1) Bioremediation uses biological processes to eliminate, attenuate, or transform polluting substances. Traditional techniques like biopiling and landfarming rely on microbial degradation of contaminants in soil. Phytoremediation uses plants and their rhizospheres to uptake or degrade contaminants. 2) Phytobial remediation combines phytoremediation and bioremediation by using microbes like Trichoderma harzianum colonized in plant roots to efficiently degrade toxicants while providing an energy source from plant root exudates. 3) Initial experiments found T. harzianum could detoxify cyanides and metallocyanides in soil, allowing plant

1. Annals of Applied Biology (2005), 146:217–221
*Corresponding Author Email: jim.lynch@forestry.gsi.gov.uk
© 2005 Association of Applied Biologists
217
Bioremediation – prospects for the future application of innovative applied
biological research
JAMES M LYNCH* and ANDREW J MOFFAT
Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK
Summary
It has been estimated that there may be as much as 300 000 ha in the UK where contamination from
previous industrial land use has occurred. The ‘Source-Pathway-Receptor’ model is used to evaluate
these risks. Traditional engineering approaches have dominated remediation technology, but biological
methods have become increasingly important in recent years. ‘Bioremediation’ has been defined as
‘the elimination, attenuation or transformation of polluting or contaminating substances by the use of
biological processes’. Techniques to treat soil materials include biopiling, windrowing, landfarming
and bioventing, all of which depend on microbiological degradation. However, increasingly it also
includes the use of vegetation to take up and/or degrade contaminants (phytoremediation) or restrict
contaminant movement (phytostabilisation). Phytoremediation can be encouraged by manipulation of
the rhizosphere, using selected fungal isolates in a process now defined as phytobial remediation.
Key words: Bioremediation, brownfield, contamination, landfill, phytoremediation, rhizosphere,
trees, Trichoderma
Introduction
As well as the use of the land for agriculture
and forestry, the land surface has had to support
a growing world population for housing, industry
and other needs of the urban infrastructure. Here,
pressures on the soil have been intense, and most
has been destroyed or severely degraded physically
or nutritionally. However, contamination with
inorganic and organic compounds as a result of
industrial (and transport) pollution is probably the
most serious issue to contend with today, because of
the proximity of the urban population and the need
to maintain its health and well being. In addition,
economic pressures are increasingly driving the
need for ‘land recycling’, so that a new use can be
made of old land in advantageous positions within
the town or city. As well as hard-end uses, soft-end
uses involving vegetation are of particular interest
in the urban context (Moffat, 2001), to provide
greenspace for recreation, landscape enhancement
and to encourage inward investment.
Land remediation is an essential response to
the principle of sustainable development. Most
definitions embrace the need to be equitable not
just to people alive today, but to future generations.
So it is important not just to cease or minimise
the degradation of land – in a world of ever-larger
population,landremediationwillbeincreasinglyseen
as an appropriate measure. In developed countries,
remediation activity is currently focussed in urban
areas for reasons given above. What follows is a
brief assessment of the scope for land remediation,
a brief definition of bio/phytoremediation rather than
a review of the subject, and the outline of the novel
concept of phytobial remediation.
Land Remediation in the UK
The UK has a large legacy of land detrimentally
affected by industrial use. In addition, it is one of
the most heavily populated countries in the world,
and a large proportion (15%) of the land is defined
as urban. In the UK, the term ‘brownfield land’
is used as a synonym for ‘previously developed
land’ (Department of Environment, Transport
and the Regions, 2000a), and there is no mention
of potential contamination (in contrast to the US
definition). Nevertheless, it is clear that many UK
brownfield sites have suffered contamination by
previous industrial activity and may be regarded as
‘contaminated land’ (Department of Environment,
Transport and the Regions, 2000b). Some estimates
suggest there may be as much as 300 000 ha in the
UK where contamination from previous industrial
land use has occurred (ENDS, 1999).
In order to raise land quality to meet new
expectations, remediation is increasingly required
on brownfield land. Remediation differs from
‘restoration’ and ‘reclamation’ because it involves
addressing the issue of potential or actual risk of
harm from ‘sources’ of chemical contaminants to
defined‘receptors’. Thesemayincludehumanswho
live on or visit the land, water draining into surface
or groundwaters, or sensitive ecosystems where
species could be lost. Remediation can take several
forms: dealing with the source of contamination,
preventing or breaking the ‘pathway’ between
source and receptor, or removing the sensitive
receptor, either permanently by changing land-use,

2. J M LYNCH & A J MOFFAT218
or temporarily until the degree of contamination
falls below a danger threshold. The source-
pathway-receptor model is now the cornerstone of
British contaminated land remediation policy and
practice.
Until recently, the most popular method of land
remediation was to remove contaminated materials
to licensed landfill premises, i.e. to remove the
‘source’. However, this ‘dig and dump’ procedure
has been criticised as exporting the problem
rather than dealing with it more fundamentally.
Another common approach has been to ‘contain’
the source of the contamination, by sealing it from
receptors using engineered impermeable caps or
walls, or to combine it physically or chemically
to reduce its reactivity or mobility, i.e. break the
pathway. However, these technologies have also
been attacked as taking too short a view of the
likely long term fate of contaminants. Remediation
technologies are increasingly focussing on ways in
which contaminants can be destroyed, or removed
sensitivelywithlowriskofsecondaryenvironmental
problems, taking a holistic and life cycle analysis
approach. In the past, these have been largely
based on physical and chemical technologies.
Bioremediation
‘Bioremediation’ has been defined as ‘the
elimination, attenuation or transformation of
polluting or contaminating substances by the use
of biological processes’ (BBSRC, 1999). The
subject has been a fallow area for the application
of the principles of microbial ecophysiology and
molecular biology. For example, a classic study on
catabolism of the herbicide Dalapon in soil showed
that microbial communities formed to bring about
the catabolism and that catabolic enzymes were
produced across the members of that community
(Senior et al., 1976). This principle probably applies
to the catabolism of many organic pollutants which
enter the soil environment. Similarly, some of the
first studies on soil molecular biology showed how
plasmid DNA segments specified hydrocarbon
degradation as a bioremediation target (Chakrabarty
et al., 1978). A multitude of such studies followed
with the characteristic feature of using pure or
defined mixture culture studies of microorganisms
in vitro. Subsequently some investigators developed
microcosms, which more closely mimicked the
natural environment, but the value of these in
relation to true in vivo applications of bioremediation
has been questioned. In more recent years, there
have been attempts to practice bioremediation in
situ (Crawford & Crawford, 1996). Sometimes
innundative approaches have been used by the
application of microbial cultures to soils and water,
but the common approach has been to elevate the
useful native populations by management of the
ecosystem with such approaches as forced aeration
or nutrient additions. Most commonly the target
has been pollutants such as ‘BTEX’ hydrocarbons,
petroleum, polycyclic aromatic hydrocarbons,
microaromatics, PCBs, chlorinated phenols and
aliphatics. Much less attention has been paid to
metals. Considerable emphasis has been paid
to measurement of bioavailability, and new
chemical and sensor technologies (Valdes, 2000).
Biogeochemists have focussed particularly on
mineral interactions and the remediation of metals,
including radionuclides (Hattori, 2003).
Today, bioremediation technologies such as
windrowing, biopiling, land farming and bioventing
have secured a significant proportion of the market
for remediation. They are mainly concerned
with microbial processes to break down organic
contaminants, and the technologies involve the
provision of ideal environmental conditions for
the growth and activity of the microorganisms
concerned. However, the useful introduced or native
organisms are frequently overtaken or outgrown by
other soil microbes, especially when glucose or other
nutrients are added. This may necessitate their use
in a bioreactor. In addition, introduced organisms
may be pathogenic to plants or produce toxicants
and this seriously limits their usefulness if the land
is to be returned to a soft end-use involving the
establishment of vegetation.
Asasubtypeofbioremediation,‘phytoremediation’
uses living plants to remove, degrade, immobilise
or contain the contaminants in situ (Nathanail &
Bardos, 2004). The greater emphasis has been on
metal as opposed to organic remediation. Reed-bed
systems to clean and polish mine and quarry effluents
are a good example of a phytoremediation system
that has been in operation for several decades. In
more recent times, short rotation coppice systems
of willow or poplar have been investigated and
reviewed (Bardos et al., 2001; Britt & Garstang,
2002) for their potential to take up and remove useful
amounts of some ‘heavy metals’, and other interest
has focussed on so-called ‘hyperaccumulators’such
as some ferns and brassicas. In all these systems,
scientific investigation has proceeded largely in the
ignorance of the plant–microbial interface.
Phytobial Remediation
Phytobial remediation aims to combine
phytoremediation and bioremediation using
microbes (Harman et al., 2004b). Plants are grown
whose roots are colonised by symbiotic microbes
that efficiently create stable microbial communities
that degrade toxicants and assist plants in taking up
toxic materials. It thus combines the best of both
traditional bio- and phyto-remediation. One of the

3. 219Bioremediation
potential reasons why bioremediation fails is that
the microbes bringing about the remediation do not
have an energy source to promote their growth other
than the toxicant which in itself may not be an energy
source but instead be a transformation substrate.
The whole concept of the phytobial process is that
it utilises the rhizodeposition products of the plant
root as the energy source for the microorganisms to
function. In quantitative terms this can account for
up to 40% of the photosynthate produced or the dry
matter production by the plant (Lynch & Whipps,
1990). In order to make maximum use of that carbon
and energy pool the microorganisms need to be
rhizosphere competent. Symbionts such as rhizobia
and mycorrhiza are the best illustrations, but other
free-living saprophytic organisms can also form
stable associations with roots which approach the
symbiotic relationship. One such organism which
has been identified by several research groups as
satisfying the conditions of rhizosphere competence
is Trichoderma harzianum. However, there are
only specific strains which exhibit this quality
and some of them have been developed using the
technique of protoplast fusion. One such strain of T.
harzianum is T22 which has already been exploited
as a biocontrol agent against root diseases (Harman
et al., 2004a). During the registration process for
that product developed by a spin-out company
from Cornell University known as Bioworks, the
toxicology and pathogenicity was evaluated and
was shown to have no deleterious effects on plants,
animals (including humans) or to the environment
if used at recommended rates. It therefore has full
clearance of the regulatory authority in the USA,
the Environmental Protection Agency (EPA). It
is rhizosphere competent, stable and robust. It is
effective on all plants tested, ranging from ferns to
conifers, monocots to dicots. It is effective across
a wide range of soil pH (acidic to alkaline) and soil
conditions (sandy to organic to gravel to very fine
soils). The use of its effectiveness in biocontrol
improves its competitive ability in the rhizosphere
and its robustness. It has also been through an
extensive process of production and deliverance
systems which give the product a long shelf life for
which a very satisfactory quality control procedure
has been developed. An added advantage of this
strain of the fungus is that it is one of the few strains
of Trichoderma which increase seedling vigour
and translate to an improvement of plant growth
by as much as 30% (Lynch, 2004) (Fig. 1). It also
increases deep rooting long after application.
The phytobial remediation concept is of course not
limited to Trichoderma spp. As already mentioned
we can expect the symbiotic groups to be within the
remit of phytobial remediation. However, there may
be other organisms which could enhance the activity
of the small defined microbial communities which
can develop in such rhizosphere ecosystems.
As an initial target for the catabolic potential of
Trichoderma spp. in remediation, cyanides and
metallocyanides, which are produced by industrial
sources, were studied. These toxicants can come
from the electroplating industries, manufactured gas
plants, recovery of precious metals, synthetic fibre
production, processing of cyanogenic crops and
paint manufacturing industries. It was found that
certain strains were both resistant to cyanide and
could in fact detoxify it. They could be resistant to
as much as 2000 ppm cyanides and could catabolise
the cyanide utilising its formamide hydrolase or its
rhodanese. The characterisation of the enzymes
involved has been published in two papers (Ezzi
& Lynch, 2002, 2003). The microbiology of the
decomposition process has also been reported (Ezzi
& Lynch, 2004). In some hitherto unpublished
observations by M Ezzi and J M Lynch, soil was
treated with 50 or 100 ppm cyanide and it was found
that wheat and pea seeds failed to germinate. When
the seeds were coated with T. harzianum the seeds
germinated and the plants grew the same extent as
in untreated soil (Fig. 2). This clearly showed the
capability of the detoxification by the phytobial
Fig. 1. Stimulation of lettuce growth by Trichoderma
harzianum (WT) compared to untreated control
(cont).
Fig. 2. Detoxification of potassium cyanide by
Trichoderma harzianum. A, Uninoculated with
50 ppm CN; B, Uninoculated with 0 ppm CN; C,
Inoculated with 50 ppm CN; D, Inoculated with 100
ppm CN.
A B C D

4. J M LYNCH & A J MOFFAT220
process of the potassium cyanide added to the
system. It was also demonstrated by D Redmond
and J M Lynch (unpublished), that Trichoderma
grown in a solution of Prussian blue (a classic
example of a metallocyanide which could enter the
ecosystem) could be rapidly absorbed by the fungus
and spread throughout the cytoplasm with eventual
detoxification using the cyanide metabolising
enzymes.
In some recent unpublished experiments at Cornell
University (G Harman), it has also been shown that
the arsenic hyperaccumulating fern Pteris vittata
inoculated with T22 have larger roots and more
root hairs than non-treated plants. They also take
up more heavy metals and arsenic, and for arsenic
there is 140% increase in uptake. In other work it
has also been shown that the uptake of nitrates can
be promoted by the phytobial system which could
be valuable in land where nitrate leaching is a
problem. In studies at the University of Naples by M
Lorito (unpublished), it has been demonstrated that
Trichoderma can remove phenolic pollutants from
olive oil waste water.
These results suggest a potential opportunity to
use the phytobials technology to help remediate both
inorganic and organically contaminated materials,
though more work is obviously needed to extend
the range of contaminants tested, especially more
complex organics.
In the most recent work by P Adams, F De
Leij and J M Lynch (unpublished) that has been
developing between the University of Surrey and
Forest Research atAlice Holt Research Station using
willow, the Trichoderma has given a substantial, up
to 30%, increase in tree growth. We would expect
the increased tree growth to contribute to the land
remediation qualities of the tree. In the work on
brownfield sites such as former landfills where trees
have been planted generating the concept of urban
and peri-urban forestry, this quality would seem to
be eminently exploitable.
Whereas the initial description and illustration
of the phytobial remediation concept has used
a rhizosphere-competent free-living fungus
(Trichoderma harzianum), there are likely to be
applications with symbiotic fungi (mycorrhizae).
In this respect it is interesting to note that the
endophytic arbuscular mycorrhizae show potential
in bioremediation (Oliveira et al., 2001; Gonzalez-
Chavez et al., 2002a,b). With trees, it might be
expected that ectotrophic mycorrhizae would also
be useful.
Discussion
Opportunities for further development and use
of phytobial remediation technologies are likely
to increase in the future. There is already a move
towards forms of remediation that themselves
pose a small risk of environmental damage,
and this favours bioremediation over chemical
technologies. One traditional method of dealing
with contamination, the ‘dig and dump’ approach,
is likely to be reduced significantly in the UK as
a result of recent changes in landfill legislation
following the EU Landfill Directive (1999/31/EC)
which has reduced the number of British licensed
landfills permitted to accept hazardous material
to a very small number. Nevertheless, in situ
bioremediation technologies remain unproven
in some quarters, partly because there remains
uncertainty over the degree of homogeneity
achieved by bioremediation treatment and thus
residual risk. Bioremediation requires that the soil
conditions (chemical, physical and biological) are
above a minimum threshold to support the chosen
vegetation and desired microbial communities,
and this is not always easily achievable across the
whole site. In addition, there is the perception
that working with plants, remediation can only be
achieved to the depth of effective rooting, and thus
to about 1 m in many circumstances. However,
certain tree species may permit greater depths to
be remediated.
Further research, both fundamental and applied,
is therefore necessary. Microbiological ecological
research is needed to understand more fully the
ability of microbes to degrade toxicants within the
rhizosphere, and when, how and over what time
periodssuchprocessestakeplace. Thedevelopment
of further rhizosphere competent symbionts, and
their testing on a range of host plants and types of
contaminated substrate is necessary to extend the
usefulness of this technology and help to define the
appropriateness of its use. The ability of phytobial
systems to degrade complex organic compounds
needs to be established, given that industrially
contaminated land usually contains a large range.
Modelling phytobial systems may be an important
way of exploring their further potential, once more
basic ecological research has been undertaken.
Applied research is required to establish how
effective phytobial systems are in remediation at
field scale, and what the operational constraints are.
To what extent is phytobial remediation likely to
rely on its use in conjunction with more established
clean up technologies? Comparison with traditional
remediation approaches should be undertaken
to establish relative clean up effectiveness and
economic cost. Such work would benefit from
involvement with waste regulatory authorities
in order to maximise the confidence with which
these novel technologies are viewed. Phytobial
remediation should also be placed in the context of
the urban greenspace agenda. To what extent can
this type of remediation be used to create soft-end

5. 221Bioremediation
uses that can be used safely by the public, and could
it help to advance the time when public access is
permitted? In fact, can it be used for remediation
at the same time as public access is allowed? And
will the public be content, or even supportive of
bioremediation techniques over more traditional
ones which have a larger ecological footprint?
From a multitude of communication networks
it is clear that the feasibility of bioremediation of
contaminated land has been established. Reliability,
however, is more questionable and in addition the
magnitude of the remediation needs to be fully
evaluated. There is a massive impetus to develop
these concepts further.
This paper has focussed on our own research
activity. The various approaches to bioremediation
can be accessed through a variety of texts (e.g.
Crawford & Crawford, 1996; Valdes, 2000; Hattori,
2003). In addition, following an excellent EU
InterCOST meeting on bioremediation in Sorrento,
Italy in November 2000, a selection of the presented
papers was published in Minerva Biotechnology
(Journal of Biotechnology and Molecular Biology)
Vol. 13, pp. 1–152.
Acknowledgements
We are grateful for inputs to this work from
Gary Hartman (Cornell University), Matteo Lorito
(University of Naples), Muffadel Ezzi, PaulAdams,
David Redmond and Frans De Leij (University of
Surrey) and Tony Hutchings (Forest Research).
References
Bardos P, French C, Lewis A, Moffat A, Nortcliff S. 2001.
Marginal Land Restoration Scoping Study: Information
Review and Feasibility Study. exSite Research Project Report
1. Nottingham: LQM Press.
BBSRC. 1999. A Joint Research Council Review of
Bioremediation Research in the United Kingdom. Swindon:
BBRRC, EPSRC and NERC.
BrittC,GarstangJ.2002. BioenergyCropsandBioremediation
– a Review. Final Contract Report to Defra. http://www.defra.
gov.uk/farm/acu/research/reports/NF0417.pdf
ChakrabartyAM, Friello DA, Bopp LH. 1978. Transposition
of plasmid DNA segments specifying hydrocarbon
degradation and their expression in various microorganisms.
Proceedings of the National Academy of Sciences of the USA
75:3109–3112.
Crawford R L, Crawford D L. (Eds) 1996. Bioremediation
Principles and Applications. Cambridge: Cambridge
University Press.
Department of Environment, Transport and the Regions.
2000a. Housing. Planning Policy Guidance Note 3. London:
The Stationery Office.
Department of Environment, Transport and the Regions.
2000b. Contaminated Land: Implementation of Part IIA
of the Environmental Protection Act 1990. DETR Circular
02/2000. London: The Stationery Office.
ENDS. 1999. April targets for new contaminated land regime in
England. ENDS Report 297:45–48. London: Environmental
Data Services.
Ezzi M I, Lynch J M. 2002. Cyanide catabolising enzymes
in Trichoderma spp. Enzyme and Microbial Technology
31:1042–1047.
Ezzi M I, Pascual J A, Gould B J, Lynch J M. 2003.
Characterisation of the rhodanese enzyme in Trichoderma
spp. Enzyme and Microbial Technology 32:629–634.
Ezzi M I, Lynch J M. 2004. Biodegradation of cyanide by
Trichoderma spp. and Fusarium spp. Enzyme and Microbial
Technology (In Press).
Gonzalez-Chavez C, D’Haen J, Vangronsveld J, Dodd J C.
2002a. Copper sorption and accumulation by the extraradical
mycelium of different Glomus spp. (arbuscular mycorrhizal
fungi) isolated from the same polluted soil. Plant and Soil
240:287–297.
Gonzalez-Chavez C, Harris P J, Dodd J, Meharg A A.
2002b. Arbuscular mycorrhizal fungi confer enhanced
arsenate resistance on Holcus lanatus. New Phytologist
155:163–171.
Harman G E, Howell C R, Viterbo A, Chet I, Lorito M.
2004a. Trichoderma species – opportunistic, avirulent plant
symbionts. Nature Reviews Microbiology 2:43–56.
Harman G E, Lorito M, Lynch J M. 2004b. Uses of
Trichoderma spp. to alleviate soil or water pollution.
Advances in Applied Microbiology 56:313–330.
Hattori T. (Ed.) 2003. Biogeochemical Aspects of Earth
System and Bioremediation of Polluted Environments. 16th
International Symposium on Environmental Biogeochemistry,
Sendai, Japan.
Lynch J M. 2004. Plant growth-promoting agents. In Microbial
Diversity and Bioprospecting, pp. 391–396. Ed. A T Bull.
Washington DC: ASM Press.
Lynch J M, Whipps J M. 1990. Substrate flow in the
rhizosphere. Plant and Soil 129:1–10.
Lynch J M, Wilson K L, Ousley M A, Whipps J M. 1991.
Response of lettuce to Trichoderma treatment. Letters in
Applied Microbiology 12:59–61.
Moffat A J. 2001. Increasing woodland in urban areas in
the UK – meeting ecological and environmental standards.
International Forestry Review 3:198–205.
NathanailCP,BardosRP.2004. ReclamationofContaminated
Land. Chichester: J Wiley.
Oliveira R S, Dodd J C, Castro PM L. 2001. The mycorrrhizal
status of Phragmites australis in several polluted soils and
sediments of an industrialised region of Northern Portugal.
Mycorrhiza 10:241–247.
Senior E, Bull A T, Slater J H. 1976. Enzyme evolution in
a microbial community growing on the herbicide Dalapon.
Nature, London 263:476–479.
Valdes J J. (Ed.) 2000. Bioremediation. Dordrecht: Kluwer.
(Revised version accepted 3 February 2005;
Received 10 September 2004)