Appl Microbiol Biotechnol (2001) 56:8187

DOI 10.1007/s002530000587

MINI-REVIEW

G. McMullan C. Meehan A. Conneely N. Kirby

T. Robinson P. Nigam I. M. Banat R. Marchant
W. F. Smyth

Microbial decolourisation and degradation of textile dyes

Received: 12 October 2000 / Received revision: 22 November 2000 / Accepted: 24 November 2000 / Published online: 19 May 2001
Springer-Verlag 2001

Abstract Dyes and dyestuffs find use in a wide range of

industries but are of primary importance to textile manufacturing. Wastewater from the textile industry can contain a variety of polluting substances including dyes. Increasingly, environmental legislation is being imposed to
control the release of dyes, in particular azo-based compounds, into the environment. The ability of microorganisms to decolourise and metabolise dyes has long been
known, and the use of bioremediation based technologies
for treating textile wastewater has attracted interest.
Within this review, we investigate the mechanisms by
which diverse categories of microorganisms, such as the
white-rot fungi and anaerobic bacterial consortia, bring
about the degradation of dyestuffs.

Introduction
Dyes and dyestuffs are widely used within the food,
pharmaceutical, cosmetic, textile and leather industries.
Over 100,000 commercially available dyes exist and
more than 7105 tonnes of dyestuff are produced annually (Meyer 1981; Zollinger 1987). The human health
impact of dyes used in the food industry, especially
azo dyes and their degradation products, has caused concern for a number of years, with legislation controlling
their use being developed in a variety of countries
(Hildenbrand et al. 1999). Increasingly, the environmental and subsequent health effects of dyes released in
textile industry wastewater are becoming subject to
scientific scrutiny. Wastewater from the textile industry
is a complex mixture of many polluting substances rangG. McMullan () C. Meehan A. Conneely N. Kirby
I.M. Banat R. Marchant
School of Environmental Studies, University of Ulster, Coleraine,
County Londonderry, BT52 1SA, UK
e-mail: g.mcmullan@ulst.ac.uk
Tel.: +44-28-70324755, Fax: +44-28-70324911
T. Robinson P. Nigam W.F. Smyth
School of Biomedical Sciences, University of Ulster, Coleraine,
County Londonderry, BT52 1SA, UK

ing from organochlorine-based pesticides to heavy

metals associated with dyes and the dyeing process
(Correia et al. 1994).
During textile processing, inefficiencies in dyeing result in large amounts of the dyestuff being directly lost to
the wastewater, which ultimately finds its way into the
environment. The amount of dye lost is dependent upon
the class of dye application used, varying from only 2%
loss when using basic dyes to a 50% loss when certain
reactive dyes are used (for a comprehensive review of
this area, the types of dyes found in textile effluent and
colour discharge consent limits see ONeill et al. 1999).
Due to increasingly stringent environmental legislation,
the textile industry in the UK and elsewhere is seeking to
develop effective wastewater remediation technologies,
especially those that allow colour removal that is largely
unaffected by conventional treatment systems (ONeill
et al. 2000). Despite the existence of a variety of chemical and physical treatment processes, bioremediation of
textile effluent is still seen as an attractive solution due
to its reputation as a low-cost, environmentally friendly,
and publicly acceptable treatment technology (Banat
et al. 1996).
A number of biological processes such as biosorption
have been proposed as having potential application in
removal of dyes from textile wastewater (Bustard et al.
1998); however, this review will focus upon the decolourisation and degradation of textile dyes by both mixed
and axenic cultures of bacteria and fungi.

Bacterial decolourisation and degradation

of textile dyes
Actinomycetes
Actinomycetes, particularly Streptomyces species, are
known to produce extracellular peroxidases that have a
role in the biodegradation of lignin. These prokaryotic
peroxidases are involved in the initial oxidation of lignin
to produce various water-soluble polymeric compounds.

82

Actinomycetes have also been shown to catalyse

hydroxylation, oxidation, and dealkylation reactions
against various xenobiotic compounds (Goszczynski
et al. 1994).
The ability of actinomycetes to decolourise and mineralise textile dyes was initially investigated by three
groups. In 1989, Ball et al. screened 20 strains of actinomycetes, representing a wide range of genera, for
their ability to decolourise the polymeric dye Poly R
(Ball et al. 1989). Only three of the 20 strains were observed to significantly decolourise the dye: Streptomyces
badius 252, Streptomyces sp. strain EC22, and Thermomonospora fusca MT800. Subsequently, Zhou and
Zimmermann (1993) embarked on an even larger screening process in which the decolourising capabilities of
159 actinomycetes were investigated. Of particular interest in this study was the investigators use of actual
textile effluents in the screening process. Five separate
effluents each containing a single dye of known concentration were used. Each dye was structurally distinct,
ranging from the azo compound Reactive Red 147 to the
phthalocyanine Reactive Blue 116. The widespread
ability of actinomycetes to bring about dye decolourisation was demonstrated by positive results being obtained
for 83 of the isolates. The finding that actinomycetes are
capable of the aerobic decolourisation and degradation
of azo dyes was significant given the recalcitrance of the
compounds to degradation by other bacteria under such
conditions.
Finally, but most significantly, a group based at the
University of Idaho and led by Don Crawford initiated
an investigation into the ability of ligninolytic microbes,
both white-rot fungi and Streptomycetes, to mineralise
and decolourise textile dyes. Initially, 14 streptomycetes
were investigated for their ability to decolourise two
polymeric dyes, Poly B-411 and Poly R-478, as well as
the azo dye Remazol Brilliant Blue R (RBBR) (Pasti and
Crawford 1991). With two of the dyes, RBBR and Poly
B-411, identical results were essentially obtained, with a
strong correlation between the ability of the isolate to
decolourise dyes and its ligninolytic capability. This observation, coupled with their finding that enhanced dye
decolourisation could be achieved as a result of extracellular H2O2 production when strains were grown in the
presence of glucose, suggested the involvement of peroxidases in the decolourisation process. Previously, extracellular peroxidases had been shown to be produced
by Streptomyces species and the enzymes were shown to
have substrate specificities similar to the Mn(II)-peroxidase of P. chrysosporium (Pasti and Crawford 1991). Interestingly, with the third dye, Poly R-478, no correlation
between decolourising activity and ligninolytic activity
(as measured by oxidation of veratryl alcohol, utilisation
of syringic or coumaric acid, and acid-precipitable polymeric lignins) was observed. The enzymatic processes
involved in the decolourisation of this dye remain unexplained.
Further work was carried out by Crawfords group to
investigate the mechanism by which Streptomyces

chromofuscus A11 decolourised and mineralised azo

dyes. Initial work confirmed that decolourisation was related to the ligninolytic capabilities of the isolate but that
the bacterial enzymatic systems responsible for degradation of azo dyes had a different specificity than those
of the white-rot fungus P. chrysosporium (Paszczynski
et al. 1992). Subsequently, the actual decolourisation
and degradation pathway for two azo dyes by both S.
chromofuscus A11 and P. chrysosporium was elucidated
in a series of elegant experiments (Goszczynski et al.
1994). It was proposed that the peroxidases of both
organisms converted the azo dye to a cation radical that
was susceptible to the nucleophilic attack of water or hydrogen peroxide. This resulted in the simultaneous split
of the azo linkage both symmetrically and asymmetrically to produce intermediates which subsequently undergo
several redox reactions before producing more stable intermediates (Fig. 1).
Recently, Burke and Crawford (1998) partially purified a novel extracellular peroxidase from S. viridosporus T7A in an effort to identify the class of peroxidase involved in dye decolourisation by Streptomyces
species. The peroxidase was found to have a substrate
specificity similar to that of fungal Mn-peroxidases
and was not inhibited by KCN, an inhibitor of hemeperoxidases (Magnuson 1996). In addition, N-terminal
amino acid sequences of the peroxidase were found to
share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Further purification
of the peroxidase in question is required to finally confirm its true biochemical nature. The molecular mechanism by which this peroxidase is induced has also
recently been investigated; with the gene oxyR, which
encodes an oxygen stress regulatory protein, apparently
having some regulatory role (Ramachandran et al.
2000).

Other aerobic bacteria

With the notable exception of the actinomyctetes, the
isolation of bacteria capable of the aerobic decolourisation and mineralisation of dyes, especially
sulfonated azo dyes, has proven difficult. A number of
reports exist suggesting the aerobic conversion of
specific azo dyes, including one notable report on the
greening of instant chocolate puddings (Dykes et al.
1994). Certain carboxylated analogues of sulfonated
azo compounds are also utilised aerobically as sole carbon and energy source by preadapted bacteria. Indeed,
oxygen-insensitive azoreductases, Orange I azoreductase[NAD(P)H:1-(4-sulfophenylazo)-4-naphthol oxidoreductase] and Orange II azoreductase [NAD(P)H:1(4-sulfophenylazo)-2-naphthol oxidoreductase], were
purified and characterised from a Pseudomonas strain
K24 (Zimmermann et al. 1982, 1984). Both these
enzymes have now been classified as being the same
enzyme, known correctly as azobenzene reductase
(EC 1.6.6.7).

83
Fig. 1 Proposed pathway for
peroxidase-catalysed degradation of sulfonated azo dyes.
The compounds in parentheses
have not been found, but their
existence is rationalised as
necessary intermediates for the
observed final products. The
compounds represented by
numbers in brackets have been
found in reaction mixtures.
Substitution pattern a (as in I),
R1=R2=O and B=O; substitution pattern b (as in II), R1=H,
R2=OCH, and B=NH. [2a]
2,6-dimethyl-1,4-benzoquinone,
[4a] 4-nitrosobenzenesulfonic
acid, [6b] 2-methoxyhydroquinone, [7b] 2-methoxy-4-aminophenol, [8a] sulfanilic acid,
[8b] sulfanilamide, [9a] 4-hydroxybenznesulfonic acid, [9b]
4-hydroxybenzenesulfonamide,
[10a] benzenesulfonic acid,
[10b] benzenesulfonamide,
[11a] azobenzene-4,4-disulfonic acid, [12] ammonia.
(Reproduced from Goszczynski
et al. 1994)

Despite this it has been argued that unequivocal evidence for aerobic bacterial mineralisation of these compounds is absent from the literature (Blhmel et al. 1998).
Recently, Blhmel et al. (1998) reported the isolation of
an unidentified bacterial strain, S5 (Genbank accession
number AF019037), capable of utilising the model sulphonated azo compound 4-carboxy-4-sulfoazobenzene
(CSAB) as sole carbon and energy source. Elucidation of
the degradation pathway demonstrated that the azo linkage of CSAB undergoes an initial reductive cleavage to
form 4-aminobenzoate and 4-aminobenzenesulfonate,
which are subsequently metabolised by conventional
aromatic catabolic pathways (Blhmel et al. 1998). Attempts to characterise the enzyme responsible for the
azo-bond cleavage in crude cell extracts have so far
proven unsuccessful.
In addition to azo dyes, the ability of bacteria to aerobically metabolise other dye classes has also attracted interest but yielded little success. Recently, however,
Sarnaik and Kanekar (1999) described the aerobic mineralisation of the triphenylmethane dye, methyl violet,
by a strain of Pseudomonas mendocina MCM B-402.
Methyl violet, which has some commercial applications
in addition to its recognised use as a bacteriological and
histological stain, was used by the isolate as sole carbon
and energy source. Preliminary studies identified that
P. mendocina degraded the dye via a number of unidentified metabolites to phenol, which then entered the
-ketoadipic acid pathway.

Anaerobic bacterial decolourisation and degradation

of textile dyes
Under anaerobic conditions many bacteria have been reported to readily decolourise azo-dyes. Initially, the bacteria bring about the reductive cleavage of the azo linkage, which results in dye decolourisation and the production of colourless aromatic amines. The potential
toxicity, mutagenicity, and carcinogenicity of such compounds is well-documented and has been reviewed elsewhere (Chung et al. 1992). Whilst concerns have long
been expressed over the threat that such compounds
have on human health, their environmental impact is
now also troublesome. Baughmann and Weber (1994)
demonstrated that in anoxic sediment environments uncharged azo dyes readily undergo biologically (and presumably microbial)-mediated reduction to the corresponding amines.
The initial step in bacterial azo dye metabolism under anaerobic conditions involves the reductive cleavage
of the azo linkage. This process is catalysed by a variety
of soluble cytoplasmic enzymes with low-substrate
specificity which are known trivially as azoreductases. Under anoxic conditions, these enzymes facilitate the transfer of electrons via soluble flavins to the
azo dye, which is then reduced. The role that such cytoplasmic enzymes have in vivo is, however, uncertain.
Reports indicate that intestinal bacteria decolourise certain azo dyes and their polymeric derivatives at roughly

84

equivalent rates (Brown 1981). As it is unlikely that

these polymeric dye molecules, or highly charged sulphonated azo dyes, can actually pass through the bacterial cell membrane, then the possibility of non-cytoplasmic azoreductase capabilities exists (Keck et al.
1997). This subject will be returned to below in the section Current state of the art. Whilst the anaerobic reduction of azo dyes is relatively easy to achieve, complete mineralisation of the molecule is difficult. Donlon
et al. (1997) reported the partial mineralisation of the
azo dye Mordant Orange 1 by a methanogenic granular
sludge in a continuous-upflow anaerobic sludge blanket.
In this study, however, complete mineralisation of only
one of the azo cleavage products, 5-aminosalicylic acid,
was possible, with the other product (1,4-phenylenediamine) accumulating in the reactor.
In addition to azo dyes, the bacterial metabolism of
other dye molecules under anoxic conditions has also
been studied. Henderson et al. (1997) demonstrated that
a range of axenic bacterial cultures which are commonly
found in the human gastro-intestinal tract, as well as
consortia of microbes from human, mouse, and rat intestines, all readily reduced the triphenylmethane dye malachite green. The metabolite produced, leucomalachite
green, is a suspected carcinogen and raises concerns over
the continued use of malachite green in aquaculture in
various regions worldwide.
Anaerobic-aerobic biodegradation of dyes
To overcome the problem of the relative recalcitrance of
azo dye breakdown products under anoxic conditions, a
number of groups have used a two-stage treatment process (Oxspring et al. 1996; ONeill et al. 2000). In the
first anaerobic stage, the azo dye is readily reduced to
the corresponding colourless aromatic amines, which are
then metabolised relatively easily under aerobic conditions. A detailed review on the anaerobic treatment of
textile effluents can be found elsewhere (Delee et al.
1998). In 1997, Kudlich et al. described the complete
mineralisation of the sulphonated azo dye Mordant
Yellow 3 by the bacterium Sphingomonas sp. BN6 coimmobilised with an uncharacterised 5-aminosalicylatedegrading isolate in alginate beads. Although the beads
were maintained under aerobic conditions, their centres
maintained anoxic conditions and it was here that cells
of Sphingomonas sp. BN6 reductively cleaved Mordant
Yellow 3 to produce 5-aminosalicylate and 6-aminonaphthalene-2-sulfonate. At the aerobic surface of the
beads, 6A2NS was also converted to 5AS by Sphingomonas sp. BN6 before being mineralised by cells of the
isolate 5AS1. These observations suggest that it may be
possible to develop biofilm-based reactors for the complete mineralisation of industrial-dye-contaminated
wastewaters.

Fungal decolourisation and degradation

of textile dyes
White-rot fungi
By far the most widely studied of dye-decolourising microorganisms are the white-rot fungi. This group of organisms is central to the global carbon cycle as a result
of their ability to mineralise the woody plant material
lignin, which has a complex polymeric structure. In addition to their natural substrate, white-rot fungi have been
found to be capable of mineralising a diverse range of
persistent organic pollutants, which distinguishes them
from biodegradative bacteria that tend to be rather substrate-specific (Reddy 1995). The ability of these fungi
to degrade such a range of organic compounds results
from the relatively non-specific nature of their ligninolytic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP) (EC 1.11.1.13), and laccase.
These enzymes and their catalytic properties are reviewed elsewhere (Hattaka 1994), but briefly, LiP catalyses the oxidation of non-phenolic aromatic compounds
such as veratryl alcohol, whilst MnP oxidises Mn2+ to
Mn3+ which is able to oxidise many phenolic compounds
(Glenn et al. 1986). Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is a copper-containing enzyme
that catalyses the oxidation of phenolic substrates by
coupling it to the reduction of oxygen to water (Edens
et al. 1999).
The decolourisation of dyes by white-rot fungi was
first reported by Glenn and Gold (1983), who developed
a method to measure ligninolytic activity of Phanerochaete chrysosporium based upon the decolourisation
of a number of sulfonated polymeric dyes. Subsequently,
the decolourisation of dyes has been used by others to
rapidly assess the biodegradative capabilities of diverse
white-rot fungi (Field et al. 1993). In 1990, the first report appeared in the literature of sulfonated azo dyes being decolourised, again by P. chrysosporium (Cripps
et al. 1990), and a degradation pathway for this isolate
was elucidated (Goszczynski et al. 1994; Figure 1).
Whilst P. chrysosporium remains the most widely studied of white-rot fungi, Trametes (Coriolus) versicolor,
Bjerkandera adusta, Pleurotus and Phlebia species, as
well as a variety of other isolates are increasingly being
studied (Conneely et al. 1999; Heinfling et al. 1998;
Kirby et al. 2000; Pointing et al. 2000; Swamy and
Ramsay 1999).
Unfortunately, due to the inherent complexity both of
the dye molecules themselves and the enzymatic mechanisms involved, the degradative pathways utilised by
white-rot fungi other than P. chrysosporium remain unstudied, despite the exploitation of powerful analytical
techniques (Smyth et al. 1999). Recently, our own group
began attempts to elucidate the degradation pathway of
copper-phthalocyanine dyes by P. chrysosporium using
such analytical tools as HPLC, atomic absorption spectrometry, and polarography (Conneely et al. 1999). Initial data indicated that the dye structure was readily de-

85

graded, resulting in decolourisation; however, both free

copper and organo-copper breakdown products were
found in culture supernatants and further work is needed
to elucidate their identity.
Whilst it is clear that enzymes such as LiP, MnP, and
laccase play a significant role in dye metabolism by
white-rot fungi, care must be taken not to exclude the
possibility of the existence of other degradative mechanisms. In many early studies, dyes were only added to
culture media when conditions for the production of ligninolytic enzymes prevailed, thus excluding the potential
discovery of other, non-ligninolytic, processes, (Cripps
et al. 1990). For example, Pasti and Crawford (1991)
have suggested the existence of alternative systems or
enzymes in dye decolourisation by white-rot fungi. The
plasma membrane redox system of white-rot fungi has
been proposed as a potential mechanism for dye decolourisation, and it is interesting to draw parallels between
this and the redox-mediated mechanisms utilised by bacteria under anaerobic conditions. Supportive evidence
was obtained by Stahl and Aust (1993), who found that
such a mechanism was responsible for the degradation of
2,4,6-trinitrotoluene in P. chrysosporium. Kirby (1999),
however, found little evidence to suggest that such a
mechanism was involved in decolourisation of Remazol
Black B by strains of P. chrysosporium, T. versicolor, or
Phlebia tremellosa.
Vyas and Molitoris (1995) discovered that Pleurotus
ostreatus produced an unusual enzyme during the solidstate fermentation of wheat straw that was capable of
Remazol Brilliant Blue R (RBBR) decolourisation. This
activity was distinct from the MnP, laccase, and manganese-independent peroxidase activities also produced by
the isolate, and from LiP and veratryl alcohol oxidase
activities, which were not detectable in P. ostreatus but
are well-known in other white-rot fungi. The activity
was named RBBR oxygenase as it possessed a catalytic
metal centre (possiblely haeme) and was inhibited by
a number of known oxygenase inhibitors (Vyas and
Molitoris 1995). The work of Vyas and Molitoris is also
of note as it documents a phenomenon seen by many
researchers of white-rot fungal dye decolourisation: the
sequential change of blue dyes to colourless through
a rainbow of intermediates (Kirby 1999). Vyas and
Molitoris (1995) proposed that such observations indicate that decolourisation is not a single-step reaction and
that these intermediate steps can be missed if enzyme
activities are high.
Non-white-rot fungi
Whilst degradation pathways utilised by these fungi
are not described in the literature, it is expected that
they would be similar to those reported to be involved
in the metabolism of other aromatic hydrocarbons
(Wunderwald et al. 1997).
Detailed investigations have been carried out on one
such isolate, a strain of Geotrichum candidum Dec1 iso-

lated from soil and capable of decolourising a number of

anthraquinone dyes (Kim et al. 1995). The broad substrate specificity exhibited by this isolate led Kim and
Shoda (1999a) to propose the existence of an extracellular peroxidase-type enzyme. Subsequently, a glycosylated haeme-based peroxidase (DyP) was purified from G.
candidum which had properties distinct from LiP, MnP,
horseradish peroxidase, and other peroxidases previously
reported (Kim and Shoda 1999a). The potential importance of this isolate for dye decolourisation rests with its
robustness in comparison to P. chrysosporium and other
white-rot fungi. DyP is produced constitutively and, unlike the activity of P.chrysosporium ligninolytic enzymes, is unaffected by the shear forces of shake flasks
or stirred tank reactors (Kim and Shoda 1998, 1999b).
Such properties should allow a number of G. candidumDec1-based bioreactors to be developed for the treatment
of diverse organic pollutants including dyestuffs. Recently the gene (dyp) encoding DyP was cloned into the heterologous expression host Aspergillus oryzae to allow
greater protein production in a safe host (Sugano et al.
2000). Molecular analysis of the protein confirmed that
DyP is distinct from other peroxidases in the plant
peroxidase superfamily, having a novel haeme-binding
region.

Current state of the art

The role of bacterial cytoplasmic and extracellular azoreductases in azo dye reduction in vivo has recently
been clarified by investigators at the University of
Stuttgart, Germany (Keck et al. 1997; Kudlich et al.
1997; Russ et al. 2000). Their work focused upon the
gram-negative isolate Sphingomonas sp. BN6, which has
recently, through molecular analysis, been shown to represent a novel species named Sphingomonas xenophaga
after its ability to eat foreign compounds (Stolz et al.
2000). It was demonstrated that an extracellular mechanism of dye reduction existed in addition to the nonspecific cytoplasmic enzymes which functioned as azoreductases by transferring electrons via reduced flavin
groups to the dye molecule and thus bringing about a
purely chemical reduction. Keck et al. (1997) demonstrated that certain quinone-based compounds generated
during metabolism of specific substrates acted as mediators shuttling redox equivalents to azo dye molecules
from the bacterial membrane (Keck et al. 1997; Kudlich
et al. 1997) (Fig. 2). To investigate if cytoplasmic enzymes played any role in dye decolourisation in vivo, a
flavin reductase [NAD(P)H:flavin oxidoreductase] was
cloned and overexpressed in both E. coli and S. xenophaga (Russ et al. 2000). In cell extracts the strains with
overexpressed flavin reductase demonstrated elevated
azo reductase activity; however, in whole cell studies
these strains showed little improvement in their dyedecolourising capabilities. These findings demonstrate
that the cytoplasmic azoreductases previously described
in the literature are likely to be a laboratory artefact and

86

Fig. 2 Proposed mechanism for the redox mediator (RM)-dependent reduction of azo dyes by strain BN6. (Reproduced from Keck
et al. 1997)

play no significant role in dye decolourisation in vivo.

Finally, Russ et al. (2000) also provided evidence that reports of aerobic azo reductases could be explained by the
isolates in which such a phenomenon was described having elevated flavin reductase activities.
Whilst many groups have demonstrated the ability of
white-rot fungi to decolourise and degrade textile dyes,
the development of potential remediation solutions for
the textile industry based upon these microorganisms has
been slow. A range of reactor systems aimed at optimising production of ligninolytic enzymes has been developed, including stirred tank reactors (Linko 1988),
packed-bed reactors (Feijoo et al. 1995), and rotating
disk reactors (Kirk et al. 1986). Few of these studies
have focused upon the ability of such reactors to remediate pollutants, with the exception of Allemann et al.
(1995), Zhang et al. (1999), and Kapdan et al. (2000),
who investigated pentachlorophenol, Orange II azo dye,
and Everzol Turquoise Blue G dye degradation, respectively. Our own group utilised the rotating-tube bioreactor system described by Allemann et al. (1995) to investigate the remediation of actual textile effluent by P.
chrysosporium (Kirby 1999). This reactor configuration
proved to be robust enough to continuously decolourise
three different mixed azo dye effluents by greater than
90% over the 38-day operating period (Kirby 1999).
Whilst such findings are encouraging, many problems
still face researchers in this area due to the lack of reliable, robust, and economic white-rot-fungi-based reactors. In addition, the volumes of effluent generated by
many of the larger dyeing plants worldwide needs to be
considered, and it may be that extraction and concentration of dyes and other pollutants is required before bioremediation is feasible (Nigam et al. 2000).

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