the environment. Much of
the recent work in the development of GMMs for use in the field have focused on the
modification of microorganisms capable of surviving in the target environment, as
well as being able to degrade the target contaminant [94, 101].
3.16 Crozat et al., (1987) [235] reported that the inoculation of a bacterial population (GM
or non-GM) into soil is often followed by a decline in viable numbers until the
population reaches a ‘survival’ population. This reduction occurs irrespective of the
initial inoculum size and therefore needs to be taken into account when calculating the
size of the population of GMMs required to bioremediate the pollutant. The rate of
reduction is reported to vary between different taxa of microorganisms, depending on
their ability to survive in the environment [232]. Enteric microorganisms such as GM
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E. coli are likely to exhibit a rapid reduction in cell numbers down to an undetectable
level within three days of inoculation into the contaminated environment.
Microorganisms such as GM Bacillus subtilis exhibit an exponential decrease over a
7-10 day period down to a population density of 1-100 cells g-1 of soil, and GMMs
such as Rhizobium sp exhibit a rapid exponential decrease within 1-14 days of
inoculation [232].
3.17 The initial decrease in microbial numbers leads to a reduction in the population of the
inoculated microorganisms, by between one and four orders of magnitude, and in
some cases may be followed by a complete and progressive decrease of culturable
cells. Studies involving GM pseudomonads were reported to be more variable, with
the inoculated population decreasing by 0.2 to 1 orders of magnitude in 10 days [232].
However, no single model has been applied successfully to determine the survival of
inoculated microorganisms (including GM strains) in a particular environment. This
is due to the successful colonisation of an environment by a GMM being dependent
on a wide variety of biotic (competition and predation) and abiotic (temperature, pH,
moisture and adsorption) factors [23]. Those which can or may be controlled such as
strain selection, the genetic modification, soil aeration and moisture content should be
addressed as part of the initial design of the bioremediation strategy [90].
3.18 Blumenroth and Wagner-Döbler (1997) [96] found that the use of microorganisms
indigenous to the target environment for bioremediation and as hosts for genetic
modification did not provide any inherent advantage over microorganisms isolated
from other environments, and that the ability of the microorganism to compete
successfully for available nutrients was the principal factor in determining
survivability. Blumenroth and Wagner-Döbler (1997) [96] concluded that the use of
well characterised strains (taxonomically, physiologically and pathologically) as hosts
for genetic modification would present a lower level of risk to human health and the
environment, compared to the isolation and genetic modification of microorganisms
indigenous to the contaminated site. Any microorganisms selected for genetic
modification would however have to be able to compete successfully for available
nutrients. However, where the genetic modification enables the GMM to utilise a
nutrient source that is not available to the indigenous microflora, for example the
target pollutant or in the case of FAVs, the surfactant [88], then the ability to compete
for available nutrients may not be as significant a factor in the survival of the GMM in
the environment.
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Transfer of Genetic Material
3.19 Transfer of recombinant genetic material from the GMM to other organisms in the
environment, is not desirable. This is due to the knock-on effects the transfer might
have on other organisms and biological processes. The level of hazard posed by the
transfer of genetic material is dependent on the properties conferred by the
modification on the GMM, and on any recipient organisms in the environment. The
properties that may have the greatest potential to cause adverse effects to other
organisms or biological processes are those that increase the virulence, pathogenicity
and survival of the GMM so that it is able to out compete other organisms for
nutrients and/or habitat. The transfer of reporter genes such as lux are unlikely to be
hazardous to the other organisms or biological processes, as they do not encode for
any selective advantage in the environment.
3.20 Therefore, the important factors in the assessment of the risk posed by the transfer of
genetic material are the potential for the genetic material to be transferred, and the
likely effect that the transfer might have on other organisms and biological processes.
Potential for genetic material to be transferred
3.21 Three basic mechanisms exist whereby genetic material can be transferred from the
GMM to other organisms [232, 236]. Whilst the potential for transformation,
transduction and conjugation between GM and non-GM microorganisms, and
between GM microorganisms and non-GM plants has been addressed extensively in
other publications [232, 233], some information that is relevant to the use of GMMs
in bioremediation is included in this review. With the exception of the Ti plasmid of
Agrobacterium tumefaciens that can enable the transfer of genetic material from
microorganisms to plants, the transfer of genetic material from GMMs is restricted to
other microorganisms in the environment.
3.22 Both transformation and transduction of genetic material between microorganisms
relies on good homology between the ‘foreign’ DNA from the GMM and the DNA of
the potential recipient [232]. The transfer of DNA by transduction is mediated by
bacteriophages with a narrow host range, and is therefore restricted to microorganisms
of the same species or between closely related species [232]. This has potential
implications to the use of GMMs in bioremediation, where in order to improve the
survival of the GMM in the target environment, a microorganism that is indigenous to
that environment is used as the recipient for the genetic modification. In such cases
the potential for transduction should be addressed, although the risk is likely to be low
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due to the narrow host specificity range of bacteriophages capable of transduction
[237].
3.23 The potential for transformation to result in the transfer of ‘foreign’ DNA from a
GMM into the indigenous microbial community is restricted both by the requirement
for good DNA homology and the restriction-modification systems operated by
individual cells. These are reported to reduce transformation efficiency in laboratory
systems by a factor of 104 [232].
3.24 Conjugation represents the most ecologically significant mechanism to transfer
plasmid encoded genetic material between microorganisms, and in the case of the Ti
plasmid from Agrobacterium tumefaciens, between microorganisms and plants [232].
The transmission of recombinant genes from the GMM is therefore increased if the
gene(s) is encoded in the GMM on a transmissible plasmid vector [10, 37]. However,
the transfer frequency of large degradative plasmids such as pJP4 (confers resistance
to mercury and the degradation of 2,4-D) between microorganisms is lower in sterile
soil than in liquid and solid growth media, and is further reduced by the biotic stresses
encountered in non-sterile soil [238].
3.25 The transfer of conjugative or mobilisable plasmids between microorganisms is most
likely to occur in environments where the plasmid encodes some ability to improve
survival in that environment. Horizontal transfer of genes of selective value in a
polluted environment has been found to occur up to six years after the introduction of
GMMs to a phenol contaminated site [239]. Because many of the genes responsible
for the degradation of organic pollutants and the resistance to inorganic pollutants are
plasmid encoded [22], then the selective pressure for transfer of such plasmids is high
in contaminated environments [232, 240]. The frequency of plasmid transfer is
however lower if narrow-host range plasmids are used instead of broad-host range
plasmids. Because narrow-host range plasmids can replicate in a restricted number of
microbial taxa then such plasmids are more biologically contained than those with a
broad-host range [30]. Narrow-host range plasmids also confer improved structural
and segregational stability in the host strain [241].
3.26 Conjugative plasmid transfer is reduced in environments where the microorganisms
are in a nutrient limited state. In this state, which is characterised by virtual absence
of protein synthesis, the replication of plasmids and expression of plasmid genes are
reported not to occur [232]. Therefore the addition of various substrates to
contaminated sites to support the growth of the GMM or to induce expression of the
modified genes, may also reduce any nutrient limitations in the environment and
consequently increase the potential for plasmid transfer [232].
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3.27 The transfer of chromosomal genes between microorganisms is reported to be very
rare and is only likely to occur between organisms of the same specie or between
closely related species with a high degree of DNA homology [232].
Effect of transfer of genetic material to other organisms or biological processes
3.28 The transfer of genetic material from the GMM is therefore only likely to occur
through conjugation, and only then if the recombinant genes in the GMM are located
on a mobilisable plasmid. Assuming that transfer of genetic material does occur
between the GMM and the indigenous microflora then the potential for any adverse
effects occurring (and therefore the level of risk) depends on the properties or
characteristics conferred by the transferred genes.
3.29 The most significant effects are likely to be incurred through the transfer of genes that
alter the virulence, pathogenicity or survivability of the recipient organism. Because
virulence or pathogenic properties are unlikely to provide any useful purpose in
bioremediation applications then they are not expected to be present in GMMs used
for bioremediation and are therefore unlikely to be transferred. Although the ability
to grow at 37 ºC is not assessed to be a pathogenic trait, it would enable the
microorganism to grow in the human body. It may therefore be preferable to avoid
the use of microorganisms that possess such traits, for bioremediation. .
3.30 However, as described previously in this report, the GMMs used for bioremediation
are usually designed to be able to survive in the environment. The most significant
‘survival’ traits are those that would give the recipient microorganism such a
significant advantage against other microorganisms in the environment that it would
be more competitive than other species for nutrients and/or habitats, and consequently
alter the microbial diversity of the resident microflora. This may subsequently affect
certain biological processes. The addition of any new population of microorganisms
to an environment containing an existing microflora may be expected to alter the
composition of that environment’s microflora to some degree.
3.31 In assessing the effect the inoculation of GMMs may have on the existing microflora,
the state and biodiversity of microorganisms in contaminated sites should first be
addressed. In environments, not exposed to pollutants, the composition of the
microbial community is relatively stable and the microorganisms present are
specialists in terms of their nutritional and physiological requirements [242]. Because
of the specialised nature of the microorganisms present, then the addition of a new
population of microorganisms is likely to have an effect on the composition of the
microflora.
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3.32 In polluted environments microbial communities are stressed due to the presence of
the pollutant, and changes in physiological factors such as pH and the types of energy
sources available [243, 244]. These changes select for opportunistic microorganisms
with more general nutritional and physiological requirements [242, 244]. Therefore,
the microbial community in contaminated sites is less specialised and more adaptable
to change. The addition of a new population of microorganisms is therefore likely to
have less of an effect on microbial biodiversity than in a non-contaminated
environment.
3.33 The level of risk associated with the transfer of genetic material from GMMs to
members of the indigenous microbial community is therefore assessed to be low.
Accumulation of Toxic Compounds
3.34 The use of GMMs designed to accumulate toxic compounds can result in the
localisation of contaminants at significantly higher concentrations than those likely to
have been present throughout the untreated contaminated site. Therefore, whilst the
original level of contamination may have been too low to cause significant effects to
human health and the environment (particularly adverse acute effects), the localisation
of the contaminant(s) through the use of pollutant-accumulating GMMs may result in
the concentration of toxic pollutants at levels sufficient to have a significant adverse
effect to biota.
3.35 It should be noted that the accumulation of toxic compounds by GMMs may be the
only mechanism to remove the pollutant from the contaminated environment, and is
usually only a transient stage in the overall bioremediation strategy. After
accumulation of the contaminant, the GMM-pollutant ‘complex’ has to be removed
from the contaminated site.
3.36 The level of risk associated with the accumulation of toxic compounds depends on the
ability of the GMM to accumulate toxic compounds, the state in which those
compounds are accumulated and the environment in which the GMM is applied.
3.37 As discussed in Chapter 1 in this report, there has been limited application of GMMs
for the accumulation of toxic compounds such as heavy metals. GM-based
bioremediation strategies designed to accumulate toxic pollutants have utilised GM
plants rather than microorganisms. The most likely reason for this is that metalaccumulating
plants are more suitable for use in in-situ bioremediation strategies.
Although metal-accumulating GMMs can be (and are) applied directly to the
contaminated environment, their small size means that further procedures are required
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to remove the microorganism-pollutant ‘complex’ from the site. GM plants,
particularly those designed to accumulate the toxic compound in above-ground plant
material allow significantly easier removal of the toxic compound from the
environment after accumulation.
3.38 The use of GMMs for accumulation of toxic compounds has therefore been confined
largely to bioreactors or other types of physically contained systems. These are used
to remove heavy metals from wastewater or groundwater (pump and treat technology)
[120], although the majority of the GMMs reported to date are still only at the
laboratory design stage [105-107]. Because GMMs used in these systems are
physically contained from the wider environment then there is likely to be only
negligible exposure of the biota to localised increases in concentrations of toxic
pollutants.
3.39 Metal-accumulating GMMs are also used in bioprotection applications, as well as in
direct bioremediation processes. In the bioprotection strategy the GMM is released
into the contaminated environment specifically to accumulate or degrade a particular
contaminant, so that it does not affect the activity of other organisms present. Such
microorganisms may have applications in protecting agricultural crops from the
inhibitory effects of any particular contaminant present in the environment [112], or
the protection of the microflora in wastewater treatment plants [124]. Because the
GMMs are released directly into the environment in bioprotection applications, then
there is a risk of localised higher concentrations of toxic compounds affecting other
organisms present. However, it should be noted that any pollutants that are
accumulated would be present in the environment anyway, and it is only the
concentration of pollutant that varies.
3.40 The risk posed to other organisms depends on the bioavailable concentration of the
toxic compound that is accumulated by the GMM, and how this compares with the
threshold tolerance concentration of the surrounding biota to that compound.
Production of Toxic Metabolites or By-products
3.41 The use of bioremediation strategies to remove pollutants from the contaminated
environment may result in the formation of toxic metabolites or by-products during
the degradation or biotransformation of the pollutant. The level of risk incurred is
dependent on the toxicity and concentration of any metabolites formed, and also the
length of time that any toxic metabolites are likely to persist in the environment.
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3.42 From the information obtained from the review of the scientific literature, no GMMs
have been identified whose use in the bioremediation of pollutants is likely to result in
the production of toxic metabolites or by-products.
3.43 The only exception to this is the use of GMMs as FAVs for the biodegradation of
hydrophobic pollutants such as PCBs. Although toxic metabolites or by-products are
not produced from the degradation of the target pollutant, the incomplete degradation
of the surfactants used to support the growth of the FAV and improve the
bioavailability of the pollutant may result in the formation of compounds that may
have a toxic effect to other organisms in the environment [82]. For example, the
degradation of alkylphenol polyethoxylates results in the formation of persistent
short-chain mono-, di- and triethoxylates. These degradation products are more toxic
than the parent compound, particularly to aquatic fauna and are potentially
oestrogenic [245].
3.44 The level of risk, posed by degradation of the surfactant used with the FAV, depends
on the type of compound used as the surfactant. The possible adverse effects caused
by the accumulation of surfactant degradation products may be avoided by
maximising the degradation of surfactants in the environment in which they are used.
Alkyl ethoxylate surfactants are more readily biodegradable in the environment than
alkylphenol ethoxylates and their degradation products are less likely to accumulate
[82]. The P. putida IPL5::TnPCB used in the FAV designed to degrade PCBs in the
environment [88] was able to degrade both alkylphenol ethoxylate and alkyl
ethoxylate surfactants as growth substrates. However, the GMM was only able to
degrade the ethoxylate moiety of these compounds, leaving potentially toxic
metabolites [88]. The Ralstonia eutrophus B30P4::TnPCB FAV was able to degrade
both of the surfactants without the production of potentially toxic compounds.
Although this GMM grew more slowly than P. putida IPL5::TnPCB [82] it may be
more suitable to the bioremediation of PCBs in the environment, particularly those
where aquatic organisms may be at risk of exposure to any surfactant degradation
products.
3.45 If suitable (i.e highly biodegradable) surfactants are used with the FAVs, then the risk
posed to the biota is assessed to be negligible. No other GMMs have been identified
whose use in bioremediation applications is likely to result in the formation of toxic
metabolites or by-products.
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Disruption of Other Organisms and Biological Processes
3.46 A number of studies have addressed the effect of the inoculation of a GMM in a
terrestrial environment on the numbers and activities of the indigenous microflora
[246-248]. In all three studies, soil microcosms (with or without 500 µg g-1 2,4-D)
were inoculated with P. putida PPO301(pRO103) which had been genetically
modified by the insertion of plasmid pRO103 which was expressed constitutively.
The plasmid encoded resistance to both the antibiotic tetracycline (25 µg ml-1) and
mercury (25 µg ml-1) and the ability to mineralise phenoxyacetate and partially
degrade 2,4-D to chloromaleylate. Control microcosms (with or without 500 µg g-1
2,4-D) were inoculated with the non-GM parental organism P. putida PPO301.
3.47 Both Short et al., (1991) [246]and Doyle et al., (1991) [247] found that in a nutrient
poor, semi-arid soil the degradation of 2,4-D by the GMM caused a significant
reduction in numbers of fungal propagules. The formation of the metabolite 2,4-
dichlorophenol during the degradation of 2,4-D was found to be inhibitory to the
fungal community. However, when the same GMM was inoculated into more
nutrient rich, less arid soil no degradation of 2,4-D by the GMM was observed, and
there was no effect on the fungal community [248].
3.48 Ingham et al., (1995) [248] also reported that there was no significant difference in
numbers of culturable bacteria, bacterial biomass and numbers of nitrifying and
denitrifying bacteria present in the microcosms inoculated with the GMM, compared
to the non-GM strain. Although the GM P. putida PPO301(pRO103) retained the
inserted plasmid for the duration of the 90 day trial, no degradation of 2,4-D was
detected. The continuing presence of 2,4-D in the soil had a significant effect on the
size of the microbial population (bacteria, fungi, amoeba and flagellates) compared to
the controls.
3.49 The inoculation of GMMs into contaminated sites may also have a positive effect on
the size and/or biodiversity of the indigenous microbial community. In contaminated
environments where the natural microflora is being inhibited by the presence of a
particular pollutant, or is restricted by a lack of nutrients, then the addition of a GMM
designed to degrade the inhibitory compound may allow the natural population to
grow. The degradation of previously non-utilisable pollutants into compounds that
can be used by the indigenous microflora may also alleviate the nutrient limited status
of the cells.
3.50 The inoculation of P. cepacia, genetically modified to degrade the compound 2,4,5-
trichlorophenoxyacetic acid (2,4,5-T), into soil resulted in the increase in the
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taxonomic and genetic diversity of the indigenous microbial community due to
increased nutrient availability [249]. However, if the degradation of a pollutant
results in the formation of toxic or inhibitory compounds, then an adverse effect on
the indigenous microbial community is likely to be realised [232]. This scenario can
of course be achieved following the inoculation of a GMM or non-GMM into an
environment as part of a bioremediation strategy.
THE USE OF PLANTS
3.51 The use of plants for the bioremediation of pollutants offers a number of advantages
and disadvantages to the environment and human health compared to the other
options available. Although the same basic hazards need to be addressed in assessing
the use of plants and microorganisms for the bioremediation of pollutants, the
different physiological and morphological characteristics of plants compared to
microorganisms, and the alternative approaches applied to their use in bioremediation
means that a different emphasis on the assessment of the likely hazards may be
required.
Transfer of Genetic Material
3.52 As with GMMs, the transfer of recombinant genetic material from the GM plant to
other organisms in the environment is not desirable due to the knock-on effects the
transfer might have on other plants and biological processes [208, 250]. With plants,
the properties most likely to have an adverse affect on the surrounding flora are those
that confer a selective advantage to survive, such as drought resistance, tolerance to
frost and altered pH, improved weedy characteristics (e.g rapid growth) and the
survival in the presence of particular toxic compounds.
3.53 Transfer of genetic material between plants is restricted largely to cross-pollination.
The potential for transfer to occur is therefore greater in those plants that are able to
undergo sexual hybridisation with resident flora and produce large quantities of
relatively mobile pollen, for example brassicas. The assessment of the risks
associated by the transfer of genetic material through cross-pollination (by wind or
insects) has been addressed by other reports, and is not restricted to those plants used
in phytoremediation. This subject will therefore not be addressed further in this
report. Particular reference is made towards the report on the ‘Guidance on best
practice in the design of genetically modified crops’ produced by the Best Practice
sub-group of the UK’s Advisory Committee on Releases to the Environment (ACRE)
[251].
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3.54 All of the genetic modifications identified to date that may be applied in
phytoremediation have been directed towards the sequestration, degradation or
resistance to particular compounds in the environment. The modified properties are
only of benefit to a plant within the contaminated site and are not expected to confer
any more general traits that may give the modified plant a selective advantage outside
the contaminated site [156], for example frost tolerance or drought resistance.
Therefore, expression of the modified trait is likely to be effectively restricted to the
contaminated environment. Outside the contaminated site in the absence of the target
pollutant, the modified properties transferred from the GM plant are likely to confer a
negative selection pressure and may therefore not be maintained in the population
[208].
3.55 Rugh et al., (1996) [171] reported that several of the GM plant lines expressing the
modified merA gene (conferring mercury resistance) actually grew better on a
mercury-containing medium than on the control medium without mercury. The
findings suggested that the mercury-resistant transgenic plants would not compete
well in areas that were un-contaminated with mercury, and that this would effectively
promote the self-containment of the transgenic plants within the mercury polluted
sites. Indeed, this is equivalent to the scientific view that natural metal tolerance in
plants is associated with a metabolic ‘cost’ that makes these plants less competitive
(fit) than non-tolerant genotypes when growing on uncontaminated soils [175].
Accumulation of Toxic Compounds
3.56 Any risks associated with the accumulation of toxic compounds by GM plants in
phytoremediation are restricted to those plants that are able to hyperaccumulate
pollutants. The plants capable of removing compounds such as mercury and
selenium, from contaminated sites by phytovolatilisation are not addressed in this
section, as the compounds are only accumulated transiently within the plant before
being released through the leaves.
3.57 The ability of some plants (GM and non-GM) to sequester toxic compounds from
their environment and accumulate them within their tissues, is a key application of the
use of plants for bioremediation, especially in the removal of heavy metals from
contaminated sites. However, because the target pollutants are accumulated at
concentrations that may be significantly higher than in the surrounding environment,
the plants may pose a hazard to the environment and/or human health if they are
eaten, not harvested or not disposed of correctly after harvest. For example, the
compound(s) accumulated by the plant may be lost during plant senescence and
returned to the environment. Therefore the use of plants in phytoextraction and
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rhizofiltration applications needs to ensure that the sections of the plants containing
the accumulated compounds (usually the above ground structures) [175] are removed
from the site before any plant senescence occurs. At the same time, there is good
evidence that metal hyperaccumulation acts as an effective feeding deterrent to help
protect the plant from herbivores [252], so this would help to minimise the risk of
metal transfer from such plants into food chain.
Production of Toxic Metabolites
3.58 As mentioned in the previous chapter, phytoremediation strategies may result in the
production of toxic metabolites, or lead to the greater exposure of the target parent
contaminant. These hazards are however not restricted to phytoremediation using
GM plants, although the improved properties of the GM plant compared to the wild
type strain may result in a potentially larger hazard to the environment and/or human
health.
3.59 In situ approaches to the phytoremediation of metal pollutants may involve the
conversion of the metal to a different more mobile and more bioavailable state.
Although this conversion is required in order to remove the metal from the
environment, the more mobile metal forms are likely to have a potentially greater
toxicity to other organisms [168, 171].
3.60 Potential applications for the phytoremediation of pollutants have been proposed in
which the GM plant is designed to secrete metal-selective ligands into the rhizosphere
to solubilise the target compound [19, 155]. Some non-GM plants are known to
release substances that are able to reduce certain species of metal, to facilitate their
uptake by the plant, and also to produce compounds capable of chelating metals and
promoting their mobility in the environment. The addition of synthetic chelating
agents to soil under laboratory conditions has been reported to result in the enhanced
accumulation of metals such as lead in the shoots of Indian mustard [159, 253, 254].
3.61 The conversion of metal pollutants into a more mobile and potentially more toxic
state is however only likely to result in a transient exposure of the more toxic metal to
the environment. Once the plant has sequestered the metal (either within the plant or
immobilised in the rhizosphere), further environmental exposure is unlikely to occur.
3.62 The production of toxic compounds is more likely to occur during the
phytodegradation of organic pollutants, especially where the pollutant is degraded
outside the plant. For example, the expression of a gene encoding the mammalian
cytochrome P450 2E1 in tobacco plants resulted in the increased metabolism of TCE,
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compared to the wild type variety [150]. Although this modification was not reported
to result in the production of any toxic metabolites during the breakdown of TCE, a
thorough study of these transgenic plants would need to be undertaken to verify that
no toxic intermediates were released into the environment, as a result of the
phytoremediation process conducted under field conditions [150].
3.63 The assessment of many of the potential risks that may be associated with the use of
plants for phytoremediation has been restricted to date by the lack of research being
conducted under field conditions involving GM plants designed for phytoremediation
applications. Although no releases of GM plants for phytoremediation have occurred
to date, one of the more advanced areas of research is the use of GM plants for the
phytovolatilisation of pollutants such as mercury and selenium. Plants used for
phytovolatilisation are designed to sequester a volatile metal such as mercury and
emit it from their leaves into the atmosphere in a less toxic form. Although the
mercury released from the plant is significantly less toxic than the form present at the
contaminated site, such plants may be described as releasing toxic compounds into the
environment.
3.64 However, in order to assess the potential implications of the phytovolatilisation of
mercury from contaminated sites, the relative scale of such releases compared to
atmospheric emissions of mercury from other sources must be addressed.
3.65 The residence time of Hg (0) in the atmosphere is approximately two years before it is
re-deposited onto the earth’s surface, usually through precipitation. Therefore,
mercury released through phytovolatilisation is likely to be diluted to trace
concentrations in the atmosphere before being re-deposited [174]. Any quantity of
mercury released from a contaminated site is likely to be small in comparison with the
atmospheric mercury load (~4 x 106 kg) [174] and would be negligible compared to
other sources of mercury emission, e.g burning of fossil fuels and medical waste
[156]. Even if the levels of volatile mercury produced during phytovolatilisation were
400-fold higher than normal background levels, they would still be 25 times below
most regulatory limits. Bizily et al., (1999) [209] proposed that the rate of mercury
volatilisation from the GM plants could be designed to ensure that the quantities of
mercury released from the plant were within government regulations.
3.66 Although the amount of mercury released from GM plants is likely to be relatively
low compared to other emissions of mercury to the atmosphere, it has been proposed
that the direction of prevailing winds, the location of nearby population centres, and
the magnitude of total site Hg(0) emission would need to be considered prior to
determining the level of risk posed to the environment and/or human health [208].
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3.67 Similarly, for GM plants capable of phytovolatilising selenium, it is thought that the
amount of selenium added to the atmosphere through phytovolatilisation would be
negligible when compared to the atmospheric inputs of this compound from
volcanoes, soil and non-GM plants [166].
Disruption of Other Organisms and Biological Processes
3.68 Disruption of other organisms and biological processes by GM plants used for
phytoremediation is most likely to be a consequence of the plants having a selective
ecological advantage and consequently out-competing other flora in a particular
environment, or by having a toxic effect on other organisms, particularly those likely
to ingest parts of the plant.
3.69 As discussed in the section assessing the risks posed by the transfer of genetic
material from GM plants, the transgenic species used in phytoremediation are unlikely
to have any ecological selective advantage outside the contaminated site, and are
therefore not expected to disrupt other flora or biological processes outside the
contaminated environment. It should be noted that within the polluted site, the types
of flora present are likely to be determined by their tolerance to the contaminants
present, and may therefore be less affected by any selective advantage expressed by
the GM plants.
3.70 The accumulation of toxic compounds within the tissues of GM plants designed for
phytoextraction or rhizofiltration, has the potential to cause harm to organisms that
consume parts of the plant. The uptake of heavy metals such as lead by crop plants,
for example, is reported to be a major source for the accumulation of such toxic ions
within the human body [177]. However, this can be avoided by preventing
consumption of the GM plants used [212].
3.71 Consumption of GM plants designed to phytoremediate metal contaminants is likely
to be low because of the presence of the metal within the plant. One of the main
functions of metal hyperaccumulation in non-GM plants is to prevent disease and
herbivory [154]. Therefore, the genetic modification of a plant to hyperaccumulate
metals is also likely to result in reduced herbivory. It has also been reported that
some species of insects that would normally be expected to eat certain species of
plant, avoid the plants when they contain metal contaminants in their tissues (Ensley
(pers comm) in) [166]. However, there are concerns that insects, particularly pest
species, could become adapted to feeding on hyperaccumulating plants containing
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high levels of metal which may lead to foodchain contamination [166]. To date no
research has been reported investigating these possibilities in GM plants.
3.72 Where genetic modification is used to improve an existing phytoremediation
capability of a particular plant, then the disruption of other organisms or biological
processes may be less likely. For example, the GM poplars designed to degrade TCE
are proposed to utilise this compound using the same pathway as employed by the
non-GM poplars for the degradation of TCE [150]. Consumption of leaves from non-
GM poplars exposed to TCE had no harmful effects on herbivorous insects,
suggesting that the degradation of TCE by the non-GM trees resulted in the
production of non-harmful metabolites. If the GM trees use the same pathway for the
biodegradation of TCE, then no toxic metabolites would be expected to be produced
by the GM trees [150]. However, the greater rate of phytodegradation of TCE by the
GM poplars means that higher concentrations of metabolites are likely to be present.
Such metabolites may be toxic at higher concentrations, and further research is
required to address this issue.
3.73 As well as affecting external systems, such as interactions between the plant and its
environment, the introduction of novel genes into a plant can also disrupt systems
within the plant itself. The disruption may be significant enough to prevent the GM
plant from germinating, or may only cause the plant to be more susceptible to
environmental effects. In GM tobacco plants designed to overexpress γ-ECS, it was
found that foliar levels of γ-ECS and GSH were increased, but that the increased GSH
appeared to result in greatly enhanced oxidative stress. The γ-ECS transformed plants
were reported to suffer from oxidative stress due to a failure in the redox-sensing
process in the chloroplast [255]. However similar findings were not reported when
the gene for γ-ECS was over-expressed in Indian mustard [17].
3.74 In addition to their direct application for removing metal pollutants from
contaminated sites, GM hyperaccumulating plants have been proposed as having
secondary benefits to other organisms. For example, plants could be modified to
extract desired, beneficial micronutrients such as selenium from the environment, at
specified levels. Such plants could then be fed to livestock as pellets to provide the
recommended daily requirement of the micronutrient [256]. Canola plants, grown for
the phytoremediation of selenium, were found to be safe to feed to marginally
selenium-deficient lambs and cows, in order to meet their normal selenium intake
requirements [257].
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4. POTENTIAL MANAGEMENT STRATEGIES FOR THE USE
OF GMOs IN BIOREMEDIATION
4.1 The purpose of this section of the report is to identify and review the possible
management strategies that could be employed to reduce any of the risks identified in
the previous chapter.
MICROORGANISMS
4.2 The purposes of the management strategies designed for the use of GMMs in
bioremediation are to ensure that any of the risks, identified in Chapter 3, are
minimised and that the GMM functions as intended. Of the risks identified, the most
significant are those resulting from the transfer of genetic material from the GMM to
other microorganisms, and the disruption of other organisms and biological processes.
The use of GMMs in bioremediation is not assessed to result in a significant level of
risk, in terms of the accumulation of toxic compounds and/or the production of toxic
metabolites.
4.3 The level of risk associated with the transfer of genetic material and the disruption of
organisms and other biological processes can be minimised through addressing the
biological and/or physical containment of the GMM, and ensuring that only
recombinant DNA that is required for the bioremediation application is present in the
GMM. Management strategies can be developed to prevent or minimise transfer of
genetic material from the GMM, and also to contain the populations of GMMs to a
particular location, determined by specific environmental or physiological parameters.
4.4 The presence of extraneous recombinant genetic material in the GMM is not desirable
if the GMM is intended for use in the environment. In addition to potentially
reducing the activity and competitiveness of the GMM in the environment, there is a
greater chance of recombinant DNA being transferred to other microorganisms.
Selectable marker genes are often inserted into the GMM, with the other transgenes,
to assist in the detection and selection of the microorganisms that have been
genetically modified. If possible it may be preferable to use a selectable marker gene
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that is appropriate to introduced modification, such as resistance to organic or
inorganic pollutants.
4.5 Virtually all available plasmids carry gene encoding resistance to antibiotics as
selectable markers [37]. However, such markers are undesirable for environmental
applications [4], and can be replaced with other selectable markers (Table 4.1), or
eliminated from the microorganism after gene transfer [258].
Table 4.1 - Alternative selectable genetic markers [37]
Selectable marker gene Origin Selectable phenotype
bar Streptomyces hygroscopicus Resistance to the herbicide bialophos
aroA CT7 Salmonella typhimurium Resistance to the herbicide glyphosate
merTPAB Serratia marcescens Resistance to mercuric salts/organomercurials
arsAB Escherichia coli R773 Resistance to arsenite
luc firefly Bioluminescence
lacZY Escherichia coli Growth on lactose
teh RP4 plasmid Resistance to potassium tellurite
Transfer of Genetic Material
4.6 The risks incurred by the transfer of genetic material from the GMM to other
organisms are assessed as most likely to occur if the recombinant genes are located on
a mobilisable plasmid. The transfer of plasmids between microorganisms, by
conjugation, can be reduced by using non-mobilisable or mobilisation defective
plasmids, or narrow-host range plasmids. However, transfer of the genetic material
can still occur and the use of such plasmids does not ensure containment of the
recombinant genetic material within the GMM.
4.7 To avoid the problems of transfer of plasmids between the GMM and resident
microflora, minitransposon vectors can be used to modify the host microorganism
genetically. Transposons can also be designed to contain a number of selectable
markers that can be useful to detect and select for the modified cells. The
minitransposons that are based on Tn5 and Tn10 lack the transposase gene (encodes
the enzyme that catalyses the movement of transposon) and only contain a selection
marker between the minimal inverted repeats needed for transposition. These
minitransposons are available on a plasmid suicide delivery system, and provide very
stable constructs with minimal horizontal transfer of cloned genes [10]. Tn5 based
transposons have been developed containing selectable markers encoding resistance
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to herbicides such as bialaphos and glyphosate, and heavy metals such as mercury,
arsenic and tellurite [37].
4.8 Minitransposon vectors have been applied successfully in the genetic modification of
a range of bacteria, including E. coli, Klebsiella, Salmonella, Proteus, Vibrio,
Bordetella, Actinobacillus, Rhiziobium, Rhodobacter, Agrobacterium, Alcaligenes
and several pseudomonads [37]. In addition to providing a very stable phenotype,
minitransposons can be engineered into microorganisms with a minimal number of
manipulations. This is important with respect to the development of GMMs able to
survive in the field, as keeping the amount of manipulation low minimises the loss of
competitiveness usually observed in laboratory-designed microorganisms [259].
4.9 Although the transfer of genetic material is not desirable after the GMM has been
released into the environment, the spread of conjugative or mobilisable plasmids from
the inoculated GMMs to other microorganisms has been proposed as a possible tool to
ensure more effective and/or more immediate degradation of the target contaminant
[64], and could conceivably be used as part of the development of the microorganism
that would be used in the bioremediation strategy. The transfer of the degradative
plasmid to a representative sample of the target environment’s indigenous microflora,
under contained conditions, may result in the formation of a microorganism that is
more effective in degrading the pollutant due to its greater ability to survive and
compete in the contaminated environment. The inoculation of GM P. putida UWC1
(containing plasmid pD10 which confers the ability to utilise 3-chlorobenzoate as a
sole carbon source) into freshwater sediment microcosms did not improve the
intensity of biodegradation of the 3-chlorobenzoate present. However, following
conjugative transfer of pD10 from the GMM to members of the indigenous
microflora, the degradation of 3-chlorobenzoate improved [260].
4.10 It was proposed that instead of using GMMs to degrade the target pollutant directly,
they should be used as donor organisms to introduce the necessary genetic capability
into a member of the indigenous microbial community that could then be inoculated
into the environment [260]. Transfer of the plasmid encoded genes from the GMM in
a contained environment such as a microcosm means that the recipient
microorganisms can be identified and characterised before their possible release into
the environment [64]. Due to the wide biodiversity of microorganisms in the
environment it is likely that any recipient microorganism is likely to be unknown and
therefore poorly characterised.
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Biological Containment Systems
4.11 Biological containment systems provide a mechanism to restrict the population of
GMMs (and the recombinant DNA) to a particular environment, as defined by
specific physical or chemical parameters [261]. Some biological containment systems
also enable the population of GMMs to be killed, following the addition of a chemical
trigger to the environment. Should the GMMs have a disruptive effect on other
organisms or biological processes, then the availability of biological containment
systems means that such disruption can be limited to the contaminated environment,
and potentially to a limited period [261]. As discussed in Chapter 3, microbial
communities in contaminated environments are likely to be more tolerable and
therefore less affected by such disruption.
4.12 Biological containment systems are applicable particularly as an alternative to the
physical containment systems available for GMMs used in laboratory type
applications [23]. The purpose of biological containment systems for GMMs is to
minimise or prevent the transfer of the genetic modification from the GMM to other
members of the indigenous microbial community. This can be achieved either by
reducing the likelihood of gene transfer of the inserted genes, by ensuring that they
are inserted into the chromosome of the recipient microorganism, or by designing the
GMM so that it is only able to survive in environments that have particular chemical
or physical characteristics [23, 262, 263]. For example, the recombinant genes in the
GM E. coli described by Winter et al., (1989) [68] were under the control of a
temperature inducible promoter and would therefore only be expressed at
temperatures >42 ºC.
4.13 Although the use of mini-transposon vectors for example to modify the recipient
microorganism is likely to reduce the potential for transfer of the recombinant genes,
the reliance on such systems for biological containment does mean that the GMM
may remain in the environment for a significant length of time. Although this may
not pose a risk to the environment or human health, the persistence of the GMMs after
completion of their intended function may not be desirable, both in terms of public
perception, and possibly because of the potential for unanticipated consequences
arising [264].
4.14 Two basic types of systems have been proposed that enable the GMM to become
inactivated at a specified time, and therefore effectively removed from the
environment when required. These are the attenuation of the microorganism so that
survival in the environment is dependent on the presence of a specific compound
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(usually a growth substrate), and the use of controllable suicide systems that are able
to kill the cell when expressed under specific conditions [263].
Attenuation based biological containment systems
4.15 Attenuation based containment systems are designed so that the GMM is only able to
survive in particular environments. These systems are used widely in physically
contained processes such as industrial fermenters. The GMMs are designed so that
they are unable to synthesise key compounds, which must be added to their culture
medium in order for them to survive. E. coli X1776, for example, is unable to
synthesise the cell wall component D-amino pimelic acid. Because this compound
does not occur naturally, then E. coli X1776 is unlikely to survive, should it be
released into the environment (Curtiss et al., 1977, cited by) [263].
4.16 With respect to their use in bioremediation, such attenuation systems are really only
applicable to GMMs used in physically contained facilities such as bioreactor systems
used to treat contaminated groundwater. Amending contaminated sites with key
nutrients or growth compounds is likely to be prohibitive both in terms of cost and
time, although the use of plants to provide the required compounds in the rhizosphere
is a possible option that could be applied to large scale contaminated sites. However,
because the GMM may still persist in the environment in a dormant state in the
absence of the required substrate, then attenuation systems are unlikely to result in the
removal of the GMMs from the environment.
4.17 The use of recA mutants has been proposed as an attenuation based biological
containment system that does not depend on the addition of specific compounds and
should result in the inactivation of the microorganism after a period of time. The recA
system in bacteria is involved in the repair of DNA following exposure of the
bacterium to ultraviolet light. If the recA system is mutated then the bacterium is
unable to repair the radiation damage and will eventually be eradicated. However,
although recA is easily identified, isolated and manipulated in most bacteria, most
recA mutants are often too disabled to survive in the environment for the length of
time required in bioremediation applications [263].
Controllable suicide systems
4.18 Controllable suicide systems (also referred to as active biological containment
systems) consist of a killing element, designed to induce cell death, and a control
element designed to modulate the expression of the killing element within the
modified microorganism [264]. Expression of the killing gene(s) causes the
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inactivation of the GMM and the destruction of the recombinant genetic material.
The advantages of controllable suicide systems, over the other types of biological
containment systems, are that they are designed to have no effect on the behaviour or
ability of the GMM to survive in the environment, but when activated cause the
GMM to be removed from the environment as a biological entity. This prevents the
GMM persisting in the environment in a dormant state.
4.19 Although a number of gene products have been identified that are toxic to certain
strains of bacteria and therefore potential candidates as killing elements in
controllable suicide systems, many of these products are growth inhibitors and are
only effective at high concentrations and towards a limited number of microbial taxa.
For the purposes of containing GMMs, the ideal suicide systems are based on gene
products that affect cellular functions common to most bacteria and whose toxicity is
high at low concentrations [263].
4.20 The gef gene is one of several genes that have been used as the basis of controllable
suicide systems for GMMs. Expression of the gef gene (isolated originally from E.
coli) results in the formation of the membrane protein Gef. This porin like protein
becomes inserted in the cell membrane [264], where it generates pores, causing the
membrane potential across the cell membrane to collapse and consequently killing the
microorganism [265]. The use of different promoter and regulator sequences for the
control element of the system, allows the gef gene to act as the killing element in
GMMs used to biodegrade a wide range of organic compounds. Because the gef gene
product targets the cell membrane (a common component of all bacterial cells), then it
should be able to kill most, if not all species of bacteria [263]. Some species of
bacteria have been found to be less sensitive to Gef than others, and may therefore
require a higher level of expression of gef to kill the cell.
4.21 The design of the control element determines under what conditions the killing
element is activated. To date, all of the controllable suicide systems incorporated into
GMMs designed for bioremediation applications have been developed so that the
GMM is inactivated in the absence of a particular pollutant [261, 263-265]. These
GMMs are therefore restricted to environments containing that pollutant, and
following the degradation of the pollutant, the GMM is destroyed. To date gef-based
systems have been designed that are activated in the absence of benzoate, alkyl-,
dimethyl-, chloro-, dichloro-, methyl-, ethyl- and methoxybenzoates; salicylate; and
methyl- and chlorosalicylate [266]. Such systems are therefore applicable to the
biological containment of GMMs designed to be able to bioremediate these
compounds.
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4.22 An example of a gef-based controllable suicide system is presented in Figure 4.1. The
use of the XylS regulator, as part of the control element, means that the system can be
controlled by the presence of effectors of XylS. These include the alkyl-substituted,
chloro-substituted and other halo-substituted benzoates [264]. In the system
illustrated in Figure 4.1, the regulatory gene xylS is expressed constitutively in both
(A) and (B). In scenario (A), the presence of an effector of the xylS gene causes the
product of xylS to be activated. This promotes transcription from Pm and leads to the
synthesis of the LacI repressor. The action of the LacI repressor is to prevent
expression from Plac, which is required to achieve expression of gef. However, in the
absence of an effector of xylS, the expression from Plac is not repressed and Gef is
produced, killing the cell [261].
Figure 4.1 - Elements and functioning of a biological containment system [261]
xyl S Pm : lac I
(+)
Plac : gef
(-)
3MB
Control element Killing element Survival of cell
NO
YES
(B)
(A)
xyl S Pm : lac I Plac : gef
In both (A) and (B) the regulatory xylS gene is expressed constitutively. However, in the presence of 3-
methylbenzoate (3MB) (A) the product of xylS is activated and causes transcription of the lacI repressor (() that
forms tetramers that prevent expression from Plac and formation of Gef. In the absence of 3-methylbenzoate
(B) expression from Plac occurs and Gef is formed thus killing the cell.
4.23 Ronchel et al., (1995) [261] inserted the gef system illustrated in Figure 4.1 into
Pseudomonas putida EEZ29. The recombinant pseudomonad (designated EEZ30),
was modified so that the killing element was located on the chromosome and the
control element on a mobilisable plasmid (pCC102). The inserted genes were found
to be stable genetically, and pCC102 was not mobilised from the GMM during the
transfer of the TOL plasmid from the GMM to other pseudomonads in soil
microcosms. Both contained (gef+) and uncontained (gef-) versions of P. putida
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EEZ29 survived well in soils amended with 0.1 percent (w/w) m-methylbenzoate.
However, in the absence of the m-methylbenzoate numbers of the contained GMM
(EEZ30) declined markedly, compared to the gef- strains [261].
4.24 The genes hok and relF have also been proposed as the ‘kill’ genes for controlled
suicide systems [266, 267], as has the streptavidin gene (stv) isolated from
Streptomyces avidinii [268]. In the presence of a particular growth substrate or target
pollutant the stv gene is repressed using a similar system to the one described for gef.
However, in the absence of the particular substrate, the stv gene is expressed and
streptavidin is produced. This compound has a particularly high binding affinity for
the ubiquitous essential prosthetic group, D-biotin (vitamin H). Inactivation of Dbiotin
by streptavidin kills the microorganism [268]. Because the streptavidin system
targets cell metabolic processes, then it should complement cell suicide systems
which target cell membranes and walls, or nucleic acids [268].
4.25 Destruction of the cell will expose the DNA to extracellular nucleases that are likely
to inactivate the recombinant genetic material. The use of nucleases as the killing
agent in controlled suicide systems will result in the destruction of the DNA and the
inactivation of the GMM [263].
4.26 The nucleases produced by Staphylococcus aureus and Serratia marescens are
reported to be suitable, as they have sufficient activity to override the ability of the
GMM to repair the damage to its DNA. However, the use of nucleases to control
GMMs in the field may be limited by the intracellular stability of the enzymes (halflife
of two minutes for S. marescens and two hours for S. aureus) and the conflicting
activities of the GMM’s DNA repair mechanisms [263].
4.27 A limitation of all suicide systems is that they are significantly inefficient. Even
under optimal laboratory conditions, up to 10-4 microorganisms in a population are
not killed [8]. The primary reasons for the low effectiveness of suicide systems are
mutational inactivation of the suicide gene and the selection of mutants with a
defective suicide system [267]. Although suicide systems are designed not to affect
the normal growth of the microorganism, even a small basal level of expression of
these genes is enough to confer a selective advantage to cells that have a mutated and
therefore non-expressed suicide system [263, 269]. If suicide systems are intended to
control large populations of GMMs then the basal level of expression of the killing
gene must be as low as possible [263].
4.28 Insertion of the suicide genes into the GMM in multiple copies was found to reduce
the mutation rate, especially when the insertions were made so that no single mutation
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event, particularly a deletion, would result in the inactivation of both copies of the
suicide system [269]. A single copy of a suicide system is reported to have a
mutation rate of approximately 10-6 per generation, whereas a double system, both
using the same control element will have a mutation rate of 10-8. With the double
system, if it is designed with independent control elements then the rate may be
reduced to as low as 10-12 per generation [269]. The mutation rate is also reduced if
the killing element and control elements are located on the chromosome of the GMM
[261, 264]. The reason for this was reported to be unknown, although it was possibly
due to the overall basal rate of transcription of the Plac::gef being lower on the
chromosome than on a high copy number plasmid [261].
4.29 Atlas (1992) [262] reported that variations in the stability of the vector, used to insert
and maintain the suicide constructs in the GMM, may also effect the effectiveness of
the biological containment system. Improvements in vector stability, through the use
of stably-inherited plasmids or chromosomal insertion, in combination with tightlycontrolled
promoters and duplicate/multiple insertions is proposed as the solution for
using suicide elements for the biological containment of GMMs in the environment.
Application of Microorganisms to the Contaminated Site
4.30 The method used to apply the GMMs to the contaminated site may have implications
to the immediate containment of the GMMs to the target area. Microorganisms are
most easily applied to contaminated soils in solution as a spray or mist. Although this
method allows an even distribution of the microorganism across the target area, it
does have the potential to form bacterial aerosols, and may result in the dispersion of
the microorganisms beyond the designated site. Ford et al., (1999) [270] reported that
the meteorological conditions encountered during the inoculation of the field
lysimeters with the GM P. fluorescens HK44 did affect the survival and dispersion of
the GMMs.
4.31 A relative humidity of 50 percent has been reported to produce a far higher death rate
of microorganisms in aerosols than either higher or lower humidities, and the death
rate was especially high during the first 5-20 min of exposure (Dunklin and Puck,
1948; cited by) [270]. High ultraviolet light also reduces bacterial counts in aerosols
(Barnthouse and Palumbo, 1986; cited by) [270]. Therefore to reduce ‘spray drift’ of
the GMMs from the application area, it was proposed that the optimum humidity for
spray inoculating contaminated soils was 45-60 percent, and that the GMMs should
be dispersed in a saline solution [270].
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PLANTS
4.32 To date, a large number of research trials and commercial growing of GM plants have
taken place worldwide in the field. The majority of these GM plants have been crop
plants grown for agricultural purposes. No releases of GM plants for bioremediation
have so far taken place in the field. However, due to the possibility that many of the
transgenic plants designed for agricultural applications may be used for
phytoremediation, particularly where rapid growth or ease of harvest is required, then
the management strategies developed and/or proposed for GM plants in agriculture
are applicable to GM plants in phytoremediation.
Transfer of genetic material
4.33 The management strategies that have been developed or proposed to limit the transfer
of the recombinant genes between the GM plants and other flora in the environment
have largely been addressed for GM plants designed for agricultural purposes [251],
and are therefore not covered in more detail in this review. However, as discussed in
the risk assessment, the transfer of genetic material from GM plants designed for
phytoremediation may not confer any selective advantage to other plants, and will
therefore be effectively self-contained within the area of the contaminated site. If this
biological containment is effective, and the GM plant only contains transgenes to
confer the desired trait (and no other ‘superfluous’ recombinant material such as
herbicide tolerant selective marker genes), then no other management strategy is
likely to be required.
Accumulation of toxic compounds
4.34 The requirement for management strategies for GM plants designed to
hyperaccumulate pollutants depends on the concentration and potential toxicity of the
pollutant that is accumulated, and the location in the plant where the accumulation
occurs. The hazards associated with GM plants designed for rhizofiltration or
phytoextraction are only likely to be realised if the GM plant is consumed. Therefore,
any release of such plants should consider the potential for herbivory of the plants and
take steps to prevent this from occurring, such as physical containment or using nonpalatable
or inedible plants.
4.35 If herbivory was a possibility, then one management strategy to reduce exposure to
the grazer is to produce GM plants with organ-specific overexpression of the
protein(s) involved in phytoaccumulation. This could lead a reduction of the
potentially toxic metals in the consumable parts of the plant, and the partitioning of
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the hazardous metals into non-consumable sections, such as below ground structures
[103, 196]. Yeargen et al., (1992) [195] demonstrated that tobacco seedlings
expressing the mouse metallothionein I gene, exhibited a difference in the tissue
partitioning of cadmium within the plant, compared to the wild type. This was
reported to demonstrate the potential for reducing the cadmium content of above-root
tissues of certain plants. This would however only be of use in reducing the potential
exposure of organisms feeding on the above-ground parts of the plant.
4.36 Alternative suggestions to prevent organisms from feeding on GM hyperaccumulating
plants have included the use of ‘clean crops’ adjacent to GM plants, along with fences
and/or other repellents. The non-accumulating ‘clean crops’ are designed to attract
organisms away from consuming the transgenic plants, with the use of repellents and
fences to further reduce the attractiveness of the hyperaccumulating plants [208].
4.37 Because the pollutant is only accumulated within the plant and not degraded, then
plants used for phytoextraction or rhizofiltration will need to be harvested to remove
the accumulated pollutants from the contaminated site. After harvesting, it may be
possible and commercially viable to employ a biomass processing system to recover
the metal contaminant from the plant material. Possible marketable metals include
nickel, zinc and cobalt [173]. If this were not possible or cost-effective, then it may
be beneficial to reduce the harvested plant material by weight and/or volume by
thermal, microbial, physical or chemical means. This step would decrease the
handling, processing and potential subsequent landfill costs [160].
4.38 Salt et al., (1995) [159] investigated methods for the further concentration of metals
in plant tissues, to reduce the weight/volume of material that would ultimately need to
be disposed of. Possible methods included sun, heat or hot-air drying;
environmentally-safe ashing or incineration; composting; pressing or compacting and
leaching.
4.39 The type of plant species used and the time of harvest also need to be considered for
plants used for phytoextraction. For example the use of annual plants or deciduous
tree species for phytoextraction would result in the return of accumulated compounds
to the soil if the plant was not harvested prior to the end of the growing season and the
start of senescence (e.g through leaf fall) [157]. For phytoextraction processes a fast
growing plant that is easily harvested would be preferable, making sure that the plant
is harvested prior to senescence and/or leaf-fall. This would ensure the maximum
possible recovery of the target contaminant from the soil, and could be designed to
involve more than one harvest per growing season [160].
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Production of toxic metabolites
4.40 The production of toxic metabolites by GM plants developed for phytoremediation is
not desirable. Where possible the breakdown of the contaminant by the transgenic
plant should be followed completely to ensure that no toxic metabolites are produced
during the reaction pathway [165]. The production of a toxic metabolite that is
subsequently converted into a non-toxic compound is likely to be less of an issue than
the formation of a dead-end toxic compound, although this will depend on the toxicity
and persistence of the transient toxic metabolite within the plant.
4.41 However, it should be noted that with GM plants designed to phytovolatilise toxic
pollutants such as mercury, the objective of the phytoremediation process is for the
plants to produce a toxic compound (although one that is significantly less toxic than
the target contaminant), and to result in the overall abatement of the environmental
hazard represented by the metal. With these plants, the potential management options
may include reducing the residence time of the mercury in the plant, and the design of
plants from which the quantities of mercury released can be predicted in advance.
Disruption of Other Organisms and Biological Processes
4.42 From the information presented in the risk assessment, the most likely mechanisms by
which GM plants developed for phytoremediation may disrupt other organisms or
biological processes is following the transfer of genetic material, or by toxicity caused
by ingestion of hyperaccumulating plants. The transgenic traits applicable to
phytoremediation strategies are unlikely to provide the GM plant with any
characteristics that would confer a selective ecological advantage over other plants
outside the contaminated area. Whilst the GM plant may become the dominant floral
specie within the contaminated site, this dominance would only be expected to persist
for the duration that the target contaminant remained on the site.
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5. REPORT OF THE WORKSHOP
INTRODUCTION
5.1 This section of the report consists of the technical report of a one-day workshop held
at Magdalen College, Oxford on 31st March 2001.
5.2 The purpose of the workshop was to create a technical forum to discuss the
applications, advantages, limitations, risks and potential management strategies
associated with the use of GMOs in field-based and contained bioremediation
programmes. By bringing together researchers in the range of disciplines that
encompass plant and microbial-based bioremediation strategies, the workshop was
designed to build on the information compiled in the previous sections of this report,
and in particular to discuss the most likely applications for this technology, the major
issues involved and to make recommendations for future research.
5.3 The meeting was comprised of six paper presentations followed by a general
discussion on the main issues raised. The morning session, which covered the use of
genetically modified (GM) microorganisms in bioremediation was chaired by
Professor Chris Knowles (Oxford Centre for Environmental Biotechnology,
University of Oxford). The afternoon session, which addressed the use of GM plants
in phytoremediation applications, was chaired by Professor Chris Leaver (Department
of Plant Sciences, University of Oxford). Professor Leaver also acted as overall chair
for the workshop and led the general discussion following the afternoon session.
5.4 The six papers presented at the meeting were:
• Prospects and challenges for bioremediation with genetically modified
microorganisms (GMMs)
− Professor Kenneth N Timmis, National Research Centre for
Biotechnology, Braunschweig, Germany.
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• Reporter gene based biosensors – risk-based management support for
remediation of contaminated land
− Professor Ken Killham, Department of Plant & Soil Science, University of
Aberdeen, UK.
• Field release of Pseudomonas fluorescens HK44:. Long term persistence and
field performance of a bioremediation bioluminescent bioreporter
− Prof Gary S Sayler, Center for Environmental Biotechnology, University
of Tennessee, Knoxville, Tennessee, USA
• Metal accumulation by plants
− Professor Andrew Smith, Department of Plant Sciences, University of
Oxford, UK
• Phytoremediation of toxic chemicals in our environment
− Professor Richard B. Meagher, Department of Genetics, University of
Georgia, Athens, Georgia, USA.
• Defusing the environment: engineering transgenic plants to degrade explosives
− Dr Neil Bruce, Institute of Biotechnology, University of Cambridge, UK
5.5 Copies of the abstracts for each of the paper presentations are provided in Appendix A
at the back of this report. A list of the delegates at the workshop is in Appendix B.
PROSPECTS AND CHALLENGES FOR BIOREMEDIATION WITH GMMs
Presentation by Professor Kenneth N Timmis
5.6 The presentation focused on the wider issues associated with the use of
microorganisms in bioremediation. Because the challenges posed by the use of
genetically modified microorganisms (GMMs) in bioremediation were proposed as
being no different from the other biocatalysts carrying out useful activities in polluted
environments, then understanding the role and application of microorganisms in
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contaminated environments would provide the information required to address the use
of GMMs in bioremediation applications.
5.7 It was recognised that there is an enormous knowledge deficit in our understanding of
what is going on in polluted environments, and that this constitutes a major barrier in
applying the information obtained from studies optimising a single bioremediation
process to developing a more generic understanding of how to optimise
bioremediation processes.
5.8 A significant area of knowledge deficit is in understanding how underlying processes
such as the microbe-microbe and microbe-contaminant leads to bioremediation in the
environment. Information is required on the interactions that are important in certain
processes and those which are not. This is an important area that needs to be resolved
if what is happening in bioremediation processes can be understood and subsequently
optimised or improved.
5.9 The lack of knowledge in these areas is partly a consequence of the understanding of
microbiology and microbial processes being based on the study of pure homogenous
cultures, and the view that catabolism occurs as a series of linear reactions taking
place in a single cell. In the environment, microorganisms are likely to inhabit
surfaces and exist in heterogeneous communities such as biofilms.
5.10 In addition to improving our understanding of basic microbial processes, another
major issue that needs to be addressed is bioavailability. It is recognised that all life
takes place in aqueous systems, and that this limits the biodegradation of many
pollutants due to their extremely hydrophobic nature. How microorganisms extract
nutritional benefits from these hydrophobic compounds in the environment is,
however, poorly understood.
5.11 Results were presented from work performed on a polychlorinated biphenyl (PCB)
contaminated site in Germany that had soil concentrations of PCBs from 0-50 g kg-1.
The lack of vegetation on the site and the low carbon content of the soil suggested that
whatever microorganisms were present and active in the site were dependent on the
PCBs present. The community was described as ‘PCB-driven’. Analysis of the
metabolically active component of the microbial community through the generation of
rRNA libraries identified a surprisingly diverse range of microorganisms, including
Burkholderia sp and Sphingomonas sp (both of which are known degraders of
aromatic compounds). However, microorganisms such as Acetobacter sp that have no
previous record as aromatic degrading organisms were also detected at the site.
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5.12 During the study of the interaction between the microorganisms and PCBs in soil
samples taken from the site, the microorganisms were observed to form biofilms
directly on the PCB droplets (when exposed to them in an aqueous environment), and
also to collate clay particles and PCBs as a composite biofilm in which the clay
particle was proposed to act as a nutrient shuttle between the microorganism and the
PCB.
5.13 Although information on the role of biosurfactants and bioemulsifiers was not
presented, these are likely to be involved in interactions between microorganisms and
PCBs, for example in altering the toxicity and solvent properties of the PCB, and in
changing the structure of the interaction faces between the microorganism and this
hydrophobic substrate.
5.14 The basis of the diversity of microorganisms at PCB contaminated sites was proposed
to be due to a number of different possibilities. The primary carbon source at the site
was PCBs. However, PCB contamination consists of a large number of different
congeners, none of which can be degraded by a single microorganism. Therefore the
diversity of microorganisms present may have been due to:
• specialist substrate utilisation with individual microbial taxa using a different
group of congeners as a carbon source;
• competition between different microbial taxa for the same substrates;
• sharing of substrates or metabolites between microorganisms; or
• a diversity of physiological or metabolic optima between different microbial
taxa towards PCB congeners.
5.15 The basis of the observed diversity of microorganisms was addressed by chemostat
culture that consisted of a stable community of four bacteria isolated from a PCB
contaminated site. One of the isolates degraded eighty percent of the 4-
chlorosalicylate substrate, and generated large quantities of 4-chlorocatechol and
proteoanemonine. When this isolate was grown on its own, production of the 4-
chlorocatechol and proteoanemonine ultimately killed the cell. However, in the
community with the other three microorganisms, this isolate survived as two of the
other organisms sequestered and metabolised either 4-chlorocatechol or the
proteoanemonine. This system was proposed as an example of substrate sharing
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between microorganisms and demonstrated that parent substrates, in this case 4-
chlorosalicylate are not metabolised linearly but rather as a network.
5.16 Therefore in order to further understand and utilise the abilities of microorganisms in
the bioremediation, it was concluded that a number of key areas need to be
investigated:
• what is the ‘face’ of the substrate in the environment from the perspective of
the microorganism?. This is likely to be in a less well defined physiochemical
form to that in the laboratory;
• what is the ‘face’ of the microorganism in the environment?. Interactions
between microorganisms and hydrophobic pollutants may involve the
production of biosurfactants. These have been identified as being produced in
a free form or bound to the surface of the outer membrane. Microorganisms
producing bound biosurfactant are likely to have a different ‘face’ and
consequently a different interaction with a pollutant than those that do not
produce a biosurfactant or secrete one in a free (non-membrane bound) form;
• what is the ‘face’ of the microbial community?. The pollutant is likely to have
an effect on the structure and physiology of the community in the
environment; and
• what is the ‘face’ of the catabolic route in the environment?. Although there is
a relatively good level of understanding of the enzymatic reactions involved in
the degradation of compounds, there is relatively little knowledge of the routes
taken by individual metabolites in microbial communities during the
mineralisation of the parent substrate.
Questions
5.17 The questions following this presentation addressed the potential benefits of
bioaugmentation and the survival of DNA in soil.
5.18 With respect to bioaugmentation, this was suggested to only improve the
biodegradation of a particular compound if the biology associated with the
degradation is in some way limiting. Although bioaugmentation was recognised as a
highly promising technique it is currently limited because of the lack of knowledge
regarding the survival of microorganisms in the environment. Sometimes a
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microorganism introduced into a particular environment will survive very well, whilst
in other cases it will not, even if it originated from the same site. Improved
understanding of the survival of microorganisms in the environment is required to
increase the success of bioaugmentation as a bioremediation strategy.
5.19 Regarding the fate of DNA in soil, much of the work reported to date has studied the
persistence of DNA in unnatural substrates. Where more natural systems have been
used, the half-life of DNA is much lower.
REPORTER GENE BASED BIOSENSORS – RISK-BASED MANAGEMENT
SUPPORT FOR REMEDIATION OF CONTAMINATED LAND
Presentation by Professor Ken Killham
5.20 The presentation focused on the use of reporter gene based biosensors as a tool for
providing key information on contaminant bioavailability. Such information is
required to develop an informed risk based management strategy for a contaminated
site, from assessment through to cleanup.
5.21 The main driver for the biosensor technology was proposed to be new legislation,
namely Part IIA of the Environmental Protection Act 1990. Such legislation requires
the development of new (preferably rapid and reliable) methods that enable
contaminant availability within a site to be determined, and allow an assessment to be
made as to whether site contaminants are risk chemicals and how they map into the
source-pathway-receptor model.
5.22 To date, assessments have relied largely on chemical models that have been devised
from data generated from appropriate analyses. Although such techniques are
powerful and well-proven technologies, they can be time consuming and do not
measure hazard directly. Chemical analyses are particularly time consuming if
information on the processes and the likely pollution occurred on a site are not
available at the time of the assessment. The lack of such information means the
chemical analysis of a site cannot be targeted towards particular areas or particular
compounds.
5.23 In addition, because chemical analyses are unable to address toxicity issues directly, it
is more difficult to use them to derive an informed remediation strategy for the
contaminated site.
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5.24 The reporter gene based biosensors addressed in this presentation involved the use of
lux-modified bacteria to monitor the presence of toxic contaminants in soil and
groundwater. Such biosensors are designed to work as complements to chemical
analyses, primarily to determine both the spatial and temporal characteristics of the
pollutant in the site, and assist in the development of a sensible site-specific
remediation strategy. Because the remediation of any contaminated site in a
commercial context is likely to be conducted by ‘non-scientists’, any technique must
be suitable for operation or application by ‘non-scientists’.
5.25 Although lux is only one of the biosensor systems available that have good in-situ
application (others include luc (luciferase) and the unstable green fluorescent protein
(GFP)), the key to all such systems is not just the type of biosensor but the method of
its application. Importantly, the means by which the sensor is placed in contact with
the contaminants present in a site, in particular when there are many compounds with
different physiochemical and toxicological characteristics present, has a real bearing
on site characterisation. For contaminants that are associated closely with solid
substrates it is necessary to ensure that the biosensor is added to the sample so that it
comes into contact with the contaminant in an environmentally relevant way.
5.26 The lux system used in the bacterial biosensor consisted of the CDABE genes and
therefore includes the lux structural genes and those required for the synthesis and
recycling of the enzyme. The gene cassette was placed downstream of a strong
general constitutive promoter, which ensures that the metabolic activity of the
biosensor microorganism can be tracked reliably and quickly. The non-specific status
of the promoter means that the system can be used against metals and organic
pollutants.
5.27 The two main approaches to generating biosensors containing lux, are to mark the
microorganisms chromosomally with the appropriate cassette or to use plasmids to
introduce the genetic material. Chromosomal marking was presented as the preferred
choice of the regulatory authorities, in order to reduce the risk of spreading genes.
The bacteria used as biosensors are all designated as Class I GMOs under the current
regulations, and therefore require a license for use at a particular location. This was
proposed as potentially restricting their use in online operations, at individual
contaminated sites, as a license would be required for each site. It was questioned
whether this requirement should be reviewed for what is now a proven technology.
5.28 The advantage of the biosensor microorganisms is that once they have been produced
they can be freeze-dried and stored on the shelf until required. The system used with
these biosensors is described as ‘lights-off’ as there is a reliable dose-response
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relationship between the biosensor and the contaminant, with a diminution of light
output from the reporter gene biosensor with increased concentration of the toxic
contaminant(s). The biosensors can be used in batch assay to provide information on
acute toxicity by exposing the biosensor to the sample for a brief period.
Alternatively they can be used online to monitor site contamination temporally to
assess the success of a specific remedial approach.
5.29 The reporter gene based biosensors addressed in this presentation have been
commercialised using a range of different microorganisms, selected on the basis of
their intended application. For example, for environmental or ecological assessments
lux modified Pseudomonas fluorescens or Rhizobium leguminosarum biovar trifolii
are likely to be the most relevant, whereas the sewage sludge microorganism
Escherichia coli HB101 may be more applicable for assessments in wastewater
treatment plants.
5.30 As discussed at the start of this presentation, the key application of biosensor
technology is that it is able to provide information on contaminant bioavailability. In
the environment, this is determined by a wide range of biological, physical and
chemical factors. Because of the variety of parameters involved, it is very difficult to
model bioavailability across a contaminated site using chemical analyses. Microbial
biosensors, however, respond to all of the parameters operating at a particular site and
integrate these into a single signal (light output).
5.31 To date, biosensor technology is well developed for addressing environmental risk
assessment issues and has achieved good correlation with other analytical processes.
However, the technology is less well developed to address human risk assessment,
and there is still some way to go before it is able to address all human toxins, although
it was suggested that GM biosensors may eventually be developed for all types of
human risk assessments.
5.32 Although this presentation focused on the use of microbial biosensors, the choice of
biosensor organism is important in order to achieve relevant information to address
such issues as ecosystem toxicity. However, the simplest and therefore the easiest
biosensor to apply, including molecular and cellular systems, may not always be
relevant to address toxicity at an ecosystem or macropopulation level.
5.33 The specific properties of particular organisms, either in terms of their ecological
niche or ecophysiological predisposition, can be exploited to address specific issues of
contaminant toxicity. For example the high sensitivity of Rhizobium sp to organic
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compounds means that this microorganism is an ideal choice as a biosensor to
determine the toxicity of organic contaminants in environmental samples.
5.34 The case study used to demonstrate the application of reporter gene based biosensors
in a contaminated environment was an 8-9 hectare paint manufacture site. The site
was still partly in operation at the time of assessment and was located on top of 30 m
deep sediments, some of which were contaminated with BTEX and chlorinated
pollutants. The depth of the sediments and on-going usage of the site meant that some
form of in-situ remediation strategy was required. The biosensors were used initially
as a primary screen to produce a toxicity map of the site. On completion this map was
found to include an additional toxic hotspot not identified by previous chemical
analyses. This was due presumably to this area not being sampled previously because
of a lack of documentary evidence that contamination had occurred at that location.
5.35 The biosensors were then used to identify the possible constraints to intrinsic
bioremediation. This was performed by exposing environmental samples to a range
of remedial procedures designed specifically to remove any potential bioremediation
bottlenecks. For example, air sparging samples removed the volatile organic
compound fraction, and muffle furnacing removed the non-volatile organic
compounds present. By comparing the biosensor response in the sample before and
after each process the significant constraints to bioremediation were identified.
5.36 In this case air sparging was found to have the greatest effect and this result enabled
the subsequent management of the remediation process to be more focused. This
example was used to demonstrate biosensors as an effective management tool for
contaminated sites.
5.37 The latest development with biosensor technology has been to design online
monitoring of contaminated sites. This allows sites to be monitored permanently to
study the process of a bioremediation campaign, by continual sampling through
groundwater wells of lysimeters. The hardware involved is now down to a more
manageable benchtop size and offers great potential for the remote monitoring of
contaminated sites.
5.38 In conclusion, the new contaminated land legislation that is now in force requires
innovative techniques that are capable of assessing contaminants in relevant
bioavailable forms. Biosensors are able to detect available contaminants and can
therefore assess the potential risk directly as prescribed in Part IIA of the
Environmental Protection Act.
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Questions
5.39 The questions following the presentation focused on what was actually involved with
online sensing and the promoter used to control expression of the lux cassette.
5.40 The online sensing system that has been used to date was described as involving some
automated mechanism that takes samples from across the site, for example a pump
linked to the boreholes on the site to extract samples of groundwater. After the
sample has been retrieved it is mixed with a resuscitated biosensor sample and the
result recorded. Therefore, although the process is described as online there is a delay
of a few minutes between sample extraction and sample analysis.
5.41 Regarding the control of the lux cassette within the GMM, the microorganisms used
are generated by minitransposon based modification of the organism’s chromosome.
This results in the random insertion of the lux genes and the generation of a large
number of isolates that are then screened to identify the most suitable biosensor.
Because the biosensor is only exposed to the contaminant for a short period (no more
than 5 minutes) then suitable modified microorganisms are selected from the initial
screen on the basis of the enzyme activity of the lux expressed system rather than
gene expression. Therefore, the inserted lux cassette is not under the control of a
prespecified promoter, although the screening system ensures that whichever
promoter is involved, it is a strong general promoter, resulting in constitutive
expression.
FIELD RELEASE OF P. fluorescens HK44:. LONG TERM PERSISTENCE
AND FIELD PERFORMANCE OF A BIOREMEDIATION
BIOLUMINESCENT BIOREPORTER
Presentation by Professor Gary S Sayler
5.42 The purpose of the presentation was to demonstrate how a research/laboratory based
study, using genetic engineering technology, would scale up to a large-scale field
application.
5.43 The overall aim of the study was to assess how effective the application of GMMs
would be for monitoring bioremediation activity in the field. The specific issues that
were addressed using GMM containing bioluminescence genes included (1) the
physiologically state of introduced cells, (2) the ability of the introduced population to
promote biodegradation of the target compound, (3) the effectiveness of
bioluminescence for monitoring biodegradation within the environment and (4) the
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identification of the environmental conditions that optimised biodegradation rates.
Improving the rate and effectiveness of biodegradation was proposed as the key goal
of bioremediation strategies.
5.44 The organism used in the study was Pseudomonas fluorescens HK44. The donor and
recipient strains used in the generation of this microorganism were isolated originally
from a heavily contaminated town gas site. The organism that was the fundamental
donor strain for HK44 contained a NAH7-like plasmid which was almost identical to
the archetypal NAH7 plasmid known to confer hydrocarbon degradation capability.
5.45 The NAH plasmid is a fairly large plasmid and transmissible at low rates.
(Transmission was however was not addressed in this study). Within the plasmid the
genes are arranged into an upper and lower pathway, with the nah regulatory gene in
the middle.
5.46 In order to use the GMM as a process-monitoring tool, the lux gene cassette
(comprising of lux CDABE genes) was inserted into the lower pathway in the NAH
plasmid using transposon Tn4431. This of course knocked out the lower pathway,
and required several more mating experiments (with other pseudomonads also
isolated from the contaminated gasworks site) to generate strain HK44, that was able
to degrade naphthalene and salicylate and had a good bioluminescent response to the
target compound.
5.47 The GM Pseudomonas fluorescens HK44 was reported to be able to degrade a variety
of mono-, di- and tricyclic aromatic compounds including naphthalene, although not
all of the compounds were degraded to completion. (The incomplete biodegradation
of compounds by microorganisms was highlighted as an issue that should be
addressed as part of the risk assessment for microorganisms, both the GM and non-
GM for use in field trials). When exposed to naphthalene, strain HK44 exhibited a
linear production of light with increasing naphthalene concentration within the range
of 50 µg/l to 10 mg/l. The GMM also responded to pulsed exposure to naphthalene or
salicylate when immobilised, and also when in dirty samples in the presence of other
compounds.
5.48 Having generated the desired microorganism, the next stage of the research was to
obtain funding from the Department of Energy to use facilities at Oakridge National
Laboratory for the field trial. These facilities consisted of five lysimeters, each of
which extended five metres into the ground. The lysimeters were intended for use in
this study to simulate the subsurface environment and to determine degradation of
PAHs in the vadose (unsaturated) zone of the soil.
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5.49 Following agreement with the Department of Energy, a proposal was submitted to the
USEPA17 for the release of the GMM. This proposal was submitted in the form of a
full-scale review (termed pre-manufacturing notification (PMN) application), which is
the same as that required by the USEPA for new industry-based activities. The reason
for this was that in addition to the research objectives of introducing and maintaining
a GMM in the environment, the programme was also intended to overcome a
perceived regulatory barrier within the USEPA towards GMMs release into the
environment. A final objective was to develop a field site for use by other
investigators18.
5.50 The PMN application was submitted to the USEPA in July 1995 and approval was
granted in March 1996, with work finally starting in October that year. Because the
GMM contained an antibiotic resistance gene and is part of a group of
microorganisms that contains opportunistic plant pathogens (as determined by RNA
classification), the USEPA granted a consent order for the work. This was not a full
and unlimited approval of the use of the microorganism, but instead required the work
to be conducted exactly as specified in the PMN.
5.51 Each of the five lysimeters were packed with a base layer of gravel/aquifer matrix, a
layer of clean soil, then soil (1 m) contaminated with hydrocarbons (except in
lysimeters 3 and 4) and a finally layer of clean soil (1 m). The GMMs were added to
lysimeters 1 to 4, with lysimeter 5 acting as the negative control (containing
hydrocarbons but no GMMs).
5.52 Each of the lysimeters were also modified by the addition of a number of sensors and
manipulation systems that allowed the microorganisms and chemical conditions
within the lysimeter to be studied remotely for the duration of the programme, and
also to alter the conditions if required. During packing of the lysimeters, an aeration
system was inserted into the gravel matrix to allow bioventing; oxygen and soil
moisture sensors19 were added to the soil layers, and fibre optic cables were inserted
into the contaminated layer to allow the direct observation of the microorganisms on
the soil surfaces. The addition of a sub-surface irrigation system within each
lysimeter meant that additional nutrients and more contaminants could be added to the
soil if required.
5.53 The soil used in the experiment was artificially contaminated specifically for the trial.
(Soil was mixed with the hydrocarbons and aged for several months prior to use).
17 United States Environmental Protection Agency.
18 The site is still available for use for other investigations.
19 The availability of oxygen was found to be more important than soil moisture in this study.
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The GMMs used were grown up in a 500 l fermenter and then added to the soil by
spray application. The original application submitted to the USEPA had proposed to
mix the microorganisms and soil outside the lysimeters and then pour it into the
lysimeter. However, due to the height of the lip of the lysimeter above ground level
this was not possible and so the contaminated soil and GMMs were added to the
relevant lysimeters in layers.
5.54 At the time of the loading the wind speed at the trial site was 25 mph, and although
the GMMs were added by spray, none of the modified microorganisms could be
detected outside the lysimeters by either Anderson sampling of the air or from nasal
swabs from the people present.
5.55 With respect to the population dynamics of the GMMs added to the soil, the initial
inoculum density was approximately 1x107 GMMs g-1 soil. The population declined
fairly rapidly and this was found to be due to a loss of the hydrocarbons from the soil.
The addition of further naphthalene, anthracene and phenanthrene to the soil increased
the population of GMMs back up to near inoculation levels. After fourteen months
the GMMs could still be detected in the soil at a population density of 1x102 GMMs
g-1 soil, and could still be recovered and cultivated by standard selective isolation
techniques.
5.56 The heterogeneity and relatively low population density of the GMMs in the soil
meant that the original fibre optic system added to the lysimeters during loading did
not work. The insertion of additional fibre optics20 to the soil after 300 days enabled
light production to be detected directly from the soil. The level of light production
was responsive to the addition of further spikes of naphthalene and was visible to the
naked eye when viewed via boreholes cut into the soil.
5.57 The GMMs added to the soil were found to utilise the hydrocarbons present, and
further degradation occurred following the addition of more naphthalene.
Interestingly, the GMMs remained within the layer of contaminated soil for the two
year duration of the project and were not detected in either the upper or lower layers
of clean (non-contaminated) soil. There was however no hydraulic gradient through
the lysimeter.
5.58 In conclusion, studies of microorganisms inoculated into a field environment typically
results in the decline of population densities. In those cases where the aim is to
sustain desired degradation rates then steps may have to be taken to maintain
20 Vapour phase sensor and downhole photomultiplier.
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introduced populations. A population density of 1 x 108 cells g-1 soil has been
proposed as a minimum requirement to achieve biodegradation of target
contaminants. Levels of 1x105 g-1 or 1x106 g-1 are unlikely to result in a level of
biodegradation significantly greater than that achieved naturally.
5.59 In terms of obtaining approval from the USEPA to conduct such work, it is important
to provide as much information on the GMM as possible. For the strain used in this
study the USEPA required the full 16S RNA sequence homology and absolute
ancestry of all of the transposon fragments. As much microcosm data should also be
provided with the application, including information on the lowest
detection/monitoring level for the microorganism in its intended environment.
5.60 Because of concerns by regulators with the possible use/release of pathogenic
microorganisms, it was recommended that any potentially pathogenic phenotype
should be avoided. For example, the ability to grow at 37 ºC and able to produce
exudates could be considered as phenotypic traits indicative of human and plant
pathogenicity, respectively. It was also recommended that GMMs designed for field
use should not contain mobile elements or antibiotic resistance selective marker
genes.
Questions
5.61 The questions following the presentation addressed the issue of gene transfer from the
GMMs during the period of study, and the advantages of bioaugmentation over in-situ
biostimulation. Gene transfer, was not studied during the trial as the focus of the
investigation was the bioremediation of the PAHs and monitoring of the activity of
the GMMs. However, additional funding is now being sought to analyse some of the
samples taken during the study to address whether gene transfer had occurred.
5.62 Questions were also raised on whether the addition of transposon Tn4431 into strain
HK44 conferred transposon immunity, and the current position of the USEPA towards
GM based field studies. The insertion of Tn4431 was thought not to confer
transposon immunity, and it was suggested that following the work reported in this
presentation it would now be easier to get approval from the USEPA for this type of
work, and that the USEPA were very keen for further studies to take place, even at the
level of a full field release.
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METAL ACCUMULATION BY PLANTS
Presentation by Professor Andrew Smith
5.63 The presentation was intended as an introduction to the application of plants in
bioremediation. Compared to microbially based strategies, the use of plants is still
developing and was described as a relatively young field of expertise. However,
many plants have been identified that demonstrate a high degree of tolerance to
potentially toxic metals, and some of these plants are able to accumulate metals to
high concentrations. Such plants, could in principle, be used to remove metals from a
contaminated soil.
5.64 Although metal-tolerant and metal-accumulating plants have been identified, further
information is now required on the natural basis of plant adaptations to metals in their
particular environments. It was proposed that only through a detailed and mechanistic
understanding of how plants deal with and survive exposure to toxic compounds, can
a rational understanding of the potential for genetic engineering technologies be
developed. A report by the US Department of Energy in 1994 identified a number of
areas of further research requirements, some of which are still relevant today:
• greater understanding of the uptake, transport and accumulation of metal ions
by plants;
• greater use of genetic screens to identify the variability within
hyperaccumulation traits. The purpose of this is to identify whether natural
breeding could improve hyperaccumulation capabilities of naturally occurring
plant species;
• better understanding of the interactions between plant root systems and the
immediate rhizosphere; and
• greater number of trials of potential phytoremediating plants under relevant
field conditions.
5.65 Naturally occurring metal-tolerant and metal-accumulating plants are thought to have
developed in response to the presence of particular metals in the environment. These
have been generated from the erosion and weathering of natural deposits as well as
through pollution from anthropogenic activities. Examples of such naturally
occurring plants include the lead violet (Viola lutea) which is able to tolerate quite
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high concentrations of lead, zinc and cadmium, and can be found in areas with long
histories of mining for metal ores. The plant Alyssum bertolonii (a member of the
Brassica family) was identified growing on serpentine ultramafic soils in Tuscany,
and has been found to accumulate nickel at concentrations of three to four percent of
plant dry biomass.
5.66 The highest concentrations of metal hyperaccumulation by plants have been reported
for zinc, nickel, manganese and more recently for cadmium, with concentrations of
these elements capable of exceeding one percent of plant dry biomass.
Hyperaccumulation of copper, cobalt and lead has also been reported but at lower
concentrations.
5.67 Although a number of plants have been identified that are capable of growing in the
presence of relatively high concentrations of metals, only a minority of these plants
are able to hyperaccumulate the metal(s). The majority of the plants identified are
able to grow in the presence of the metal because of their ability to exclude the metal
from their cells. Such plants are described as metal excluders and include the Viola
lutea described earlier. These plants are able to exclude the metal(s) up to a relatively
high concentration, above which acute toxicity occurs and the plant is unable to
survive. Work with arsenate tolerance in grasses found that genetic variations in the
plant’s phosphate transport system21 conferred differential resistance to arsenate.
5.68 In contrast to metal excluders, metal hyperaccumulating plants are very effective at
sequestering and accumulating metals, even when the concentrations of that metal(s)
in the environment are relatively low (although saturation of the metal is reached
eventually). The general consensus within the scientific community is that metal
exclusion and metal hyperaccumulation are distinct strategies for growth in
environments with high metal concentrations, rather than opposite ends of a
continuum.
5.69 With respect to the application of metal hyperaccumulation to the treatment of metal
contaminated sites, there are a number of traits expressed by metal hyperaccumulators
that may be of interest. These include:
• effective mobilisation and transport of the metal from the soil into the root
system. The mechanism(s) involved are however poorly understood due
21 Arsenate is thought to enter plant cells through the phosphate transport system because of similarities between
the ions.
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partly to a lack of understanding of the soil system and factors determining the
bioavailability of different metals;
• very effective root-to-shoot translocation system, since most of the metal
accumulated by these plants is concentrated in above-ground structures (stem
and leaves). This has potentially advantageous implications for the removal of
the metal from contaminated sites, through harvesting/cropping of the
hyperaccumulating plants (and subsequent retrieval of the metal from the plant
matter22). However, the mechanistic basis of this translocation is poorly
understood; in particular, it is not yet known why certain metal ions can be
transported from the roots to the shoot so effectively in different species; and
• the mechanism by which the plants chelate the toxic metal within the plant
cells. There is still inadequate information on the cellular basis of metal
tolerance in plants, although it is accepted that this must rely on complexation
or chelation of the metal ion within the cell. The most toxic form of most
metals is likely to be the free or hydrated metal cation, and the key to the
detoxification of this compound within the plant is the chelation or binding of
the ion to a particular ligand. Further identification of the ligands involved
and an improved understanding of their production will provide better
knowledge of metal tolerance at a cellular level. The metals
hyperaccumulated by the plant are thought to be stored predominantly in the
central cell vacuole (which can occupy ninety percent of cell volume) and also
in the apoplastic phase of the cell wall outside the cell membrane.
5.70 Many of the metals hyperaccumulated by plants are essential micronutrients, required
by the plant for effective growth, albeit at much lower concentrations.
Hyperaccumulation may therefore be the overexpression of a trait possessed by all
plants for the acquisition of essential nutrients.
5.71 In addition to the hyperaccumulation applications discussed so far, there are a number
of other phytoremediation strategies that are envisaged. These include rhizofiltration
which has applications particularly in the treatment of aqueous environments, and
may be more appropriate with metal-tolerant plants rather than metal-accumulating
plants, due to their greater biodiversity and growth rate. Metal-tolerant plants would
22 Where the harvested material is incinerated to reduce the quantity of plant matter and extract the metal, the
ash produced may contain up to 30 percent by mass of the metal. Where the plant material is burnt in a waste to
energy system, the entire process becomes a highly sustainable and environmentally friendly strategy for the
treatment of contaminated land.
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potentially be employed to adsorb the metal(s) onto their roots, either through the
sediment or onto the cells directly.
5.72 Other strategies include phytovolatilisation and plant-assisted degradation of organic
pollutants. Metal tolerant plants also have potential application in land reclamation
and landscaping programmes on contaminated sites. The mining sector for example
is now required to direct significant proportions of their budget to site rehabilitation
and revegetation.
5.73 To date a number of field trials have been conducted using plants to remediate
contaminated sites. None of these trials to date, however, have involved GM plants.
5.74 The ideal metal-hyperaccumulating plants for field application are those with a high
growth rate/biomass production, good accumulation of the target compound within
the biomass and a high planting density. Whilst naturally occurring metal
hyperaccumulators have the advantage of being able to accumulate high
concentrations of particular metals in their biomass, the majority of them have low
growth rates. Poor growth rates are a consequence of these plants being adapted to
growing on relatively poor and infertile soils, typically with low levels of organic
matter and particular nutrients as well as the presence of metal contaminant(s).
5.75 The exceptions to the slow-growing metal hyperaccumulators are some members of
the Brassica family, which whilst not true hyperaccumulators, exhibit good growth
rates and reasonable metal accumulation. These plants were proposed as representing
the best targets for genetic modification for metal phytoremediation.
5.76 The only example of a metal-hyperaccumulating plant in the UK is the slow-growing
and low biomass forming alpine pennycress (Thlaspi caerulescens), that is able to
hyperaccumulate between five or six different metals. This species is unusual
amongst metal hyperaccumulators, which usually are only able to target one or two
specific metals. For example, nickel hyperaccumulators usually demonstrate some
capacity to tolerate and accumulate cobalt, but are very sensitive to copper. Multiple
metal accumulation, such as that exemplified by Thlaspi caerulescens, is likely to be
an important trait in plants developed for phytoremediation applications.
5.77 Most metal hyperaccumulators are tropical species, and in the case of nickel
hyperaccumulators the greatest diversity are present on the island of New Caledonia.
Most of these New Caledonian plants are trees, one of which, Sebertia acuminata (the
blue sap tree) has in its sap the highest nickel content of any biological fluid, with
nickel concentrations of up to 26 percent by dry weight of nickel. The nickel in this
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tree is complexed with carboxylic acids, mainly citrate, which gives the sap its
distinctive blue/green colour.
5.78 Work with other nickel hyperaccumulators has identified the importance of the free
amino acid histidine in chelating the nickel within the plant immediately after uptake
from the soil23. With the hyperaccumulating members of the genus Alyssum a linear
relationship has been identified between the level of nickel exposure and production
of histidine by the plant. This system is currently being applied on a field scale using
Alyssum sp for the phytoextraction and subsequent recovery of cobalt from
contaminated soil. The increased production of histidine by this plant has been found
to be specific for nickel and cobalt, and was not observed in the case of
hyperaccumulation of other metals by different species (e.g zinc hyperaccumulation
by Thlaspi caerulescens).
5.79 A further advantage of using hyperaccumulating plants to remove metals from soil,
compared to non-accumulating plants is that the hyperaccumulating plants are able to
mobilise and sequester the metal at a localised level, so avoiding subsequent
environmental contamination issues. This was demonstrated during a field trial on a
former lead battery production site in the USA, where the metal chelator EDTA24 was
added to the site at the start of the trial to improve the bioavailability of lead in the
soil. The increased availability of the lead meant that the plants could sequester and
take up this metal. Although this resulted in the death of the plants, the plants were
harvested within several days of EDTA application, so that the accumulated lead was
removed from the site before it could leach back out of the decaying plants.
However, the increased availability and therefore the mobility of the lead meant that
there was increased risk of leaching of the metal to runoff or groundwater. Use of
hyperaccumulator plants able to accumulate lead without EDTA applications would
be a more desirable strategy for soil clean-up by phytoremediation.
5.80 The final issues that have to be addressed regarding the use of metal
hyperaccumulating plants in bioremediation are the potential risks of grazing animals
consuming plants containing high concentrations of metals, and the potential for the
metal subsequently to enter the foodchain. Although this could occur should such
plants be grown in the field, previous studies have shown that high metal
concentrations within the plant actually deter grazing herbivores. Indeed, it seems
likely that metal hyperaccumulation has evolved naturally as a mechanism by which
the plants can protect themselves from grazing animals.
23 Immediate chelation is important to prevent any toxic effects of the metal to the plant being realised.
24 Ethylenediaminetetraacetic acid.
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Questions
5.81 The issue of foodchain contamination was addressed during the questions that
followed the presentation. The question was raised as to whether it was less
hazardous to the environment and human health to have a bare untreated but
contaminated site, or to introduce metal hyperaccumulating plants to that site with the
risk of some metal entering the food chain through herbivores grazing on the plants.
This issue was addressed further during the general discussion at the end of the
workshop.
5.82 Other questions addressed the number of genes likely to be involved in
hyperaccumulation; the phytoremediation of aluminium, and the potential for mutant
screens to help identify genes involved in metal hyperaccumulation using wellcharacterised
genetic models such as Arabidopsis thaliana. Although work is known
to be underway to address the latter point using mutant screens, no details have yet
been published, probably because mutations are more likely to cause an impairment
of metal tolerance rather than produce a hyperaccumulator phenotype.
5.83 Regarding the number of genes involved, this was currently unknown and would pose
a challenge to genetic modification technology. It was likely that the system used by
plants to chelate metals and to translocate them through the plant was under the
control of many different genes.
5.84 With respect to aluminium, this metal has a distinct chemistry and is characteristic of
acid soil. Work is currently being conducted in Mexico using GM plants designed to
overexpress the citrate synthase gene; these plants secrete elevated amounts of citrate
from their roots, which serves to chelate and detoxify the soil aluminium.
PHYTOREMEDIATION OF TOXIC CHEMICALS IN OUR ENVIRONMENT
Presentation by Professor Richard B Meagher
5.85 The basis of the presentation was the application of plants for the bioremediation of
pollutants in the environment. The presentation focused on the use of GM plants for
the treatment of heavy metal contaminated environments.
5.86 Compared to inorganic pollutants, strategies to degrade organic contaminants are able
to mineralise or reduce the organic pollutant into smaller constituents. Naturally
occurring plants have already been used at a number of sites to degrade (or stimulate
the degradation of) organics, and even where the plant does not degrade the
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compound directly, the plant may have an indirect role in stimulating the rhizosphere
microorganisms to break down the contaminants.
5.87 Results from trials at two contaminated sites using naturally occurring plants were
presented. At a coal gas manufacturing site, trials with 40 plant species found that
two to three plant species showed good activity against the principal contaminant
benzopyrene (present at concentrations of 50 ppm). The trials were conducted by
growing the plants in pots containing soil from the site. Degradation of the
benzopyrene to below detectable limits was found to coincide with the plant’s roots
filling the pot. Trials using soil from a floor tile manufacturing site that contained 0.4
percent phthalates and 25 percent casein identified four plant species that were able to
grow successfully and reduce the phthalate content in the soil significantly.
5.88 The major limitation with both of these trials was that the identification and screening
for suitable plants was an extremely time consuming and labour intensive activity,
taking four months to select the final plants. Although the work with naturally
occurring plants has shown promising results, the use of plants genetically modified
with genes from other organisms may offer a potential advance in the use of
phytoremediation at contaminated sites.
5.89 Metal contaminants cannot be mineralised and are effectively immutable by any
available remediation strategy. According to the USEPA, mercury is now the number
one metal pollutant, with large numbers of contaminated sites across the USA. In the
environment, mercury is present in the environment in one of three states:
• elemental mercury (Hg(0)) - this is the least toxic of the three forms of
mercury. It is volatile and accumulates in the atmosphere where it has a
residence time of several years and is not subject to wet deposition;
• ionic mercury (Hg(II)) - this has moderate toxicity and has a transit time in the
atmosphere of several weeks. Hg(II) is subject to wet deposition; and
• methylmercury - this is the most toxic of the three forms, and is approximately
1000 times more toxic than Hg(0). Methylmercury is produced from ionic
mercury in the environment by microbial activity, particularly in wetland sites.
The compound is also bioaccumulated through the foodchain, with a 50-fold
increase in toxicity at each trophic level. As with ionic mercury,
methylmercury is also subject to wet deposition from the atmosphere where it
has a transit time of only a few weeks.
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5.90 The potential application of bioremediation for the treatment of mercury contaminated
sites has developed from the identification the mer operon in bacteria. Two of the
genes merB and merA are known to be able to confer on bacteria the ability to convert
methylmercury to ionic mercury (merB) and Hg(II) to elemental mercury (merA).
5.91 The ability of bacteria to detoxify methylmercury and ionic mercury was proposed to
have developed as a consequence of the prokaryotic nature of bacteria and the fact
that their electron transport chain, which is particularly sensitive to mercury, is
located in the outer membrane of these organisms. In order to avoid the toxic effects
of the mercury, bacteria are proposed to use a mer operon encoded pump to sequester
and pump Hg(II) into the bacterial cell where it is converted into the less toxic Hg(0),
which then diffuses passively out of the cell.
5.92 By extending this biotechnical hypothesis to plants, it was proposed that because the
electron transport chains in plants are located within organelles, protected by a layer
of cytoplasm, then plants are likely to be less immediately sensitive to the toxic
effects of mercury. Plants may therefore be more suitable to bioremediate mercury,
using the plant’s systems to sequester and extract the mercury from the environment
and recombinant bacterial merA and merB genes to detoxify the mercury within the
plant to Hg(0) which would then be volatilised from the plant into the atmosphere.
5.93 Initial work with GM plants has focused on the development of plants modified with
the bacterial merA gene. Due to the high guanine/cytosine (GC) content of this gene
and the strange codon usage, the bacterial gene had to be reconstructed before it could
be expressed in the plant. However, GM (merA+) tobacco were found to be able to
grow in soil containing Hg(II) at 500 ppm. Growth of the GM tobacco plants was
slow initially, although this was attributed to the small size of the plants at this stage.
Once the plants initiate operation of photosystem II, their growth improved
significantly and levels of Hg(II) were reduced. Similar findings were obtained using
merA+ yellow poplar.
5.94 Although the GM plants were shown to be successful against high concentrations of
ionic mercury (the non-GM control plants did not survive), the majority of mercury
contamination in the environment is low level. For example, light bulb manufacturing
plants have mercury in their waste streams at levels of 1 ppm.
5.95 Tobacco plants modified to express merA and developed to grow hydroponically were
found to be able to remove 70 percent of the Hg(II) from a 1 ppm solution within one
week. Both the merA+ plants and the non-GM control plants reduced the mercury
content in the solution to below detectable levels within several hours. This was a
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consequence of the strong sorption of mercury to the root systems. Although they
were able to sequester the mercury, the control plants did not survive the experiment.
5.96 Although these experiments demonstrated the potential for GM plants to convert ionic
mercury to the less toxic elemental mercury, the environmental contamination of
methylmercury is a more significant pollution issue. It was proposed that by
converting methylmercury to ionic mercury, this would prevent the more toxic
methylmercury from entering the foodchain.
5.97 In laboratory mesocosm studies, tobacco genetically modified to express merB were
able to survive in the presence methylmercury at concentrations approximately 20
times greater than those detected in the environment, and much higher than the 0.1
µM concentration that was sufficient to kill the non-GM control plants. In the
absence of methylmercury, the GM plants grew almost as fast as the non-GM plants.
5.98 Where the tobacco plants were modified to express both merA and merB they were
able to grow at even higher concentrations of methylmercury than plants expressing
just merB. The merA+merB+ plants were also observed to be healthier than the merB+
plants in the presence of the same concentrations of methylmercury. Alteration of the
level of expression of the merA gene in the merA+merB+ plants was found to have no
effect on the detoxification of methylmercury by the modified plant. However,
increasing expression of merB did increase the detoxification, although not in a linear
fashion. A ten-fold increase in merB activity only produced a two-fold increase in the
merA/merB coupled reaction.
5.99 In the original experiments, the merB gene was expressed in the plant cytoplasm.
When the plants were modified to express the gene either in the endoplasmic
reticulum (ER) or in the cell wall, no changes were observed in the coupled reaction.
However, the level of expression of merB in either the ER or cell wall was 20 to 100
times lower than in the cytoplasm, indicating a much greater specific activity of the
merB enzyme following expression of the gene in the ER or cell wall. Studies to
investigate the broader implications of these findings are now underway.
5.100 With respect to applying all experimental studies using plants to treat contaminated
sites, it is essential that the range of plant species studied to date is extended to
include plants other than tobacco and Arabidopsis. Because of the high
bioaccumulation of methylmercury within wetland environments, the goal of any
plant selection needs to be to identify plants that can thrive in a wetland habitat.
Therefore work is now underway to trial GM rice, cottonwood, willow, sweetgum and
yellow poplar.
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5.101 It was concluded that by using these types of plants it should be possible to harness
GM technology to target ionic and methylmercury in the environment and to block
the formation of methylmercury and its subsequent bioaccumulation within the
foodchain. Although this conclusion had yet to be supported by field data, the results
from the laboratory and mesocosm work certainly suggest that it should be possible.
Because of the severity of the pollution of many sites with extremely recalcitrant
compounds such as mercury, the use of non-GM plants is unlikely to be sufficient to
deal with the contamination. Therefore, for such compounds, GM plants pose the
most realistic in situ remediation system.
5.102 In addition to the work on the phytovolatilisation of mercury, a number of other
applications of GM plants for phytoremediation were also presented, including the:
• use of plants for phytomining - the merA gene for example has good activity
towards gold;
• development of above ground accumulators for mercury - such plants would
hyperaccumulate mercury rather than volatilise it off during transpiration. The
merA+ plants have little mercury in their above ground biomass as they
volatilise it. However, by grafting a wild type plant onto a GM merA+ root
system, reasonable hyperaccumulation was achieved in the above ground parts
of the plant. It was proposed that the level of accumulation could be improved
further by generating a bigger mercury sink in the above ground part of the
plant through for example, the addition of a phytochelatin system.
The use of grafted plants was reported to be unsuitable for field use. Using
non-grafted plants would require root specific promoters for example; and
• development of GM plants to target arsenic and cadmium - initial work in this
area has focused on the generation of GM plants expressing the gammaglutamyl
cysteine synthetase system (thought to be the rate limiting step of the
three part phytochelatin system). The GM plants were slightly retarded
compared to the wild type in the absence of arsenite, but were able to survive
exposure to arsenite, unlike the wild type.
5.103 It was concluded that different strategies may be expected for different metal
pollutants, but that based on the work conducted to date within the field, then even six
gene systems such as the one involved in arsenic detoxification could now be
addressed.
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Questions
5.104 The questions raised after the presentation addressed the importance of indigenous
rhizosphere microorganisms in the sequestration and detoxification of the mercury
and arsenic, and also the identification of plant homologues to the merA and merB
genes. These have been identified in plants using genomic screenings, but are not
sequence homologues.
5.105 The studies presented were all based on non-sterile soils. Rhizosphere
microorganisms were present in each experiment and thus may have been expected to
have played a role in the phytoremediation process at some level. Non-sterile soil
was used in the experiments in order to replicate field conditions.
DEFUSING THE ENVIRONMENT: ENGINEERING TRANSGENIC PLANTS
TO DEGRADE EXPLOSIVES
Presentation by Dr Neil Bruce
5.106 The presentation addressed the work that has been conducted to date on the use of
enzymes, microorganisms and plants to degrade explosives. Explosives were
described as true xenobiotic compounds and in some cases have been introduced to
the biosphere for only tens of years. This is an insufficient period of time for
microorganisms to evolve metabolic pathways to utilise and consequently degrade
these compounds.
5.107 To date microorganisms have been isolated that are able to grow on all of the major
classes of explosives, usually using the explosive as a nitrogen source. However, due
to their high recalcitrance and toxicity, explosives are proposed as one group of
pollutants that may require genetic engineering techniques to remediate them.
5.108 The most significant sources of explosives contamination are the ordnance
manufacturing and disposal sites. An ordnance manufacturing plant for example
generates 500,000 gallons of explosive contaminated waste water a day which is
discharged to on-site reed beds. The relatively short shelf-life of ordnance means that
levels of disposal are potentially high. Some contaminated sites are so polluted with
explosives that they are bare of vegetation.
5.109 The initial interest in identifying microorganisms capable of growing on explosives
was the development of biosensors for these compounds. By isolating microbial
degraders, the relevant enzymes can be identified and used as the recognition
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components in the sensors. These enzymes can however also be used as the basis for
bioremediation strategies.
5.110 Microorganisms such as Rhodococcus sp have been isolated that grow on RDX
(Royal Demolition Explosive) as a sole nitrogen source and an Enterobacter cloacae
has been isolated that can use PETN (pentaerythritol tetranitrate) and TNT
(trinitrotoluene) as a sole source of nitrogen. The structures of the enzymes involved
in these pathways have been determined and this information can potentially be used
to engineer the enzyme and improve their activity against the explosive.
5.111 Enzymes similar to those isolated from Rhodococcus sp and Enterobacter cloacae are
present in a wide range of microorganisms. Due to the short time explosive
compounds have been present in the biosphere, these enzymes could not have evolved
in response to explosives and are thought to have been developed as part of the
organism’s antioxidant defence mechanism. All of the enzymes show activity against
nitrate ester explosives such as GTN (glycerol trinitrate), but only those very closely
associated to PETN reductase will degrade TNT.
5.112 The potential for direct evolution using gene shuffling to improve activity against
explosives was also investigated. This technique involves cutting, mixing and
reannealing genes from different organisms to generate chimaeric mutants. Gene
shuffling is a very powerful technique, but is limited by the screening system
available to select the mutants generated. Gene shuffling was described as a means of
effectively speeding up the evolutionary process. Once potentially useful genes have
been identified these can be reinserted into organisms for use in the environment.
5.113 As has been discussed in the previous two presentations, plants and phytoremediation
offer a number of advantages (scientific and ‘presentational’) over the use of bacteria
to degrade pollutants. Plants generate a large quantity of biomass, have large root
systems, are cheap to grow and have a relatively good public perception. It was
proposed that the public were more likely to accept the release of plants for
bioremediation than microorganisms. It was also suggested that in the short term at
least there was a higher chance of releasing GM plants for bioremediation than GM
microorganisms. Rhizosphere interactions mean that plants grown on a contaminated
site have the added advantage of being able to stimulate the natural microflora to
assist in the bioremediation process.
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5.114 However, although plants such as pondweed (Lemna sp.) have been used to degrade
organic pollutants25, the phytoremediation of organics is limited by the relatively low
metabolic diversity of plants towards these compounds, certainly when compared to
microorganisms. Therefore, the genetic modification of plants with bacterial genes
may offer the best solution to the phytoremediation of organic pollutants.
5.115 The final part of the presentation focused on the development and potential of plants
modified with the PETN reductase system or the nitro-reductase system. To date only
GM tobacco26 has been produced, although trials with more robust plants such as
poplar are planned.
5.116 Studies have shown that modification of tobacco with PETN reductase resulted in
high levels of expression of the enzyme. Germination of the GM tobacco was
unaffected by exposure to 1 mM nitroglycerine. The GM plant grew as well in the
presence of nitroglycerine as the wild type plant did in the absence of this compound.
The non-GM wild type control plant did not germinate in the presence of 1 mM
nitroglycerine.
5.117 Tobacco modified with the nitro-reductase system grew well in the presence of TNT
at concentrations ranging from 0.1 mM to saturation. At sites contaminated with high
levels of explosives, compounds such as TNT can be present at saturation levels,
highlighting the application of this technology to bioremediating explosives waste in
the environment.
5.118 The results of studies with plants modified with the nitro-reductase system were
reported to be surprising, as they were expected to be less resistant to TNT than those
modified with the PETN reductase. This is because the degradation pathway of TNT
by nitro-reductase is reported to generate toxic metabolites. However, on exposure to
0.25 mM TNT both the wild type and PETN reductase modified plants died, whilst
the transgenic nitro-reductase plants remained healthy.
5.119 The results with tobacco plants were used as an indication of the potential for GMbased
phytoremediation strategies to target sites contaminated with explosives. To
date only incineration and landfill have been available for reducing the contamination
levels in these sites. (Techniques such as biopiling are probably too costly). In the
USA, plants used to phytoremediate explosives waste are disposed of by composting.
25 Trials in the USA have used pondweed to remove TNT from contaminated aqueous environments.
26 Tobacco is used as a model system as it is easy to transform and generates large quantities of biomass.
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Unlike metal phytoremediation, plants are able to degrade toxic organics, although
some toxic metabolites may remain in the plant.
5.120 Further work is now underway to develop, through gene stacking, plants capable of
degrading several classes of explosives. Sites contaminated with explosives typically
contain more than one class of these toxic compounds.
DISCUSSION ON THE USE OF GMOs FOR THE BIOREMEDIATION OF
POLLUTANTS
Chaired by Professor Chris Leaver
5.121 The final session of the workshop was intended as a general discussion forum on the
use of GMOs in bioremediation. The issues discussed in this session can be
summarised as:
• the development of bioremediation applications incorporating both plant and
microbial based strategies;
• the potential for plants employed in phytoremediation strategies to have
secondary effects on the environment and/or human health;
• the perception of the risk of bioremediation strategies by the general public;
• the advantages of GMOs over non-GMOs for bioremediation strategies; and
• the information that is required for the further development of bioremediation
applications.
Incorporation of plant and microbial based strategies
5.122 In the environment, terrestrial plants do not exist in the absence of a rhizosphere
microbial community. However, although the importance of the rhizosphere is
accepted, there is only very limited understanding within the scientific community of
the interactions between plants and microorganisms, even at the most basic level.
Because of the range of different processes and reactions involved, the interactions
between plants and microorganisms in the rhizosphere are likely to be complex. It
was suggested that experiments were needed that would identify these interactions
and assess how important they were in phytoremediation.
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5.123 The introduction of plants to the environment will increase the metabolic activity of
the rhizosphere microbial community by the exudation of carbon into the soil. By
introducing the appropriate plant into an environment, the rhizosphere may effectively
be ‘fine tuned’ so that the rhizosphere community operates to its greatest potential in
terms of remedial activity. A potential application of genetic modification technology
is to develop plants that are able to introduce specific carbon substrates, for example,
benzoate analogues into the environment. This will have the effect of increasing the
relative abundance and activities of those microbial populations that are effective at
degrading the target pollutant(s). Because naturally occurring microorganisms such
as those in the rhizosphere are more suited to survival in their particular environment,
then the combination of GM ‘supporter’ plants and non-GM microorganisms may be
a successful approach for phytoremediation.
5.124 For example, field trials are now underway in Mexico involving plants genetically
modified to secrete citrate from their roots as a mechanism to block uptake of
aluminium by the plants, and ensure their survival in acidic soils (which tend to be
high in aluminium). A naturally occurring aluminium tolerant plant has also been
identified with a mutated hydrogen ion pump that causes an increase in the pH of the
rhizosphere by 0.7 units. This is likely to have a significant impact on the
biodiversity of the rhizosphere community and demonstrates the potential effect
different plants could have on the soil microorganisms.
Secondary effects of organisms used in bioremediation
5.125 The organisms employed in bioremediation strategies (plants and microorganisms)
are constructed to do a specific job or have a primary effect, for example degrade or
accumulate the pollutant, or support the activities of other organisms present. Many
of the organisms designed for use in the field are, however, developed and assessed as
a monoculture in a controlled environment. In the field these GM organisms are
likely to exist as part of a heterogeneous community, so their activities should never
be thought of as one of a mono-culture, but as part of a complex and dynamic
community.
5.126 Therefore, there is the potential for the introduced organisms to have secondary
effects that were not identified during their development in a monoculture. For
example, a microorganism designed to degrade a particular pollutant may also have
activity in the environment against a second compound that is required as a growth
substrate by another organism that has a pivotal role in its habitat.
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5.127 This was recognised as a possibility by the panel of speakers, particularly because
many pollutants have not been present in the biosphere for sufficient time for
organisms to develop effective metabolic pathways to degrade them. Many catabolic
pathways therefore have kinetic bottlenecks and involve the misrouting of metabolites
to unproductive end points. The development of genetically modified organisms has
been proposed as possibly amplifying the potential for such misrouting to occur.
However, it was recognised that this should be addressed when designing the GMO.
It was important to determine what was going to happen at the target environment (or
type of target environment) before any large scale release, and not to rely solely on
the results from laboratory monoculture experiments.
5.128 With plants, the production of pollen and fruit, herbivory of the plants by other
organisms and the accumulation of toxic compounds, may all have secondary effects
on the surrounding environment and/or human health. The risks posed by the transfer
and dissemination of pollen, seed and fruit may be avoided by using, for example,
male sterile plants or plants that reproduce by vegetative means.
Perception of risk by the general public
5.129 With respect to the perception of risk by the general public of using GMOs for the
bioremediation of pollutants, there was considerable discussion on the commitment of
various groups (general public, industry, regulators, NGOs27) to remediate
contaminated sites, and at a more basic level, in answering the question “can we
afford to do nothing?” (with respect to chemical pollution). Although the use of
GMOs may have some adverse effects on the environment, the risk of these possible
effects must be balanced against the risk of leaving the contaminants untreated or
remediating the site using other technologies. It is also important to address whether
significant resources should be employed to remove every last trace of a contaminant
from a site, or to use less effort to remove the bulk of contaminants present, and then
to use the resources saved to remediate tens of other contaminated sites.
5.130 In the UK, ‘dig and dump’ treatment of contaminated material is currently the most
cost effective strategy and is encouraged by landfill tax exemptions. Therefore, the
remediation of contaminated sites may be expected to be treated in this way until the
economics change or there is pressure from NGOs or the general public, for example,
to use alternative and more sustainable technologies. Pressure to use different
technologies is likely to arise from a change in the perceived costs/benefits of the
27 Non-government organisations.
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respective methodologies and their risks to the environment and human health, as well
as concerns about sustainability.
5.131 In the USA, the driving force behind the choice of remediation strategy is economic
rather than regulatory. In particular, remediation technologies are selected on the
basis that they offer the opportunity to clean-up a site for the lowest expense.
Bioremediation has the potential to provide the cheapest option for the remediation of
contaminated sites, and on a site specific basis may offer potential benefits and/or less
risk to the environment than other technologies. However, since bioremediation is a
biologically based process and is rate limited, often being slower than alternative
approaches such as dig and dump. If bioremediation is to be a feasible option, it will
be essential to improve long term management and planning of land use. Better long
term planning should provide more time for the biological process to become a
feasible option.
5.132 At a fundamental level, irrespective of the specific purpose of the plants (GM or non-
GM) used in phytoremediation applications, the action of covering what is often a
poorly vegetated or barren contaminated site with healthy vegetation should be
expected to receive a positive public perception. If the site is intended for housing,
then it was proposed as possibly beneficial to ‘green’ the site for a period of time
before developing the housing. In the long term it may then be easier to sell the
houses if there is a less immediate link between the site as a contaminated area and
the site as a residential area. This green amenity type of phytoremediation application
is most suited to naturally occurring plants, or possibly GM plants designed to
optimise rhizosphere activity.
5.133 Regarding the field release of the GM Pseudomonas fluorescens HK44 at Oakridge
National Laboratory described at this workshop, the release encountered virtually no
adverse public reaction. The general public within the vicinity of the site were kept
informed of what the trial would entail and there was extensive communication with
the press. However, it was noted that because Oakridge National Laboratory also
make material for nuclear weapons, the local population around the site may be
relatively ambivalent to the field trial of some GMMs.
5.134 A similar situation however was reported at a contaminated site in Long Island Sound,
New York. Here there was a highly vociferous NGO trying to get the site cleaned up.
Although the regulators favoured excavation and re-burial of the contaminated
material, residents in the vicinity of the site wanted the option of using of GM plants
to bioremediate the site rather than a re-burial strategy.
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5.135 Approval for field trials of tobacco genetically modified with merA was reported to
have been received from the USEPA with relatively little resistance (six week
approval time), although the USEPA required that the site be well fenced and away
from the general public. (The GM tobacco trial was approximately 100 miles away
from the nearest commercial tobacco plantations). As with the Long Island Sound
site, the owners of the contaminated material had approached the phytoremediators as
they (the site owners) felt it was morally unacceptable to dig and dump the
contaminated sediment (although they had approval from the regulators to do so).
5.136 It was concluded that improving the public perception of all applications of GM
technology should increase the acceptance and demand for bioremediation based
strategies for the clean-up of contaminated land. This would be particularly the case
when the public was made aware that for many contaminated sites GM technology
may be the only feasible approach other that dig dump, to clean-up a contaminated
sites. GMOs have potential application for use in end of pipe treatment of
contaminated waste and even for the production of added value products such as
specific polymers from low level waste materials. Other GMO applications that may
be envisaged may include the use of plants and/or microorganisms for carbon
sequestration to combat global warming, and for biomining to recover or extract
metals such as cobalt from mining waste or other contaminated material.
Specific advantages of GMOs for bioremediation
5.137 The specific advantages and applications of GMOs in bioremediation have been
addressed in the report that accompanies this workshop report. However, it was
proposed at the workshop that recalcitrant and newly synthesised compounds
represented the most likely targets for direct bioremediation by GMOs.
5.138 In addition to the use of GMOs to directly sequester or degrade the target pollutant, it
may be more effective to manipulate the organisms already present at the
contaminated site to target the pollutant(s). These organisms are more predisposed to
bioremediating the contaminants for a wide variety of reasons compared to any
organism that is introduced into the environment (GM or non-GM). It is recognised
that the necessary catabolic genes to degrade the pollutant(s) are likely to be present
in the contaminated site. Therefore it should be possible, by manipulating the
environment, to reshuffle those genes within the biota and generate whole new
catabolic pathways so that the natural flora and fauna are able to bioremediate the
pollutant(s). Similarly, it should be possible to preferentially stimulate beneficial
activities and populations. However, it was recognised that where a large amount of
gene space is required to be manipulated then the natural biota may be expected to
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take a long time to evolve to do the job. In these situations, bioaugmentation with
GM or non-GM organisms may be required.
5.139 Bioremediation of pollutants by the natural biota is often restricted by certain key
metabolic bottlenecks. It was proposed that GMOs could be applied to identify those
bottlenecks and alleviate them in the contaminated environment.
5.140 With respect to the use of biosensors, these were described as being very good at
assessing the bioavailability of pollutants across several scales of organisms.
However, as sensors to determine human toxicity, it was recognised that microbial
biosensors should only be applied as a rapid pre-screening system. Whilst they were
useful to indicate a possible problem for human health they should not be applied
further.
What information is required?
5.141 Although a large amount of knowledge is available to develop and employ organisms
to bioremediate contaminated sites, it was highlighted several times during the
workshop that there were still a number of basic research questions that needed to be
addressed, and that the commitment to remediating contaminated land needed to be
resolved. Many of these questions are due to the fundamentally poor understanding
of the diversity and activity of microbial communities in the environment, both GM
and non-GM.
5.142 At the fundamental level, information was required on the function of microbial
processes in the environment, and in particular in the rhizosphere. It was recognised
that is was important to identify which activities were important to bioremediation
and how to optimise them. These information requirements were applicable to both
GM and non-GM organisms.
5.143 In addition to requiring further information to answer these fundamental base-line
questions, the most pressing requirement was to determine the commitment to cleanup
contaminated sites. Bioremediation applications were recognised as having the
ability to treat contaminated environments now, and that further research would
optimise existing processes and identify new ones. However, it was concluded that:
• the questions that asked whether the use of a particular organism in
bioremediation may or may not have an effect on the environment could only
be answered by conducting environmentally relevant experiments and field
trials; and
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• the potential for bioremediation to cause adverse effects to the environment
and/or human health should be addressed in the context of the alternatives,
namely leaving the contamination untreated or using other remediation
technologies. Such assessment may require a form of cost/benefit analysis
comparing the different methods available.
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6. CONCLUSIONS
6.1 The purpose of this report has been to identify current and future applications of
GMOs in bioremediation and to address the potential risks and available management
strategies related to their use in the field. From the information reviewed in this
report, it may be concluded that GMOs offer a wide number of potential applications
for the bioremediation of contaminated environments. GMOs offer the ability to
bioremediate a large range of pollutants, including organic and inorganic compounds,
in both terrestrial and aquatic environments.
6.2 Existing applications of GMOs in bioremediation may be divided into:
• GMMs designed to degrade organic pollutants, either in situ in terrestrial
environments, or in ex situ applications such as bioreactors or as part of ‘pump
and treat’ systems;
• GMMs employed as biosensors to determine the presence and potential
toxicity of pollutants present in a contaminated site (aquatic or terrestrial).
Depending on the system used, the biosensor can be designed as a general
screening system to determine the combined toxicity of all pollutants present,
or as a more targeted system to detect the presence of specific compounds; and
• GM plants designed to hyperaccumulate or volatilise metal pollutants. Such
systems also have applications in aquatic and terrestrial environments.
6.3 Although the development of other GMOs for bioremediation applications has been
reported, for example the generation of GM plants to degrade organic pollutants such
as nitroaromatic compounds, the three areas outlined above offer the most immediate
application of GM technology to the treatment of contaminated land.
6.4 Indeed, although no in situ applications of GMOs for bioremediation have been
conducted to date in the UK, field trials of GM plants and GMMs have been and are
underway in other countries (see workshop report). The use of GMMs as biosensors
in ex situ applications has been applied in the UK as, for example, a preliminary
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screening system to identify the presence of pollutants within a large contaminated
site. It should be noted that the use of GMMs in this way does not involve any release
of GMMs into the environment and is essentially risk free.
6.5 The application of GMOs for the bioremediation of pollutants offers a number of
advantages (and disadvantages) over bioremediation strategies using non-GM
organisms and compared to physical or chemical remediation strategies. An
advantage of bioremediation strategies (using GM or non-GM organisms) compared
to physical or chemical processes is one of cost. This is due, in part, to the low
maintenance costs of using the technology (once the organism has been identified and
tested), and also to the ability of bioremediation processes to target particular
pollutants selectively, without the need for the wholesale removal of all the material
from the site (irrespective of the level of contamination present).
6.6 The disadvantages of bioremediation applications are a consequence of physiological
characteristics of the organism(s) used. Phytoremediation of contaminated soils, for
example, has huge potential for the low-cost treatment of large sites where the
contaminants are present at relatively low levels, but throughout the first few metres
of the soil across the site. In such cases the pollutants will be well within the vicinity
of root systems of the plants and available for phytoremediation.
6.7 However, careful selection of the organism(s) used for the bioremediation strategy
should avoid any significant disadvantages posed by particular organisms, and should
improve the success of the bioremediation process.
6.8 Although existing applications of GMOs in bioremediation are limited largely to the
three areas highlighted at the start of this section, there are a significant number of
publications highlighting the future applications of GMOs in this field. These may be
divided into three broad areas:
• the identification of the genetic basis of the processes involved in the
bioremediation of pollutants. This includes the identification of the particular
genes responsible, for example, the degradation of a particular compound, as
well as the identification of the necessary promoter and terminator sequences;
• the application of GM technology to improve non-GM bioremediation
processes. Many of the applications to use GMOs have developed by altering
or improving existing biochemical processes. Developments include the
identification and modification of rate limiting steps of a catabolic pathway, or
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the alteration of the pathway to avoid the production of toxic or dead end
metabolites; and
• the improvement and development of existing GMOs used in bioremediation.
The initial applications of GMOs for bioremediation focused on the
development of organisms capable of bioremediating the target pollutant(s).
The effectiveness of many of these applications has, however been restricted
by the poor performance of the GMO in the environment. The environment in
which the GMO is intended to function is an important consideration as is the
nature of the target pollutant. This is now being addressed for example,
through the genetic modification of aquatic microorganisms, rather than
terrestrial ones, for the treatment of pollutants in aquatic environments; and the
development of GMOs capable of degrading the target pollutant as well as
tolerating toxic concentrations of other pollutants, such as radionuclides.
6.9 The development of each of these three areas is restricted by a lack of information,
both at the genetic and metabolic level, and also at the population and environmental
level. As discussed during the workshop, information on how microorganisms, plants
and substrates (including pollutants) interact in the environment will assist in the
development of GMOs, and indeed non-GM organisms for bioremediation
immeasurably.
6.10 The risks posed by the use of GMOs in bioremediation depend on the characteristics
of the particular organism and those of the site in which it is used. For GMMs, the
most significant risks were determined to result from the transfer of the recombinant
genes to other organisms, and the disruption of other organisms and biological
processes. In both cases the level of risk that might actually occur can be minimised
by using GMMs with stable genetic insertions, and with no superfluous recombinant
material, that may confer a selective advantage over organisms already present in the
environment.
6.11 For plants, the transfer of the inserted genes can be minimised, for example, by
managing the plants so that flowering and transmission of pollen does not occur.
Other possible risks that may be associated with GM plants, such as the accumulation
of toxic compounds may be reduced by ensuring that the herbivory of the plants does
not take place, or by developing plants where the compounds are accumulated in nongrazed
areas of the plant.
6.12 In assessing the risks posed by the use of GMOs in bioremediation, the nature of the
site and the alternatives to bioremediation should be considered. As a result of their
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contamination, sites targeted for bioremediation are relatively inhospitable habitats,
characterised by a limited number of micro and macroorganisms. In many cases, the
level of contamination is so high as to render the site barren of all vegetation.
Therefore, the implications of using GMOs on such sites, with respect to the effect
they may have on natural flora, fauna and biological processes, may not be great,
particularly where the site has a very low level of biological activity.
6.13 As discussed in the risk assessment in Chapter 3 of this report, the risks posed by the
use of GM plants in bioremediation are similar to those encountered with GM plants
used in agriculture. However, the differences between arable environments and
contaminated sites will have a significant effect on the level of risk that is likely to be
realised.
6.14 In addition to offering the potential to remove or sequester specific pollutants from a
contaminated site, GMOs also have indirect applications for bioremediation. These
may include the use of pollutant tolerant plants in phytostabilisation strategies, and
the application of GMOs (microorganisms or plants) to support and supplement the
bioremediation activities of non-modified organisms. It is these combined strategies
for bioremediation, involving a number of organisms (plants and microorganisms,
GM and non-GM) that offers very significant potential for the bioremediation of a
wide range of sites contaminated with a cocktail of pollutants.
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7. RECOMMENDATIONS FOR FUTURE WORK
7.1 The purpose of this section of the report is to identify and propose areas of future
work that are required in order to develop the application of GMOs for the
bioremediation of organic and inorganic pollutants and assess the potential risk of
their use in the environment. During the production of this report, two broad areas of
work have been identified. These relate to the development of the GMOs themselves,
and also the understanding of the processes that underlie the survival, behaviour and
activities of organisms in the environment. Although this latter area is not confined to
GMOs, information on how microorganisms and plants behave in the environment,
and interact with themselves, each other and the pollutants, will only improve the
efficiency of GMOs in bioremediation, as well as reducing the level of any potential
risk to the environment and human health.
7.2 The application of GMOs for bioremediation has, to date, focused on the use of single
species of microorganisms or plants to target and bioremediate the contaminant.
However, the use of combined strategies, involving a number of GM plants and/or
microorganisms may offer a more powerful approach for treating contaminated sites.
This is particularly the case for those sites where the contaminants present offer little
nutritional benefit on their own, either due to their high toxicity or low concentrations.
Because the effectiveness of such combined strategies will depend on maximising the
interaction between the different organisms involved, then further research into these
areas would be extremely useful. This was one of the recommendations from the
workshop. One important example of such research is:
• the investigation of how indigenous microbial communities influence the
acquisition and accumulation of heavy metal pollutants by GM plants.
7.3 With respect to the development of the GMOs themselves, there are areas for further
work at almost every stage of the developmental process. These include:
• the identification of the metabolic pathway(s) involved in the degradation or
accumulation of pollutants;
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• the identification of the genes involved and the promoter sequences and
effectors that control them;
• the identification of organisms suitable for modification with the relevant
genes; and
• the best approach to using those organisms to bioremediate the contaminated
site, to ensure that any risks to the environment and human health from the
GMO or the pollutant are minimised.
7.4 Further work is also recommended to test the activity of the GMOs in their target
environment, and to assess the effect their use may or may not have on indigenous
organisms present. Examples include:
• proof of concept studies. Are the GMOs effective at site clean-up, and what
can they achieve or perform in the field that indigenous (non-GM) organisms
cannot; and
• cost/benefit analyses of using GMOs for bioremediation, rather than
indigenous plants and/or microorganisms.
7.5 The more advanced applications of GMOs developed to date are those where the
GMO has been selected for its ability to survive and grow in the contaminated
environment, as well as expressing the recombinant genes. These include flocforming
aquatic microorganisms designed to biodegrade PAHs in wastewater
treatment systems, and wetland plants and trees modified to remove methylmercury or
Hg(II) from contaminated marshlands.
7.6 For any bioremediation strategy to work effectively, the organism must survive and
compete in the contaminated environment and be able to degrade or accumulate the
target pollutant. Many contaminated sites are characterised by a cocktail of organic
and inorganic pollutants. Any organisms released into the site must therefore be
tolerant or resistant to a wide range of compounds, in addition to being able to
degrade the intended pollutant. Further work will almost certainly be needed to
identify suitable organisms and bioremediation strategies for the more contaminated
sites.
7.7 However, based on the information already available, and the organisms and
strategies that have been developed to date, it is recognised that the use of GMOs for
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the bioremediation of contaminated sites offers and extremely powerful suite of
strategies for the low cost and potentially low-risk removal of toxic compounds from
polluted soils and aquatic systems. Further work into any aspect of the use of GMOs
in bioremediation can only improve the development of this field.
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9. APPENDICES
APPENDIX A - ABSTRACTS OF WORKSHOP PRESENTATIONS
APPENDIX B - LIST OF WORKSHOP DELEGATES
APPENDIX C - WORKSHOP PROGRAMME
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APPENDIX A - ABSTRACTS OF WORKSHOP PRESENTATIONS
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Reporter gene based biosensors – risk-based management support for remediation of
contaminated land
Professor Ken Killham, Department of Plant & Soil Science, University of Aberdeen,
Aberdeen.
The use of microbial biosensors for risk-based site assessment, monitoring and remediation is
now providing a completely new and powerful form of support for the management of
contaminated land. Construction of the biosensors is based on the insertion of luminescencebased
(lux) or unstable gfp reporter genes into a range of environmentally relevant bacteria,
and allows for both specific contaminant and overall toxicity-based contaminant detection.
The latter approach is particularly relevant to the new environmental legislation in the UK –
Part IIA of the Environmental Protection Act defines site contamination in terms of
significant harm. The toxicity biosensors offer a reliable and rapid means of diagnosing the
harm caused by site contamination.
Case studies are presented where reporter-gene based biosensors were first used to map the
sites for toxic contaminants. This was carried out for both groundwater and for
soils/sediments and proved to be able to identify all risk-based contaminants (organic and
inorganics) at the sites. The sensors were then used to identify the potential for
bioremediation and, using a series of sample manipulations coupled to biosensor-based
assays, identify any constraints to bioremediation across the site. Finally the sensors were
used to provide risk-based monitoring of the sites to provide the basis for advice to the
regulatory authorities on sign-off.
Recent incorporation of reporter genes into a range of single and multi-cell eukaryotes has
provided an opportunity to extend toxicity-based biosensing of site contaminant towards
human risk assessment. Such biosensors are insensitive to contaminants such as zinc (which
strongly inhibit biosensors based on environmental bacteria), but responsive to contaminants
such as certain PAH’s (which have no effect on, or stimulate bacterial sensors).
Reporter gene based biosensors are highly compatible with on-line instrumentation, where
biosensor response can be used for automated, and often remote, groundwater or surface
water monitoring. Mathematical analysis of the kinetic response of the sensors on-line can
even be used to diagnose the site contaminants.
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References
Hollis, R.P., Killham, K & Glover, L.A. 1999. Design and application of a biosensor for monitoring
toxicity of compounds to eukaryotes. Applied and Environmental Microbiology, 66, 1676-1679.
Shaw, L.J., Sousa, S., Beaton,Y., Glover, L.A., Killham, K., Meharg, A.A. 2000. Lux based
biosensing of 2,4-dichlorophenol biodegradation and bioavailability in soil. In: Proceedings of the
International In situ and on-site Bioremediation Symposium, San Diego, California, 5, 247-252.
Beaton, Y., Shaw, L.J., Glover, L.A., Meharg, A.A & Killham, K 1999. Biosensing 2,4-
dichlorophenol toxicity during biodegradation by Burkolderia sp RASC C2 in soil. Environmental
Science and Technology, 33, 4086-4091.
Preston, S., Hall, J.M., Rattray, E.A.S., Chaudri, A. M., McGrath, S.P., Killham, K. and Paton, G I
1999. Assessing metal bioavailability in soils using luminescence-based microbial biosensors. In: In
situ and On-Site Bioremediation, Battelle Press, Columbus, Ohio.
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Phytoremediation of toxic chemicals in our environment
Professor Richard B. Meagher, Department of Genetics, University of Georgia, Athens, GA
30602, USA.
Phytoremediation is the use of plants to extract, sequester, and/or detoxify pollutants in soil,
water, and air. At present, phytoremediation is widely viewed as the most promising,
ecologically responsible alternative or amendment to the environmentally destructive
excavation and reburial methods currently used to remediate toxic sediments. Sediment
excavation and reburial not only destroys the environment of the original contaminated site,
but create new sites that must be monitored indefinitely. Another serious drawback to
physical remediation methods is that they are proving too costly to be applied on the
imposing scale that is required. One measure of these drawbacks is that most chemical waste
and spill sites larger than a few acres have never been cleaned up.
Phytoremediation is generating great excitement, because its technologies offer a efficacious
means of restoring the hundreds of thousands of square miles of land and water now polluted
by human activities. Most phytoremediation strategies reestablish a normally functioning
ecosystem concomitant with cleansing chemical waste sites. Phytoremediation technologies
should also make it possible for manufacturing and agricultural wastes to be processed
immediately as they are leaving their point sources, preventing their entering the
environment. These same approaches may also help forestry better utilise traditionally
marginal lands that are pour in nutrients and contain toxic elements from natural or
anthropomorphic sources.
Plants have numerous natural genetic, biochemical, and physiological properties that make
them ideal agents to remediate soil and water (Meagher and Rugh, 1996). Among the many
plant attributes useful to phytoremediation, there are three specific characteristics that can be
directly manipulated for remediation purposes. First, many plant species can grow 100
million miles of roots per acre per year that normally extract nutrients from soil and water.
Clearly, this provides an enormous surface area through which contaminants can be contacted
and extracted from sediment. Second, plants are photosynthetic and thus control more than
80% of the energy in most ecosystems; this energy can be devoted to phytoremediation.
Third, plants are autotrophs with numerous biochemical pathways for degrading or storing
complex organics and elements that can be enhanced for phytoremediaton. To understand
more specificallyhow plants might be used to degrade or sequester environmental
contaminants, it is helpful to distinguish between the strategies for phytoremediating organic
and elemental pollutants (Meagher, 2000).
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Organic pollutants are composed of carbon usually complexed with nitrogen, sulfur, and/or
chlorine. Organic pollutants on every country’s priority list include polychlorinated
biphenyls (PCBs) like dioxin, polycyclic aromatic hydrocarbons (PAHs) like benzoapyrene,
nitroaromatics like trinitrotoluene (TNT), linear halogenated hydrocarbons like
trichloroethylene (TCE), and halogenated aromatic pesticides like DDT. All of these
compounds are toxic and some are also teratogenic and carcinogenic. Plants have the
potential to degrade such organic pollutants to much less toxic forms or even to completely
mineralise them into harmless constituents like carbon dioxide, ammonia, or chloride ion.
The right plant species can accelerate degradation 100- or even 100,000-fold over natural
attenuation. These plants use endogenous enzymes that are either part of their normal
metabolism or are part of the biochemical defenses they use against predators and parasites.
However, in many cases the fast growing, deeply rooted plants that would be ideal for
phytoremediation, like hybrid poplars, willow species, or grasses, do not have enough of the
right enzymes to degrade target compounds. Genetically modifying these plants with genes
from bacterial, animal, or other plant sources can greatly enhance their phytoremediation
properties. For example, trinitrotoluene (TNT) is widely distributed in the environment from
use in munitions manufacture. A particular bacterial nitroreductase gene expressed in plants
can greatly accelerate the rate of TNT degradation and enhance plant growth by reducing its
toxic effects (French et al., 1999).
Elemental pollutants include toxic heavy metals and radionuclides such as arsenic, cadmium,
cesium, chromium, lead, mercury, strontium, technetium, tritium, and uranium.
Unfortunately, elemental pollutants are essentially immutable by any biological or physical
process short of nuclear fission and fusion, and thus their remediation presents special
scientific and technical problems. Phytoremediation strategies for elemental pollutants focus
first on reducing toxicity; second on sequestering the element in roots to prevent leaching
from the site; and third on hyperaccumulation in above-ground organs for later harvest. Only
rare and generally exotic native plants have been found that can efficiently extract heavy
metals into above-ground tissues. Most of these are so fastidious in their growth habits that
they are seldom useful in phytoremediation strategies. Put another way, most ubiquitous,
rapidly growing native plants do not naturally express the right genes in the right tissues to be
effective agents for elemental remediation. However, several significant experiments
demonstrate that the phytoremediation of elemental pollutants can be greatly enhanced using
genetically modified plants (Meagher, 2000). My own laboratory’s work on the remediation
of mercury will serve as a useful example.
Mercury primarily enters the environment from industrial and defense related accidents.
Although a few hectare-sized sites have been excavated, tens of thousands of mercury sites
worldwide have never been cleaned up, and several of these cover hundreds of square
kilometers (Meagher et al., 2000). Ionic mercury Hg(II) is relatively toxic, but with rare
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exceptions neither it nor metallic Hg(0) have been involved in serious incidents of human
mercury poisoning without first being transformed into methylmercury (Keating et al., 1997).
Various mercury species are efficiently converted to methylmercury by anaerobic bacteria,
especially in anaerobic sediments (Choi et al., 1994). One of the most dangerous aspects of
methylmercury is that it is biomagnified up the food chain by several orders of magnitude
and has a greater toxicity than most other mercury compounds (Meagher et al., 2000). As a
result, the fish-eating predatory animals and humans at the top of long aquatic food chains
suffer the most sever methylmercury poisoning (Boischio and Henshel, 1996; Keating et al.,
1997). The world first became aware of the extreme dangers of methylmercury after a large,
tragic incident of human mercury poisoning in Japan in the 1950s at Minamata Bay (Harada,
1995). It is likely that a new methylmercury poisoning incident of even greater proportions is
victimizing native populations in the tributaries of the Amazon River in South America due
to the ongoing use of mercury in the gold mining industry (Barbosa et al., 1995; Cleary,
1996). Because native bacteria at aquatic sites synthesise methylmercury from other forms of
mercury, eliminating all forms of mercury contamination from lakes, rivers, and wetlands
should largely prevent methylmercury formation.
Our laboratory has used two genes from the well-characterised bacterial mer operon, merA
and merB, in order to engineer a mercury transformation and remediation system in plants
(Meagher, 2000; Rugh et al., 2000). The bacterial merB gene encodes an organomercurial
lyase that degrades methylmercury to methane and Hg(II). In plants, expression of merB
alone confers high levels of methylmercury resistance, allowing the transgenics to grow
normally on methylmercury concentrations 10 times higher than those that kill wild type
plants (Bizily et al., 1999). At levels of methylmercury as high as or much higher than found
in the environmental these transgenic plants accumulate and are able to outgrow Hg(II), the
product of the MerB catalyzed reaction. The bacterial merA gene encodes a mercuric ion
reductase that converts ionic mercury (Hg(II)) to elemental, metallic mercury (Hg(0).
Metallic mercury is nearly two orders of magnitude less toxic than ionic mercury and is
readily eliminated due to its volatility. Diverse plant species expressing merA constitutively
are resistant to at least 10-fold higher levels of Hg(II) than those that kill non-transgenic
controls (Rugh et al., 1996; Rugh et al., 1998). Plant expression of merA and merB together
results in the two-step conversion to volatile Hg(0) and produces resistance to 50-fold higher
levels of MeHg than is required to kill control plants and 5-fold higher than the levels that kill
merB plants (Bizily et al., 2000). The merA/merB plants eliminate mercury 1000 times faster
from methylmercury-containing growth medium than control plants. Neither product of these
enzymes, Hg(II) or Hg(0), are biomagnified. Because methylmercury is such an extreme
human health hazard it is important that a number of phytoremediation strategies that block
its flow into the environment be adopted.
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Phytoremediation holds great promise as an environmentally friendly approach to cleaning
sediments, soils, and water supplies of toxic chemicals. Physical remediation methods are
usually very environmentally destructive and create thousands of newly contaminated sites.
Natural attenuation of typical contaminated sites is often estimated to take 100-10,000 years,
while well-designed phytoremediation strategies function in the range of 3-20 years.
Genetically modified plants will greatly accelerate the rates of organic remediation and are
essential for the phytoremediation of most elemental pollutants. Applying modern genetic
manipulation techniques to phytoremediation will reverse the ongoing deterioration of our
environment.
Bibliography
Barbosa, A.C., Boischio, A.A., East, G.A., Ferrari, I., Goncalves, A., Silva, P.R.M., and da Cruz,
T.M.E. (1995). Mercury contamination in the Brazilian Amazon. Environmental and occupational
aspects. Water, Air, and Soil Pollution 80, 109-121.
Bizily, S., Rugh, C.L., and Meagher, R.B. (2000). Phytodetoxification of hazardous
organomercurials by genetically engineered plants. Nature Biotech. 18, 213-217.
Bizily, S., Rugh, C.L., Summers, A.O., and Meagher, R.B. (1999). Phytoremediation of
methylmercury pollution:. merB expression in Arabidopsis thaliana confers resistance to
organomercurials. Proc. Natl. Acad. Sci. USA 96, 6808-6813.
Boischio, A.A., and Henshel, D.S. (1996). Mercury contamination in the Brazilian Amazon.
Environmental and occupational aspects. Water, air and soil pollution 80, 109-107.
Choi, S.C., Chase, J.T., and Bartha, R. (1994). Metabolic pathways leading to mercury methylation
in Desulfovibrio desulfuricans LS. Appl. & Env. Microbiol. 60, 4072-4077.
Cleary, D. (1996). Mercury contamination and health risk in the Brazilian Amazon. An ethical
dilemma (editorial). Rev Inst Med Trop Sao Paulo 38, 247-8.
French, C.E., Rosser, S.J., Davies, G.J., Nicklin, S., and Bruce, N.C. (1999). Biodegradation of
explosives by transgenic plants expressing pentaerythritol tetranitrate reductase. Nat Biotechnol 17,
491-4.
Harada, M. (1995). Minamata disease:. Methylmercury poisoning in Japan caused by environmental
pollution. Critical Reviews in Toxicology 25, 1-24.
Keating, M.H., Mahaffey, K.R., Schoeny, R., Rice, G.E., Bullock, O.R., Ambrose, R.B., Swartout, J.,
and Nichols, J.W. (1997). Mercury study report to congress, I, 2:1-2:9.
Meagher, R.B. (2000). Phytoremediation of toxic elemental and organic pollutants. Curr. Opin.
Plant Biol. 3, 153-162.
Meagher, R.B., and Rugh, C.L. (1996). Phytoremediation of heavy metal pollution: Ionic and methyl
mercury. In:. OECD Biotechnology for Water Use and Conservation Workshop, eds (Cocoyoc,
Mexico: Organization for Economic Co-Operation and Development), 305-321.
Meagher, R.B., Rugh, C.L., Kandasamy, M.K., Gragson, G., and Wang, N.J. (2000). Chapter 11.
Engineered phytoremediation of mercury pollution in soil and water using bacterial genes. In:
Phytoremediation of Contaminated Soil and Water, N Terry, and G. Banuelos, eds (Boca Raton:
Lewis Publishers), pp. 203-221.
Rugh, C.L., Bizily, S.P., and Meagher, R.B. (2000). Phytoremediation of environmental mercury
pollution. In: Phytoremediation of toxic metals: Using plants to clean-up the environment, B. Ensley,
and I. Raskin, eds (New York: Wiley and Sons), pp. 151-169.
Rugh, C.L., Senecoff, J.F., Meagher, R.B., and Merkle, S.A. (1998). Development of transgenic
yellow-poplar for mercury phytoremediation. Nature Biotech. 16, 925-928.
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Rugh, C.L., Wilde, D., Stack, N.M., Thompson, D.M., Summers, A.O., and Meagher, R.B. (1996).
Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana. plants expressing a
modified bacterial merA gene. Proc. Natl. Acad. Sci. USA 93, 3182-3187.
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Field release of P. fluorescens HK44:. Long term persistence and field performance of a
bioremediation bioluminescent bioreporter
Professor Gary S Sayler, Center for Environmental Biotechnology, University of Tennesse,
Knoxville, Tennesse, USA
Under support of the US Department of Energy (DOE), the University of Tennessee received
the first US Environmental Protection Agency (EPA) Consent Order (March 1986) for
environmental test release of genetically modified bacteria for applications in bioremediation.
In October 1986, under this Consent Order, Pseudomonas fluorescens HK44 containing an
introduced stable catabolic plasmid (NAH7-like) for polyaromatic hydrocarbon
biodegradation with a bioluminescent (lux) bioreporter gene transcriptional fusion was
inoculated into replicated test and control subsurface soil lysimeters at DOE’s Oak Ridge
National Laboratory. Both donor and recipient P. fluorescens strains used to construct the
GMO were isolated from manufactured gas plant contaminated soils. Origins of the lux
genes and antibiotic resistance markers delivered by transposon Tn4431, to create the
bioreporter fusion were Vibrio fisheri and E. coli, respectively.
The focus of these field studies was to determine under what conditions the GMO could be
maintained in the subsurface soil to promote PAH bioremediation. A fundamental hypothesis
tested was that light emission from the bioreporter could be used as a real-time
bioremediation process-monitoring tool to provide on-line control strategies for system
optimization. These objectives were pursued over a two-year intensive investigation after
which time additional intermittent monitoring has been conducted to the present.
Organismal and process monitoring was conducted in a multi parametric format, including
selective cultivation, colony hybridization, PCR amplification of target genes, bioluminescent
MPN, on-line and in-situ bioluminescence, hydrocarbon decay, temperature, humidity and
respiration. Experimental manipulations, included water table and moisture adjustments,
exogenous micronutrients, re-contamination with PAH in transformer oil, and soil gas phase
aeration.
Population dynamics of the GMO followed a predicted long-term decay, but HK44 was
readily detectable and recoverable at densities of 102g-1 after two years and <102g-1 after 1100
days. Population decay could be interrupted and regrowth of the GMO to densities as high as
109g-1 could be achieved with aeration and hydrocarbon replacement. In-situ bioluminescent
in subsurface soil was measured in real-time and corresponded to experimental manipulations
and hydrocarbon bioremediation. Virtually all naphthalene components of the PAH mixture
were removed during the field test, but discrimination of abiotic and natural attenuation
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influences vs. GMO bioremediation was not clear; thus limiting absolute quantitation of the
bioremediated component.
Observations on future successful biotechnology risk assessment review and process
application include:
• Provide detailed taxonomy including 16s rRNA phylogeny of the proposed
GMOs
• Provide absolute ancestry of all genetic elements introduced
• Insure the GMO phenotype does not exhibit potential pathogenic properties,
i.e growth at 37°C, enzymes or polymers
• Avoid, if possible, mobile genetic elements and antibiotic resistance markers
• Provide sufficient efficacy data from lab or microcosm simulation on
detection, monitoring and process effectiveness
• Anticipate natural population decay kinetics and contamination attenuation by
indigenous and abiotic processes
• Strive for >108 active organisms g-1 to achieve maximal treatment efficiency
• Soil gas phase 02 concentration is a key rate limiting process for aerobic
bioremediation in the vadose
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Prospects and challenges for bioremediation with GMMs
Professor Kenneth N Timmis, National Research Centre for Biotechnology, Braunschweig,
Germany.
Microbial life on planet Earth is thought to have originated some 3,800m years ago, and by
about 3,500m years ago microbes had evolved most of the central biochemical processes we
currently know, some 2,000m years before eukaryotes appeared. Microbes have evolved a
phylogenetic and metabolic diversity that greatly exceeds all other forms of life, and that
enables them to colonise a vast spectrum of diverse environments, ranging e.g from polar ice
to thermal vents; microbial habitats thus define the biosphere. The diverse range of nutrients
and sources of energy that can be used by microbes, coupled with the rapid growth rates of
some, make them major players in the biogeochemical cycling of elements, and in the
turnover of polluting wastes in the environment. Many toxic xenobiotics represent a
nutritional opportunity to the microbial flora, which is often able to eliminate such pollutants
from the environment. However, in contrast to the millions of years of exposure and
evolution of microbes to natural polluting compounds, they have only been exposed to
xenobiotics for a few decades, a very brief period in evolutionary time. It is therefore to be
expected that metabolic activities towards such compounds are not optimal.
Modern genetic methods allow accelerated evolution in the laboratory of optimised catabolic
functions, and of important accessory functions, and represent a powerful option to accelerate
the elimination of toxic chemicals from the environment, when the rate-limiting factor is suboptimal
catabolic potential. The use of such designed biocatalysts is reasonably
straightforward in end-of-pipe applications and will not be further discussed. In-situ/ex situ
applications are, however, more ecological in nature and less subject to operator control.
Since polluted sites are often characterised by rather extreme physico-chemical parameters
that are unfavourable to colonisation by an introduced GMM, the use of genetic modules to
optimise selected site-adapted members of the habitat to be treated will become increasingly
important. In any case, effective application of GMMs will require a much better
understanding of the ecological parameters regulating the biodegradation of xenobiotics in
the environment. In particular, microbes designed to better degrade a pollutant need to
function as integrated members of the natural microbial communities colonising the polluted
habitat. Since the vast majority of the microbes active in natural habitats cannot be cultivated
and studied by traditional means, interactions of GMMs with other community members,
with the pollutants, and with the abiotic components of the habitat, must be investigated by
culture-independent methods.
Important underlying ecological issues that need to be better understood are:
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• The significance of the high diversity of degraders in natural microbial
communities
• The structural and functional organisation of catabolic communities in
biofilms
• The population dynamics of community members during catabolism of the
pollutant and the parameters regulating the population changes and community
activities
• The strategies whereby microbes access hydrophobic substrates
(‘bioavailability’ with regard to biodegrading microbes may not be the same as
‘bioavailability’ with regard to risk assessment), or: what is the face of the
bug? What is the face of the pollutant?
• The metabolic routes and networks followed during catabolism of the
pollutant, and relevant kinetic data and metabolic fluxes of the community
• The sharing of the available nutritional resources by the community, and
underlying metabolic interactions
The understanding gained from such studies needs to be underpinned by a theoretical
framework of microbial community structure and function, to be obtained by modelling the
community and its metabolic, physiologic and genetic potential and interactions. Such
modelling will identify parameters that directly or indirectly limit or regulate key community
or GMM activities, and suggest interventions that can reduce limitations.
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Metal accumulation by plants
Professor J.A.C Smith, Department of Plant Sciences, University of Oxford, Oxford
Metals are present in many soils at concentrations inimical to plant growth. Naturally
metalliferous soils have arisen from the weathering of parent geological materials, and these
substrates often support highly distinctive vegetation (which has proved a spur to fields such
as geochemical prospecting). But through the exploitation of this natural resource there are
now many anthropogenic sources of toxic metals contaminating today’s environment. These
include atmospheric discharges from industrial processes such as metal processing and
burning of fossil fuels, deposition onto the soil in the form of mine waste, sewage sludge,
animal manures, fertilisers and agrochemicals, as well as transfer to the soil through irrigation
water or run-off, and accidental discharges from the nuclear industry.
Certain plants have, however, succeeded in evolving tolerance to relatively high
concentrations of soil metals. In fact, some of these have become classic examples of
‘microevolution’ in plants in response to a particularly strong selective pressure (Antonovics
et al., 1971), e.g the occurrence of zinc-tolerant grasses under galvanised electricity pylons,
or copper-tolerant ecotypes in the vicinity of metal smelters. However, it is now clear that
different plants may show fundamentally different mechanisms of metal tolerance. At one
extreme are the metal excluders, in which metal tolerance is inversely related to metal
concentrations within the plant biomass. At the other extreme are the so-called metal
hyperaccumulators, which are the most metal-tolerant plants known and which can
accumulate certain metals (such as Zn, Mn, Ni, Co, or Cd) to over 1 % of shoot dry biomass
(Baker et al., 1999). These plants pose many interesting questions as to how such metals can
be accumulated and sequestered in biological tissues without toxic effects, as well as raising
broader issues concerning the ecological significance of metal hyperaccumulation.
For a number of years, it has been clear that hyperaccumulator plants represent a potentially
valuable resource for the remediation of contaminated soils (Salt et al., 1995; Chaney et al.,
1997). These plants possess a number of useful attributes for this type of ‘phytoremediation’
technology. For example, they extract metals very effectively out of the soil; they translocate
most of the metal out of the root system to the above-ground parts of the plant in the shoot;
and the high shoot concentrations themselves tend to deter grazing herbivores, helping to
minimise further dispersal into the food chain. It is even feasible to harvest the above-ground
parts as part of a true recycling process to recover metals from the dry biomass. Estimates
suggest that this type of phytoremediation technology could be up to 100 times cheaper than
conventional methods such as excavation and removal to landfill, as well as being less
environmentally damaging.
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On the other hand, there are a number of significant disadvantages to a phytoremediation
approach based on naturally occurring hyperaccumulator plants (Baker et al., 1999). First,
they are without exception rare plants, often with very restricted population sizes: only about
430 species are known worldwide, representing less than 0.2 % of all flowering plants.
Second, most hyperaccumulator species are rather small, slow-growing plants, so successive
harvests would be needed over an extended period of time (perhaps 10 to 15 years at
minimum) to decontaminate the soil to acceptable levels. Third, most hyperaccumulators are
rather specific to particular metals (for example, the Ni hyperaccumulators can tolerate Co,
but are very sensitive to Zn and Cu: Krämer et al., 1996), so this would not allow treatment
of mixed metal wastes with these plants. And fourth, many are adapted to special climatic or
edaphic conditions that would preclude their cultivation in particular environments.
An obvious route to the realization of an effective phytoremediation technology would be to
introduce the metal-hyperaccumulation trait into a high-biomass plant with favourable growth
characteristics and harvestability (Chaney et al., 1997; Salt et al., 1998). Theoretically, this
could be achieved by introgression using closely related genotypes, or by techniques such as
protoplast fusion. The more targeted route, however, will be to identify the essential genes
involved in metal hyperaccumulation and to introduce these specifically into the appropriate
genetic backgrounds. This will necessitate more detailed understanding of the
hyperaccumulation mechanism than achieved hitherto. Certain key components have been
identified, such as the need for appropriate metal-binding ligands to detoxify the metal ions,
and specific transporters to allow movement of metals through the plant (Krämer et al., 1996;
Meagher, 2000). But it is not yet understood at the genetic level how these processes are
uniquely regulated in hyperaccumulator plants. Production of transgenic plants exhibiting a
metal-hyperaccumulation trait has not yet been reported, but this is an area of intense activity
that will very likely produce notable advances in the near future.
References
Antonovics, J., Bradshaw, A.D and Turner, R.G. (1971) Heavy metal tolerance in plants. Adv. Ecol.
Res. 7, 1-85.
Baker, A.J.M., McGrath, S.P., Reeves, R.D and Smith, J.A.C (1999) Metal hyperaccumulator plants:
a review of the ecology and physiology of a biological resource for phytoremediation of metalpolluted
soils. In Phytoremediation of Contaminated Soil and Water, eds N Terry and G. Bañuelos,
pp 85-107 Lewis Publishers, Boca Raton, Florida.
Chaney, R.L., Malik, M., Li, Y.-M., Brown, S.L., Brewer, E.P., Angle, J.S and Baker, A.J.M. (1997)
Phytoremediation of soil metals Curr. Opin Biotechnol. 8, 279-284.
Krämer, U., Cotter-Howells, J.D., Charnock, J.M., Baker, A.J.M. and Smith, J.A.C (1996) Free
histidine as a metal chelator in plants that accumulate nickel. Nature 379, 635-638.
Meagher, R.B (2000) Phytoremediation of toxic elemental and organic pollutants Curr Opin Plant
Biol 3, 153-162.
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Salt, D.E., Blaylock, M., Kumar, P.B.A., Dushenkov, V., Ensley, B.D., Chet, I. and Raskin, I (1995)
Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants
Bio/Technology 13, 468-474.
Salt, D.E., Smith, R.D and Raskin, I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol.
Biol. 49, 643-668.
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Defusing the environment: Engineering plants to degrade explosives
Dr Neil C Bruce, Institute of Biotechnology, University of Cambridge
Over the last twenty years there has been major international concern over the contamination
of land and ground water with persistent organic pollutants. Remediation has been attempted
mainly through the economically and environmentally costly large-scale removal of soil and
water; however, large areas of contaminated land still exist and such demanding and
expensive remediation procedures are not an option for the developing world. The lack of
affordable and effective cleanup technologies demands the development of novel processes.
Much work has focused on microbial biodegradation, but results to date have been
disappointing. The effectiveness of such processes has been inhibited by factors such as poor
biomass, the requirement of an additional substrate for cometabolism and the lack of
induction of the relevant metabolic genes. These problems may be overcome through the use
of genetically modified bacteria with altered regulation of degradative genes; however, it is
not clear that such modified organisms will thrive in the environment, and furthermore,
current legislation severely restricts the release of genetically modified microorganisms into
the environment. Recent attention has, therefore, focused on phytoremediation, which is the
use of plants to remediate environmental toxicity.
Plants have potentially impressive economic benefits as a robust and renewable resource.
They produce large amounts of biomass and have a remarkable ability to extract compounds
from the surrounding environment. Their root systems are dense and extensive and promote
increased microbial activity in their rhizosphere. Plants also have a high degree of public
acceptance with an appreciation of their aesthetic quality and environmental benefits. The
reluctance by regulatory authorities to approve the release of genetically modified to approve
the release of genetically modified bacteria is partly because of the mobility of bacteria and
the ease of horizontal gene transfer among dissimilar prokaryotes. However, these problems
can be addressed by the use of transgenic plants, as by virtue of their size, plants are more
easily controlled than microorganisms. Furthermore, by selecting sterile plants and
controlling propagation by harvesting the plants prior to flowering, uncontrolled genetic
release can be prevented. A major disadvantage of plants is that their innate biodegradative
abilities are limited and often rates of uptake and metabolism can be slow. Recently,
transgenic plants were shown to germinate and grow in the presence of normally toxic levels
of ionic mercury1,2. This raised the question of whether the impressive biodegradative
abilities of certain bacteria could be combined with the high biomass and stability of plants to
yield an optimal system for in-situ bioremediation of organic pollutants in soil. We therefore
decided to combine the biodegradative capabilities of soil bacteria with the high biomass and
stability of plants to yield an optimal system for in-situ bioremediation of explosives in soil.
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Toxic explosive residues are major environmental contaminants due to the manufacture,
testing and disposal of munitions. Explosives can be broadly classified into three groups:
nitroaromatics (e.g trinitrotoluene, TNT, is probably the most persistent pollutant in military
sites), nitramines (e.g hexahydro-1,3,5-trinitro-1,3,5-triazine, RDX) and nitrate esters (e.g
glycerol trinitrate, nitroglyerin, GTN and pentaerythritol tetranitrate, PETN). We have
isolated bacteria that degrade all the major classes of explosives 3,4,5,6. Our recent studies of
the biodegradation of energetic compounds by bacteria resulted in the isolation of a strain of
Enterobacter cloacae, termed PB2, which is capable of utilising the nitrate ester explosives
PETN and GTN as the sole source of nitrogen. Liberation of nitrite is mediated by an
NADPH-dependent PETN reductase (PETNR) which has FMN bound as prosthetic group4.
The gene for PETNR, onr, has been cloned, sequenced and overexpressed in E coli and the
crystallographic structure of this enzyme has recently been determined5,7. Interestingly, this
enzyme is related in sequence and structure to Old Yellow Enzyme from Saccharomyces
carlsbergenesis. All the members of this family have Tim-barrel structures, FMN as a
prosthetic group and display various chemistries with electrophilic substrates. Activity
towards α,β-unsaturated carbonyl structure is a common property of this family. PETNR is,
however, unusual, in that it displays activity towards the highly recalcitrant aromatic
explosive TNT via a reductive pathway resulting in nitrogen liberation6. Furthermore, we
have recently demonstrated that PETNR can reductively liberate nitrite from the nitramine
explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). PETNR is therefore remarkable in
that it is able to degrade all the major classes of explosives by distinct mechanisms. Current
work is focused on relating structure and function within this growing family of enzymes
with a view to engineering novel enzymes exhibiting useful characteristics towards
explosives for bioremediation purposes.
Our work has been targeted on the use of genetic engineering for broadening and improving
the degradative capabilities of plants for phytoremediation of the major classes of explosives.
We have engineered transgenic tobacco plants that express PETNR to degrade nitrate ester
explosives and TNT7. Importantly, we have shown that seeds from transgenic tobacco plants
which express PETNR were capable of germinating and growing in media containing levels
of GTN and TNT that are toxic to the wild-type plant. Furthermore, we have shown that
transgenic seedlings are able to denitrate GTN at a much faster rate than wild-type seedlings.
We have also recently constructed transgenic plants that express the gene encoding the
aromatic nitroreductase gene from E. cloacae PB2. This small monomeric flavoprotein
reduces aromatic nitro groups to nitroso, hydroxylamino and amino groups, with oxidation of
NAD(P)H. Partially reduced nitroaromatic compounds are highly reactive, and when
produced intracellularly are known to react with cell constituents to form covalent bonds,
thus being removed from the environment. Our initial studies with nitroreductase plant lines
look very promising, with the transgenic plants detoxifying high concentrations of TNT.
Since the bacterial degradative pathways for many classes of pollutants have been elucidated,
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this may be a generally applicable method of achieving bioremediation of contaminated soil
in the environment.
References
Rugh, C.L., Wilde, D., Stack, N.M., Thompson, D.M., Summers, A.O., and Meagher, R.B (1996)
Proc Natl Acad Sci USA 93: 3182-3187.
Rugh, C L., Senecoff, J F., Meagher, R B and Merkle, S A (1998) Nature Biotech 16: 925-928.
Binks, P R., Nicklin, S and Bruce, N C (1995) Appl Eviron Microbiol 61: 1318-1322.
Binks, P R., French, C E., Nicklin, S and Bruce, N C (1996) Appl Environ Microbiol 62:1214-1219.
French, C E., Nicklin, S and Bruce, N C (1997) J Bacteriol.178:6623-6627.
French, C E., Nicklin, S and Bruce, N C (1998) Appl Environ Microbiol 64: 2864-2868
Barna ,T., Khan, H., Bruce, N C., Scrutton, N S and Moody, P.C.E (2000) (submitted for publication).
French, C E., Rosser, S J., Davies, G J., Nicklin, S and Bruce, N C (1999) Nature Biotech 17: 491494.
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APPENDIX B - LIST OF WORKSHOP DELEGATES
---------------------------------------
Dr Colin Cartwright WS Atkins Environment
Dr Ian Thompson CEH, Oxford
Dr Michelle Barclay CEH, Oxford
Prof Mark Bailey CEH, Oxford
Prof Andrew Smith Dept of Plant Sciences, University of Oxford
Dr Neil Bruce Institute of Biotechnology, University of Cambridge
Prof Ken Killham Dept of Plant & Soil Science, University of Aberdeen
Prof Rich Meagher Dept of Genetics, University of Georgia
Prof Gary Sayler Center for Environmental Biotechnology, University of Tennessee
Prof Ken Timmis National Research Centre for Biotechnology, Braunschweig
Prof Chris Leaver Dept of Plant Sciences, University of Oxford
Prof Chris Knowles Oxford Centre for Environmental Biotechnology, University of Oxford
Dr Martin Broadley Horticulture Research International, Wellesborne
Dr Ken Bruce Division of Life Sciences, Kings College London
Dr Adrian Butt DEFRA
Miss Alexandra Christopher Dept of Chemistry, University of Oxford
Dr Gillian Davis University of Oxford
Dr Richard Ellis NERC Centre for Population Biology, Imperial College at Silwood Park
Dr Dawn Field CEH, Oxford
Dr Anne Glover Dept of Molecular and Cell Biology, University of Aberdeen, Remedios
Dr Rosie Hails CEH, Oxford
Ms Judith Hann
Dr Frances Harper School of Life Sciences, University of Dundee
Dr Penny Hirsch IACR Rothamsted
Dr David Hopper Institute of Biological Sciences, University of Wales, Aberystwyth
Mr Robert Ingle Dept of Plant Sciences, Oxford
Dr Simon Jackman CEH, Oxford
Dr Theresa Kearney Environment Agency
Mr Anthony Keeling Elsoms Seeds Ltd
Ms Renatta Krasowaik School of Biosciences, University of Birmingham
Dr. Mike Larkin Questor Centre, Queen’s University of Belfast
Ms Rachel Leighton Dept of Plant Sciences, Oxford
Dr Andy Lilley CEH, Oxford
Prof Jim Lynch School of Biological Sciences, University of Surrey
Dr Lynne Macaskie School of Biosciences, University of Birmingham
Dr Sarah MacNaughton AEA
Dr Steve McGrath IACR Rothamsted
Prof Andy Meharg Dept of Plant & Soil Science, University of Aberdeen
Dr Andreas Meyer Institut fuer Forstbotanik und Baumphysiologie, Freiburg
Dr Kate Millar School of Biosciences, University of Nottingham
Ms Lucy Moore Dept of Plant Sciences, Oxford
Dr Martin Naylor CEH, Oxford
Dr Marion Rawlins DEFRA
Dr Brian Reid School of Environmental Science, University of East Anglia
Dr Jonathan Rees Dept of Plant Sciences, Oxford
Dr Gary Robinson Dept of Biosciences, University of Kent at Canterbury
GMOs for the Bioremediation of Pollutants Final Report
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Dr Natasha Smith ADAS
Mr Ben Sykes BBSRC
Dr Phillip White Horticulture Research International, Wellesborne
Dr Andy Whitely CEH, Oxford
Dr Neil Willey Centre for Research in Plant Science, University of the West of England
Dr Peter Williams School of Biological Sciences, University of Wales, Bangor
Dr Luet Wong Dept of Chemistry, University of Oxford
GMOs for the Bioremediation of Pollutants Final Report
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APPENDIX C - WORKSHOP PROGRAMME
---------------------------------------
9.45 Registration
10.20 Introduction to the workshop from project consortium
10.30 Presentation from Ken Timmis
(National Research Centre for Biotechnology, Braunschweig, Germany)
Prospects and challenges for bioremediation with GMMs
11.10 Presentation from Ken Killham
(Department of Plant and Soil Science, University of Aberdeen)
GM biosensors for monitoring contaminant bioavailability and assessing the
potential for bioremediation of contaminated land
11.50 Presentation from Gary Sayler
(Center for Environmental Biotechnology, University of Tennesse, USA)
Field release of P. fluroescens HK44: Long term persistence and field
performance of a bioremediation bioluminescent bioreporter
12.30 summary of morning session
12.45 buffet lunch
13.30 Presentation from Andrew Smith
(Department of Plant Sciences, University of Oxford)
Metal accumulation by plants
14.10 Presentation from Rich Meagher
(Genetics Department, University of Georgia, USA)
Phytoremediation of toxic chemicals in our environment
14.50 Presentation from Neil Bruce
(Institute of Biotechnology, University of Cambridge)
Defusing the environment: engineering transgenic plants to degrade
explosives
15.30 summary of afternoon session
15.45 general discussion on issues raised
16.30 end of meeting
GMOs for the Bioremediation of Pollutants Final Report
AF2032/cdc@@20120 CC 9-196
Word 97\epg 1-5-142 final report.doc

726140@Fax: ]01372] 740055

 

 




Last Updated on Wednesday, 18 June 2014 01:57  

FEATURED VIDEOS, AUDIOS, ARTICLES, EDITORIALS, BLOGS

 



marti.sandi.cssandra3.16.11show
 
 
 
 

martiSHOW3.23.2011

****

*IS THE WORLD BEING  LED TO BELIEVE THAT ONLY "NATURAL" (naturally found in the environment)  BACTERIA AND/OR FUNGUS IS ONLY BEING USED IN MICROBIAL PRODUCTS; BIOPESTICIDES, BIOINSECTICIDES, BIOFUNGICIDES ETC. WHICH ARE BEING USED ON OUR FOOD CROPS, ORNAMENTALS AND/OR USED FOR INSECT CONTROL?   THIS IS NOT ALWAYS THE CASE... "MUTANTS" ARE BEING USED THAT THE PUBLIC IS NOT AWARE OF
Are we being led to believe these microbial products are safe??
*It seems that using "mutants" and mutants created using recombinant techniques. (last 2 patents below) doesn’t qualify the claim of "naturally found" bacteria and fungus which are claimed as being the “active ingredient” in microbial biocontrol products.
*When searching the United States Patent Office for the search terms of “Agraquest” and “mutants” the results were twenty (20) United States patents; that were assigned to Agraquest alone and furthermore does not take into account International patents. These patents uses the wording; mutants, mutants thereof and/or recombinant techniques.  There are many MANY more patents that don't list Agraquest as the holder of the patent; but Agraquest's scientists were listed as "inventors" on other company patents. (these are not listed below)
*The reader is invited; NO... encouraged to view the following twenty (20) Agraquest United States patents and decide for themselves whether we can be 100% certain that only “naturally found in the environment” bacteria and/or fungus is being used on our food crops, ornamentals and/or used for insect control.... OR are mutants being used?

READ MORE HERE


AUDIO:   "Falling To Be Held" fallingTObeHELD

Song: "Control" Listen → HERE

Applicable Lyrics:

You Can’t Control Me
You Know Me
I’ve had it up to here with your lies and your ties
~WHAT ABOUT MY LIFE ?~

PRESENTATION - FRAUD IN THE COURT - COMMITTED BY JUDGE SUZANNE F. DUGAN IN CALIFORNIA WORKERS COMPENSATION SYSTEM; by Sandi Trend

VIDEO - INJURED AGRAQUEST BIOTECH WORKER, DAVID BELL TELLS HIS STORY; by Labor Video Project

VIDEO - Workers Comp, The Destruction Of Ca-OSHA/EPA And The Case Of David Bell; by Labor Video Project

VIDEO - Conflict of Interest By Judge Says Sandi Trend, Mother of Injured Agraquest Biotech Worker Bell; by Labor Video Project

VIDEO - INJURED WORKERS AND ADVOCATES DEMAND THAT INSURANCE COMP FRAUD BE PROSECUTED; by Labor Video Project

VIDEO - 12/10/2009; SANDI TREND, MOTHER OF INJURED BIOTECH WORKER DAVID BELL ASKS CALIFORNIA COMMISSION ON HEALTH AND SAFETY & WORKERS COMPENSATION WHAT "GAMING THE SYSTEM IS?"; by Labor Video Project

VIDEO - Injured Worker, DAVID BELL's Mother Charges Fraud On 9/9/2009 At the California Department of Insurance; Fraud Assessment Comission Meeting; by Labor Video Project

VIDEO - JUNE 17, 2009 SANDI TREND SPEAKS OUT ABOUT FRAUD BY DISTRICT ATTORNEYS AT THE CALIFORNIA DEPARTMENT OF INSURANCE; FRAUD ASSESSMENT COMMISSION; by Labor Video Project

AUDIO - KDRT 95.7 PODCAST of Davis California's Journalist Interview - with David Bell, Doug Haney and Sandi Trend; by Davis, CA Journalist, David Greenwald *Yolo Judicial Watch)

ARTICLE - Biotech Workers Struggle For Safety Measures; by Seth Sandrosky: The Populist

ARTICLE - Biotech canaries - Sickened workers get little relief; by Seth Sandrosky: The Sacramento News & Review

ARTICLE - MARCH-APRIL 2010: COUNCIL FOR RESPONSIBLE GENETICS "GeneWatch MAGAZINE EDITORIAL on David Bell and Agraquest titled; TeaTime In The Lab; by GeneWatch Editor, Sam Anderson

ARTICLE - The Fungus and Bacteria of Deregulation and biotech Worker David Bell; by Steve Zeltzer - LaborNet.org

ARTICLE - Cal-Osha: Going Down The Tubes?; by Larry Rose MD, MPH Cal/OSHA Medical Unit

ARTICLE - The Last Physician/Medical Officer Position is Eliminated at Cal/OSHA; by Larry Rose MD, MPH Cal/OSHA Medical Unit

ARTICLE - Blood, phlegm and tears; by Seth Sandronsky - Sacramento News & Review

ARTICLE - The Criminal Cover-up Of Pam Marrone's Agraquest Operation; www.indybay.org/newsitems Central Valley | Labor & Workers

ARTICLE - Toxic Dump Sites And Agraquest/Pam Marrone Case May Get Light In Davis, California Hearing; by David Greenwald - Central Valley | Environment & Forest Defense | Health, Housing, and Public Services | Labor & Workers

ARTICLE - Local biotech employee says health affected by work.. Officials say no threat to public health; by California Aggie - Oooja Kumar

BLOG - Biotech Worker Safety; by JEEG, The Council for Responsible Genetics - GeneWatch

BLOG - Mother of Injured Biotech Worker Speaks Out On Conflicts Of Interest; by JEEG..."This could prove sufficient evidence to reopen the worker's compensation claim." by Council for Responsible Genetics

BLOG - MAN BECOMES INFECTED WHILE WORKING AT BIOTECH, AGRAQUEST; by WatchDog on Science

BLOG - Did Davis Biotech Firm Expose Davis to Potentially Dangerous Pathogens?; by The People's Vanguard of Davis

BLOG - California Aggie Covers Issue of Agraquest; Yolo County Health Discounts Health Concerns; by The People's Vanguard of Davis

BLOG - The Vanguard's Article on AgraQuest Provokes Strong Response From both County Health Director and Building Owner; by The People's Vanguard of Davis

BLOG - Did Congressman Lungren Ignore Potential National Security Threat Posed By AgraQuest's Imporation of Foreign Soils?; by The People's Vanguard of Davis

 



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