Bioremediation of polynitrated aromatic compounds:
plants and microbes put up a fight
Juan L Ramos, M Mar Gonzalez-Perez, Antonio Caballero and
Pieter van Dillewijn
Industrialization and the quest for a more comfortable
lifestyle have led to increasing amounts of pollution in the
environment. To address this problem, several
biotechnological applications aimed at removing this pollution
have been investigated. Among these pollutants are xenobiotic
compounds such as polynitroaromatic compounds
recalcitrant chemicals that are degraded slowly. Whereas
2,4,6-trinitrophenol (TNP) can be mineralized and converted
into carbon dioxide, nitrite and water, 2,4,6-trinitrotoluene
(TNT) is more recalcitrant although several microbes can use
it as a nitrogen source. The most effective in situ biotreatments
for TNT are the use of bioslurry (which can be preceded by
an abiotic step) and phytoremediation. Phytoremediation
can be enhanced by using transgenic plants alone or together
with microbes.
Addresses
Estacion Experimental del Zaidn, Consejo Superior de Investigaciones
Cientficas, Apdo Correos 419, E-18008 Granada, Spain
Corresponding author: Ramos, Juan L (jlramos@eez.csic.es)
Current Opinion in Biotechnology 2005, 16:275281
This review comes from a themed issue on
Environmental biotechnology
Edited by Gerben J Zylstra and Jerome J Kukor
Available online 8th April 2005
0958-1669/$ see front matter
# 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2005.03.010
Introduction
Since the end of the nineteenth century humans have
developed a series of technologies that have brought
increasing comfort to industrialized societies. A side
effect of the development of new materials during this
industrialization has been the generation of wastes, with
their consequent impact on the biosphere. When wastes
are degraded at a slower rate than their appearance in the
biosphere they accumulate and become what we call a
pollutant, constituting a burden to the environment.
Among these wastes, xenobiotic compounds, which are
chemicals with structures or substituents that are rarely
found in natural products, represent a serious problem as
they are seldom mineralized by living organisms and are
often toxic.
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Nitroaromatic compounds are true xenobiotics. Although
nitroaromatics with few substituents such as mononitro-
toluenes, nitrobenzoates and nitrophenols, are easily
degraded by ubiquitous microbes, polysubstituted
nitroaromatic compounds are more difficult to degrade
[1]. Furthermore, when microbes able to deal with these
chemicals are isolated they degrade them at a very slow
rate. This slow rate of degradation can result from the
intrinsic toxicity of the compounds, as well as from their
limited solubility.
Vast sites worldwide are contaminated with 2,4,6-
trinitrotoluene (TNT) and 2-4-6-trinitrophenol (TNP),
because of the large-scale manufacture of these com-
pounds. Bioremediation is an attractive means for decon-
taminating these sites. Several microorganisms have
recently been isolated as able to use TNT as the sole
nitrogen source; however, very few cases of mineraliza-
tion have been reported [2,3]. Yet, several species of
Actinomicetales have been found to mineralize TNP
[46,7,8,9]. These findings have prompted the search
for enzymes capable of putting up a fight against poly-
nitrated aromatic compounds such as TNT and TNP. In
this review we have analyzed recent advances in the
removal of polynitroaromatic chemicals by microbes,
plants and plantmicrobe associations.
Enzymes involved in TNT and TNP
degradation
Although there are currently no known pathways for the
complete catabolism of TNT [2], this compound can be
transformed into diverse products in a wide range of
microorganisms by a variety of enzymes. Increased inter-
est has been bestowed on the enzymes involved in TNT
degradation: the best known of these enzymes are flavo-
nitroreductases belonging to the b/a old yellow enzyme
(OYE) family, such as XenA and XenB in Pseudomonas,
pentaerythritol tetranitrate (PETN) reductase in Enter-
obacter cloacae, NemA in Escherichia coli, morphinone
reductase in Pseudomonas putida M10, and OYE in Sac-
charomyces cerevisie [10,11,1216].
The OYE family of enzymes attacks TNT using either
one or both of two possible pathways (Figure 1): the
sequential reduction of the nitro groups to hydroxylamine
and amine derivatives (which can be catalyzed by any of
the six enzymes mentioned above) or the reduction of the
aromatic ring by hydride addition, with the subsequent
Current Opinion in Biotechnology 2005, 16:275281
276 Environmental biotechnology
Figure 1
CH3
NO2 NO2
H ?
A H
NO2
CH3
2
NO2 NO2
CH3
NO2 NO2
1 B
NHOH
3 NO2
Initial TNT (compound 1) degradation pathways. A: Reduction of the aromatic ring by OYE-type enzymes via a Meisenheimer complex (compound 2)
to unresolved products with the release of nitrite. B: Common reduction pathway of a nitro group to a hydroxylamine derivative (compound 3).
This product could be further reduced to amino derivatives (compound 4) or transformed to as yet unknown products, releasing ammonium
in the process.
release of nitrite (catalyzed only by XenB, NemA and
PETN reductase).
The mechanism of hydride addition to the nitroarene
aromatic ring has been studied with the PETN reductase
of E. cloacae by Khan and coworkers [11,12]. They
showed that in the reductive half-reaction of PETN
reductase NADPH binds to form an enzymeNADPH
charge transfer intermediate before hydride transfer from
the nicotinamide coenzyme to flavin mononucleotide.
Oxidation of the flavin by the nitroaromatic substrate
TNT is kinetically indistinguishable from the formation
of its hydrideMeisenheimer complex. This is consistent
with a mechanism involving a direct nucleophilic attack
by hydride from the flavin N5 atom on the electron-
deficient aromatic nucleus of the substrate. This was
further corroborated when the crystal structures of com-
plexes of the oxidized enzyme bound to TNT were
solved [12]. The hydrideMeisenheimer complex then
breaks down to form alternative products. The chemical
identities of these products are uncertain, but have been
studied in reactions catalyzed by PETN reductase of E.
cloacae and XenB from P. fluorescens [15]. It is believed that
nitrite is released in this process. The utilization of nitrite
by microbes requires further reduction to ammonium. In
agreement with this observation, several microbes that
use TNT as a nitrogen source transitorily accumulate
nitrite in the culture medium, which upon induction of
nitrite reductase can be used as a nitrogen source [17].
Current Opinion in Biotechnology 2005, 16:275281
NH4+ Incorporation into
NO2
carbon skeletons
NO2
?
CH3
NO2
NH2
4
Current Opinion in Biotechnology
All of the enzymes described above, as well as many other
nitroreductases, are able to carry out a different reaction:
the reduction of one or more nitro groups of TNT to its
hydroxylamine moiety. These derivatives tend to release
ammonium from the aromatic ring through an unknown
mechanism that could involve a Bamberger-like rearran-
gement [18]. The E. coli NfsA and NfsB flavin proteins,
which use NADPH and NADH, respectively, as a source
of reducing equivalents, and PnrA in P. putida are among
the nitroreductases that carry out this type of reaction
[1923]. These enzymes reduce TNT in addition to a
variety of structurally diverse nitroaromatic compounds,
including nitrofurans and nitroimidazoles [1921].
Ammonium released from the TNT ring can be used
as a nitrogen source by P. putida and E. coli via the
glutamine synthetase/glutamate synthase pathway [17].
Watrous et al. [24] identified a new enzyme, an Fe-only
hydrogenase, involved in TNT metabolism in the strict
anaerobe Clostridium acetobotulinicum. This enzyme cata-
lyzes the H2-dependent reduction of the nitro group of
TNT to the corresponding hydroxylamine in an acidogenic
environment. This finding is relevant not only because it
expands the range of enzymes capable of attacking TNT,
but because it also allows us to envisage biotreatments
using microbes that colonize different niches.
TNP, also known as picric acid, is structurally similar to
TNT and can be mineralized. Several bacteria belonging
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 Bioremediation of polynitrated aromatic compounds Ramos et al. 277

Figure 2

(a)

orfA orfB npdC orfD orfE npdF npdR npdG npdH npdI orfJ orfK

(b) O O O O
NO 2 NO 2 NO 2 NO 2 NO2 NO 2 NO 2 NO 2

HTII-NDFR HTI-NDFR Tautomerase

NO 2 NO 2 N+ H OH
O OH
1 2 3a 3b

NO2 Denitrase

O O
NO2 NO2

HTII-NDFR
NO2 NO2

4 5

HTI-NDFR

O
NO 2 NO2
NO 2
H
HOOC Hydrolase
H NO2
7 6
Current Opinion in Biotechnology

Gene cluster and early reactions in 2,4,6-trinitrophenol (TNP) metabolism. (a) The npd gene cluster of R. opacus HL PM-1 showing the proteins
that have been functionally identified: npdC, hydride transferase I (HTI); npdF, hydrolase; npdR, regulator; npdG, NADPH-dependent F420
reductase (NDFR); npdH, tautomerase; npdI, hydride transferase II (HTII). (b) The degradation pathway of TNP: 1, TNP; 2, H -TNP; 3a, aci-nitro
form of 2H-TNP; 3b, nitro form of 2H -TNP; 4, dinitrophenol (DNP); 5, H -DNP; 6, 2,4-dinitrocyclohexanone (2,4-DNCH); 7, 4,6-dinitrohexanoate
(4,6-DNH). (Figure modified from [5].).

to the genera Rhodococcus and Nocardioides grow aerobi-
cally on TNP or dinitrophenol and utilize these com-
pounds as their sole nitrogen, carbon and energy sources
[46,7,8,25,26].
The genes and enzymes involved in TNP degradation
have been characterized [5,7,9,27]. The TNP degrada-
tion genes of Rhodococcus opacus HL PM-1 were identified
using a messenger RNA differential display system [9].
Subsequently, the genes that encode the earlier steps of
TNP degradation were located in a cluster (Figure 2).
The npdI gene encodes hydride transferase II (HTII),
which transfers the first hydride to TNP; npdC encodes
hydride transferase I (HTI), which catalyzes the second
hydride transfer giving rise to 2H -TNP (Figure 2). Both
enzymes require an NADPH-dependent F420 reductase
(NDFR) encoded by npdG to supply the hydride ions in
the form of F420H2. The npdH gene encodes a tauto-
merase that catalyzes a proton shift between the acid
nitro and the nitro forms of the dihydrideMeisenheimer
complex of TNP, whereas npdF encodes a hydrolase that
converts 2,4-dinitrocyclohexanone (2,4-DNCH) into 4,6-
dinitrohexanoate (4,6-DNH) [5]. HTI, HTII and NDFR
have low activity with TNT and can produce 2H -TNT.
The tautomerase is able to convert 2H -TNT to differ-
ent tautomeric forms, but the nitrite-releasing enzyme
cannot apparently denitrate the complex and thus 2H -
TNT accumulates as a dead-end product in R. opacus.
The TNP degradation gene cluster (Figure 2) also con-
tains a transcriptional regulator, NpdR. In an npdR dele-
tion mutant, high activity was found for HTII and HTI
irrespective of whether they had been previously induced
[7]. Furthermore, in the wild-type background NpdR
represses the expression of npdI and npdC in R. opacus HL
PM-1, as confirmed by in vitro gel shift assays.

www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:275281

278 Environmental biotechnology
Biological treatments of TNT
Biological field treatments of TNT include composting,
bioslurry processes and phytoremediation [26,2831].
Composting and bioslurry treatments involve co-
metabolic processes that are based on the feasibility of
the reduction of the nitro groups of TNT by microorgan-
isms [30]. The final outcome of these processes is that
hydroxylamine and amine groups on the nitroarene ring
react with quinones and carbonyl groups of the humic
fraction of the soil, giving rise to immobilized TNT
derivatives that are not bioavailable and thus exhibit
decreased toxicity [30].
In composting, the soil is mixed with alternative degrad-
able organic material that is used by the microorganisms
in the soil to transform TNT [29]. The best conditions for
composting are produced in windrow composting, using
alternate anaerobic and aerobic phases. The first anoxic
step leads to the rapid reduction of TNT and condensa-
tion of the amine derivatives to the humic material. In the
second phase, the products from the anaerobic treatment
are metabolized by soil microbes to non-toxic products.
Bioslurry processes involve the mixture of contaminated
material with water and nutrients [26]. Bioslurry treat-
ment is faster than composting and a high rate of TNT
reduction can be achieved. A field-scale process, called
sequential anaerobic bioremediation, uses as combination
of anaerobic and aerobic steps. Using this technique with
14
C-labelled TNT no 14CO2 release was detected, but
15
N nuclear magnetic resonance results indicated that
TNT reduction products were eventually incorporated as
part of the soil material [29,3236]. Recently, Weiss and
co-workers [35] have studied the cleavage of 15NC
bound in soil. In addition to the production of 15NH4+
and 15NO2 , they also detected 15N2O. This finding
suggested the possible slow metabolism of the immobi-
lized products. In both types of treatment, extensive
monitoring and a final evaluation of remediation effi-
ciency are necessary for environmental safety considera-
tions [37].
Because of the limitations of the processes described
above, Schrader and Hess [38] proposed a combined
system consisting of a fast abiotic pre-treatment with high
concentrations of hydroxyl radicals followed by a bioslurry
treatment to degrade the products from the first step.
An advantage of this procedure is that TNT products,
instead of being immobilized, are mostly (97%) miner-
alized. Nonetheless, the abiotic step could lead to a
drastic reduction in the microbial populations, which
may affect the second step in some cases.
Phytoremediation, in which plants are used to remove
TNT from the soil, is considered a cost-effective alter-
native to the biological systems mentioned above
[28,39,40]. Implementation of phytoremediation techni-
Current Opinion in Biotechnology 2005, 16:275281
ques requires an understanding of how the plant interacts
with TNT and how much of the contaminant is tolerated.
Plant tolerance to TNT depends on the species, the
growth stage of the plant, TNT bioavailability, and soil
characteristics [28,39,4143]. For instance, germinating
seeds, seedlings or mature plants of the same species
tolerate different concentrations of TNT. When TNT is
bioavailable at higher levels, for example under aqueous
conditions, the plant tends to tolerate lower concentra-
tions of the xenobiotic than in soils where bioavailability
is more restricted. In general terms, aquatic and wetland
plants show growth inhibition and chlorosis (loss of chlor-
ophyll) at concentrations ranging from 15 mg TNT/L,
whereas in soil most plants can withstand between 50 mg
and 100 mg TNT/kg and some even up to 1600 mg TNT/
kg soil. However, the mechanisms of TNT toxicity
remain unknown.
Plants seem to deal with TNT as though they were a
green liver, whereby the contaminant is detoxified via
the chemical transformation of TNT, conjugated to plant
metabolites, and sequestered within plant tissues or poly-
mers rather than being mineralized to carbon dioxide and
nitrogen [28,40]. TNT can thus be transformed by reduc-
tive and oxidative processes in the plant. A wide range of
reduction derivatives (e.g. 2-amino-4,6-dinitrotoluene, 4-
amino-2,6-dinitrotoluene, 2-hydroxylamino-4,6-dinitro-
toluene and 4-hydroxylamino-2,6-dinitrotoluene) have
been found in several plant species [42]. Most of the
amino-dinitrotoluenes remain in the roots where their
concentrations usually exceed those of TNT, whereas the
TNT derivatives accumulate to a lesser extent in the
stem and leaves. In the aquatic plant Myriophyllum aqua-
ticum, TNT oxidation products have been found (e.g. 2-
amino-4,6-dinitrobenzoate, 2-N-acetoxyamino-4,6-dini-
trobenzaldehyde, 2,4-dinitro-6-hydroxybenzyl alcohol
and 2,4-dinitro-6-hydroxytoluene). The last two of these
products may be the result of ring hydroxylation together
with the removal of a nitro group. This is of interest
because derivatives with fewer nitro groups are more
susceptible to microbial degradation [44].
TNT metabolites are subsequently conjugated with
plant-derived glucose, malonate or glutathione. The nat-
ure of the intermediates changes with time into unknown
non-extractable bound material, suggesting that conjuga-
tion acts as an intermediate step between transformation
and sequestration. Sens et al. [45] reported for wheat that
43% of TNT and its derivatives were found in the
cytoplasm, whereas the remaining 57% was present in
the cell-wall fraction. Of the TNT in this fraction, 27%
was associated to lignin.
Transgenic plants bearing bacterial nitroreductases have
been shown to exhibit increased tolerance to TNT
(Figure 3). French et al. [46] and Hannink et al. [40]
obtained transgenic tobacco plants that expressed the
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 Bioremediation of polynitrated aromatic compounds Ramos et al. 279
Figure 3
Transgenic aspen hybrid exhibiting high resistance to TNT in soil
with this xenobiotic. (Figure courtesy of P van Dillewijn and
colleagues).
PETN reductase gene (onr) or the nitroreductase gene
(nfs) from E. cloacae, respectively. In wild-type plants,
growth was inhibited at 0.025 mM TNT, whereas onr-
tobacco lines germinated and grew normally at 0.05 mM
TNT. Although these transgenic plants failed to grow in
media containing 0.5 mM TNT, nfs lines germinated well
at this concentration and removed TNT from hydroponic
media faster than wild-type plants.
Recent work by Ekman et al. [47] described differential
gene expression in Arabidopdsis roots in the presence of
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TNT. Their results support the green liver hypothesis, as
among the enzymes encoded by upregulated genes were
P450 cytochromes, which oxidize many xenobiotics and
might have a role in TNT metabolism. Moreover, other
upregulated proteins included glutathione S-transferases,
which may be involved in the conjugation of these
transformation products. The authors also observed
increased expression of the genes involved in the plant
response to oxidative stress. Induction of these genes was
also observed in response to TNT in microarrays covering
about 30% of the Chlamydomonas reinhardii genome [48],
which suggests that at least part of the toxicity of TNT is
derived from oxidative stress.
Conclusions and future perspectives
The biochemical mechanisms underlying the different
modes of attack on TNT are of great interest. Very little,
if anything, is known about the reactions that lead to the
removal of the nitro groups by denitrases, and knowledge
acquisition is essential for the different reductive pro-
cesses in the aromatic ring, reduction of the carbon
skeleton or nitro groups. Further characterization of deni-
trases and their interactions with TNT should shed light
on these interesting reaction patterns.
Traditional methods to detect TNT and TNP are based
on chemical analyses; however, alternative techniques
based on immunochemical reactions (immunosensors)
or living organisms (biosensors) are being developed.
Immunosensors of TNT based on anti-TNT monoclonal
antibodies provide considerable sensitivity and selectiv-
ity for the development of field-portable systems [49].
Biosensors can be engineered by combining regulators
that recognize TNT and promoters fused to luciferase
(lux) genes.
Although research efforts regarding issues related to mass
balance in the case of TNT are desirable, new metage-
nomic approaches will aid the identification of new
enzymes with activity against nitroaromatic compounds.
These methods require a host strain free of enzymatic
activities against TNT. Such hosts are now available in
our laboratory and will be useful in exploring a variety of
niches to find new enzymes, which in addition to their
applications in the removal of explosives may be of
potential pharmaceutical interest.
Some concerns remain that need to be addressed to make
the phytoremediation of TNT more effective. The toxi-
city of sequestered TNT derivatives and the recalci-
trance of these compounds in the environment remain
unknown. Another major concern is bioavailability: if the
plants do not come into contact with the contaminant,
because it is tightly bound to the soil, their potential for
phytoremediation will be restricted. It is likely that
combined treatments with microbes and plants will over-
come the limitations of phytoremediation [50].
Current Opinion in Biotechnology 2005, 16:275281
280 Environmental biotechnology
Update
A number of recent articles have investigated the phytor-
emediation of TNT. Among these, one article used
tobacco cell suspension cultures and the identification of
conjugates of sugars with hydroxylaminodinitrotoluene
isomers to reveal a role for glycosyltransferases in TNT
phytoremediation processes [51]. In a very different study,
rotation of salicaceae and conifer trees was proposed to be
used for efficient phytoremediation [52]. Robertson and
Jjemba [53] reported that a bacterial consortium estab-
lished in soils polluted with TNT was able to mineralize
almost 50% of 14C-TNT, and identified a strain of the
genus Enterobacter as one of the key microbes in the
mineralization of TNT. This report shows promising
advances in the removal of this pollutant. Among microbes,
a marine yeast was found to be capable of removing nitro
groups from TNT and yielded 2,4-dinitrotoluene in a
process in which a hydride-Meisenheimer complex was
identified as a potential intermediate [54].
Acknowledgements
Work in the authors laboratory was supported by a grant from the
European Commission (MADOX, QLRT-2001-00345) and Ministerio de
Medio Ambiente (Ref. 059/2004/3). We thank Carmen Lorente for
secretarial assistance and Karen Shashok for improving the use of English
in the manuscript.
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 Bioremediation of polynitrated aromatic compounds Ramos et al. 281
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