ELSEVIER
Journal of Biotechnology 51 (1996) 287-295
Basic knowledge and perspectives of bioelimination
xenobiotic compounds
Hans-Joachim
of
Knackmuss
Institut Jiir Mikrobiologie, Universitiit Stuttgart and Fraunhofer-Institut fir GrenzfIiichen- und Bioverfahrenstechnik, Stuttgart,
Germany
Received 20 June 1996; revised 15 July 1996; accepted 15 August 1996
Abstract
Almost every natural product, irrespective of its molecular weight or structural complexity, is degraded by one or
another microbial species in some particular environment. The omnipotence of microorganisms also extends to the
majority of synthetic compounds, which are funneled into the natural metabolic cycles. Certain substituents such as
halogen, sulfo-, azo- or nitro-groups, particularly the accumulation of such groups and specific substitution patterns
confer xenobiotic character to a synthetic compound. Moreover, the electron-withdrawing
character of these
substituents generates electron deficiency and thus makes the compounds less susceptible to oxidative catabolism. As
a consequence, many of these chemicals tend to persist under aerobic environmental conditions.
When enzymes with low substrate specificity encounter foreign compounds, highly reactive species may be
generated. On the one hand, these can eliminate some of the xenophors spontaneously generating less persistent
metabolites. On the other hand, gratuitous metabolism of complex structures may give rise to extensive chemical
misrouting, generating dead products of high molecular weight. Biodegradation of a xenobiotic substance can be
accomplished when the catabolic activities, present in mixed microbial communities, complement each other. Thus,
syntrophic interactions can lead to complete mineralization of even complex xenobiotic compounds.
Mineralization of xenobiotics by a single organism can be achieved by taking advantage of natural or induced gene
transfer to construct hybrid degradative pathways. Mobilization of blocks of genes, encoding catabolic bottleneck
reactions, may speed up the evolutionary potential of natural communities so that under appropriate selective
conditions new multifunctional pathways are generated.
As a consequence of the electron deficiency of xenobiotic compounds such as polychlorinated arenes or ethenes, azo
dyes or polynitroaromatics,
the combination of the reductive potential of anaerobic microbes with subsequent
oxidative processes opens a hitherto largely unexploited technology for biological treatment of waste water and for
soil bioremediation. Copyright 0 1996 Elsevier Science B.V.
Keywords:
Haloalkenes;
Anaerobic/aerobic
Humification
processes; Arylsulfonates;
Azodyes; Cometabolism;
016%1656/96/%15.00
Copyright 0 1996 Elsevier Science B.V. All rights reserved
PZI SO168-1656(96)01608-2
Electron
deficient xenobiotics;
1. Introduction
( Xenobiotic
Natural communities
of microorganisms
harbor
an amazing physiological
versatility and catabolic
potential
for the breakdown
of an enormous
number of organic molecules. Almost every natural product, irrespective of its molecular weight or
structural complexity, is readily degraded by one
or another microbial
species in some particular
environment.
This omnipotence
of microorganisms also extends to the majority
of synthetic
compounds
which are funneled into the natural
metabolic
cycles. Certain
substituents
such as
halogen,
sulfo-, azo- or nitro-groups
(Fig. l),
particularly
the accumulation
of such groups and
specific substitution
patterns,
confer xenobiotic
character to a synthetic compound.
Moreover, the
electron-withdrawing
character
of these
substituents generate an electron deficiency and thus
make the compounds
less susceptible to oxidative
catabolism.
In general, oxygen is directly involved in the
initial attack
of aliphatic
and aromatic
compounds. Because introduction
of hydroxyl functions into the molecule is necessary for further
oxidative catabolism,
the initial electrophilic
attack by oxygenases of aerobic bacteria may bestep.
This
can
be
rate-limiting
come
a
demonstrated
in bacterial systems, exhibiting low
substrate specificity of oxygenases so that a strict
correlation
between the Hammett substituent
constant and the log Vre, values of the initial dioxy-
/
X
\
-t
X= C-Halogen
Halogen
C=N
COR,
S03H
N=N-RR2
NO2
Fig. I Xenophors
(X) causing biological
otic and electron-withdrawing
character
arrow) impede initial electrophilic
attack
persistence. Xenobi(indicated
by heavy
by oxygenases.
Compound
1
Fig. 2. Cometabolism
of a xenobiotic
compound
by mixed
microbial population.
Sequences of arrows indicate catabolic
routes that give rise to dead-end products (0). To some extend
these products may be further degraded by interspecies transfer (horizontal
arrows) and syntrophic
interactions
may accomplish
partial mineralization
of a xenobiotic
compound
(reaction
sequence a g). Theoretically,
a hybrid organism
harboring
a complete catabolic sequence (a-c + hhk) should
exhibit highest catabolic performance
and selective advantage
in the population.
genation reaction can be observed (Reineke and
Knackmuss,
1978).
The fate of environmental
pollutants
is largely
determined by abiotic processes such as photooxidation and by the metabolic activities of microorganisms. Since catabolic enzymes are more or less
specific, they can act on more than their natural
substrate. This explains why the majority of xenobiotics
are subject
to fortuitous
metabolism
(cometabolism).
Normally, an initial catabolic enzyme or sequence of enzymes convert a chemical
to an organic product that is not further metabolized. Thus, for a cometabolically
active organism,
the process is unproductive
because it is not coupled to energy conservation.
Therefore, in a natural mixed population,
a wide variety of dead-end
products (Fig. 2) may be accumulated
and subject
to physical
and chemical
secondary
reactions.
Since initial oxidation by mono- or dioxygenases
requires reduction equivalents or the loss of active
protein by suicide inactivation,
cometabolism
may
H.-J. Knackmuss /Journal
of Biotechnology 51 (1996) 287-295
289
Unproductive
or
Counterproductive
u’$)+O
+ &OH
_
ri_
e.g.
1 ,2- or 1 ,4-additions.
condensailons
radical
react!ons
Fig. 3. Cooxidation of 2-chlorophenol by phenol-degrading bacteria generates 3-chlorocatechol, which cannot be utilized (unproductive catabolism is indicated by heavy arrows). If 3-chlorocatechol is subject to meta-cleavage, a highly reactive acyl chloride may be
generated which destroys the meta-cleavage enzyme by suicide inactivation. As a consequence, 3-chlorocatechol is accumulated and,
by autoxidation, quinones or phenoxyl radicals may be formed. Chemical misrouting and the toxicity of the autoxidation products
make the catabolism of 2-chlorophenol even counterproductive (indicated by broken arrows).
be even counterproductive.
An example is the
cooxidation of 2-chlorophenol
by phenol- or
cresol-degrading microorganisms, which gives rise
Xenobiotic
Compound
-1
COmetabollc
activation
1
/ Reactive Metabolite
1
,
Further
degradation,
minerai~zatron
CO,, W
Fig. 4. In general cometabolic transformation of a xenobiotic
compound represents an activation of the molecule. Subsequent spontaneous reactions can therefore lead to chemical
misrouting (see Fig. 3) or anionic elimination of the xenophor
allows productive metabolism of the chemical.
to the accumulation of 3-chlorocatechol (Fig. 3)
(Schmidt et al., 1983). The latter, if subject to
meta-cleavage, generates an arylchloride which
irreversibly inactivates the ring cleavage enzyme
(Bartels et al., 1984). As a consequence, chlorocatechols are accumulated which by autoxidation
generate
highly reactive products
such as
quinones or phenoxyl radicals. These are subject
to chemical misrouting or may be toxic and thus
paralyze the entire process.
Cometabolic transformation
of a xenobiotic
compound often generates reactive metabolites
such as epoxides, dihydrodiols, aromatic diols,
aromatic amines (see below) or at least products
that are more easily oxidized than the original
chemical. On the one hand, the reactive metabolite may be misrouted by spontaneous chemical or
physical reactions giving rise to absorption, coupling or polymerization reactions (Fig. 4). On the
other hand, further chemical or biological transformation may eliminate the xenophor so that at
least part of the xenobiotic compound can be
mineralized and utilized by certain members of
the population.
H.-J. Knackmuss
290
Reductwe
: Journal of’ Biotrc,hnology 51 (1996) 287-295
Transformation
Fig. 5. Degradation
of electron-deticient
xenobiotics
such as
polynitroaromatics,
azo dyes and polyhalogenated
hydrocarbons are readily attacked by reducing agents which are harbored by anaerobic bacteria. This reductive transformation,
by
generating
aromatic
amines or dehalogenation
products,
reduces electron deficiency of the molecule so that subsequent
mineralization
by aerobic bacteria is possible.
2. Electron-deficient
xenobiotics
Because of the electron-withdrawing
character
of the xenophors
given in Fig. 1, anaerobic
microorganisms
harboring
strongly reducing nucleshould
react
with
these
structures,
ophiles
generating
less electrophilic
metabolites.
Candidates for these initial reductive transformations
are polyhalogenated
compounds
which may be
dehalogenated.
These reactions would involve the
anionic removal of a halogen and its replacement
by a hydride ion (Fig. 5). Actually,
reductive
dehalogenation
occurs most readily under strict
anaerobic conditions
( I - 400 mV) and requires
a reducing
auxiliary
substrate.
Recently,
it has
been shown that this exergonic dehalogenation
may be coupled to energy conservation
(Mohn
and Tiedje, 1992). With a pure culture of Dehalospirillum
multivorans,
growth
in mineral
medium
could be demonstrated,
with H, plus
perchloroethane
(PCE) as sole source of energy
when acetate served as a carbon source (Neumann
et al., 1994). This clearly indicates a chemiosmotic
mechanism
of energy conservation.
Since dechlorination
yields cis-dichloroethene
and vinylchloride, the highly electron-deficient
tetrachlorethene,
which cannot be attacked by aerobic bacteria, is
converted
to compounds
that are less electrondeficient and readily degradable
by cooxidation
(Ewers et al., 1990). It is obvious that the combination of a reductive and an oxidative process
offers a hitherto unexploited
possibility for complete degradation
of polychlorinated
hydrocarbons as contaminants
of soil and groundwater.
A similar situation
exists in polynitroaromatic
compounds
and in azo dyes (Fig. 5). The electronaccepting character
of the azo- and nitro-group
renders these chemicals less susceptible to oxidative processes. In contrast, with increasing number of nitro-groups
on the aromatic ring, initial
reductive transformations
are favored.
Because of the strong electron deficiency
of
polynitroaromatics,
even aerobic bacteria exhibit
reductive potentials against these xenobiotics.
An
unusual
hydrogenation
of the aromatic
nucleus
has been recently discovered for the degradation
of picric acid and 2,4,6_trinitrotoluene
(TNT)
(Rieger and Knackmuss,
1995). During coreduction of the nitro-groups
of TNT, amino-groups
are generated.
These weaken the electron deficiency originally present in the aromatic ring so
that the reaction velocities of the sequential reduction of TNT decrease with the number of nitrogroups
being
converted
into
amino-groups.
Therefore, complete reduction
of TNT to 2,4,6triaminotoluene
(TAT) requires strict anaerobic
conditions
( I - 200 mV) and a reductant
as an
auxiliary substrate such as glucose or saccharose.
TNT is thus completely
reduced to TAT by
anaerobic
sludge with aminodinitroand diaminonitrotoluenes
as intermediates
(Rieger and
Knackmuss,
1995). TAT is an electron-rich
compound
and should
be readily
oxidized
when
treated aerobically.
If TNT-contaminated
soil is
treated anaerobically,
TNT is also subject to stepwise reduction; however, the end product TAT is
not detectable.
Kinetic data clearly demonstrate
that irreversible sorption of TAT proceeds already
under strict anaerobic conditions.
Obviously,
the
acidic polyanion
structures
of the clay minerals
and of the humic substances
interact with the
basic groups of TAT. In addition,
subsequent
O,-dependent
reactions
and aerobic conditions
may give rise to the formation of polymers, which
resembles humification
of natural phenolic sub-
H.-J. Knackmuss / Journal of Biotechnology 51 (1996) 287-295
Anaerobic
291
Treatment
Aerobic Treatmenl
Fermentation
Substrate
(Alcohol,
+ co,
Products
CO,+ H,O
Organic Acids)
-t
0-24e-
Fig. 6. Biological remediation of TNT-contaminated soil by anaerobic/aerobic treatment. Glucose or saccharose was added as an
auxiliary substrate which is degraded by the autochthonous soil population. Part of the reducing equivalents were used for stepwise
reduction of TNT via aminodinitro- and nitrodiamino toluenes (ADNT and DANT). Triaminotoluene interacts irreversibly with the
clay and humic acid fraction of soil.
stances. Phenolic and quinoide structure in humus
can also covalently bind TAT and other highly
reactive species generated by autoxidation
of
TAT.
Fig.
6
summarizes
the
process
of
anaerobic/aerobic
treatment of TNT-contaminated soil. It offers a cost-effective and simple
way to clean up polluted sites and does not
require
the
establishment
of
special
nonindigenous
microorganisms
(Daun et al.,
1995). Ecotoxicological tests with aquatic and
terrestrial organisms indicate that toxic effects
cannot be detected in the remediated soil and it
was thus recommended for reuse as green land
and recreation areas (Lenke et al., unpublished).
higher degree of biodegradation and even mineralization can be expected when cometabolic activities within a microbial community complement
each other (Fig. 2, sequences a-c+ d +e-g).
Such syntrophic interactions actually exist in natural populations and in certain cases the concerted action of a two-species culture is well
understood (Fig. 7). A case in point is the degradation of naphthalenesulfonates
(Fig. 8), which
Organism
I:
Organism
II:
I/
3. Degradation of xenobiotics by mixed cultures
With increasing complexity of a xenobiotic, we
cannot expect to find complete catabolic pathways
in a single organism. Besides incomplete oxidation
and accumulation of dead-end metabolites, a
Dead end
product
---TCC
Fig. 7. Degradation of a xenobiotic compound by a synergistic
two-species culture. Organism I eliminates the xenophor as an
anion and utilizes part of the carbon skeleton, whereas organism II harbors a complementary sequence that allows complete
mineralization and productive catabolism of the dead-end
product of organism I.
292
H.-J. Knackmuss / Journal of Biotechnology 51 (1996) 287-295
SO3H
Rates (IOO~mol
OH
stra,n BN6
Fig. 8. Degradation
of naphthalene-2-sulfonate
(2NS) in syntrophic culture. Strain BN6, tentatively identified as a Sphingomonas strain, can partially degrade 2NS to salicylate as a
toxic dead-end
metabolite.
Strain BN6 can only grow with
2NS if a salicylate-degrading
strain is present.
are structural features and building blocks of azo
dyes.
Sphingomonus strain BN6 harbors the ability to
degrade naphthalene-2-sulfonate
(2NS) to salicylate. Because the latter compound
is not further
degraded and toxic to strain BN6, it can grow
with 2NS only in the presence of a salicylate-assimilating organism. The initial catabolic enzymes
of 2NS catabolism
distinguish
themselves by an
extraordinarily
low substrate specificity so that a
broad spectrum
of substituted
amino- and hydroxynaphthalene
sulfonates are transformed
into
the corresponding
amino- and hydroxysalicylates
(Fig. 9). Therefore, the functionality
of the system
can easily be expanded by complementary
organisms degrading
substituted
salicylates. Typically,
it is difficult to establish
a stable continuous
degradation
process with these mixed cultures be-
g;,,l,,”
min-‘) with
in the respective postition
or NH2 -
Fig. 9. Turnover
of amino- and hydroxynaphthalene-2-sulfonates by Sphingomonas strain sp. BN6. With the exception
of 3-substituted
isomers and 5-amino-naphthalene-2-sulfonate,
these xenobiotics
were oxidized to the corresponding
salicylates (Nortemann
et al., 1986). 5-Aminonaphthalene-2-sulfonate
is also
readily
cooxidized
to
5-hydroxyquinoline-2-carboxylate
(Nortemann
et al., 1993) as a dead-end
product.
cause interspecies transfer of the salicylates, particularly
of the aminoand hydroxysalicylates,
may be disturbed
by autoxidation
and chemical
misrouting
of these metabolites.
The instability of
the biological
system can be circumvented
by
immobilization
of the complementary
microorganisms (Diekmann
et al., 1988) or by generating
eg
pJP4.
pWR1
pAC25
Maleylacetate
4
TCC
Fig. IO. Evolution of hybrid
haloaromatic
compounds.
pathways
for the catabolism
of
H.-J. Knackmuss
Table 1
Competition
Days
0
0.94
1.93
2.83
3.84
4.9
5.9
6.9
8.1
9.6
11
12.7
between
Strains
the mixed culture
/ Journal of Biotechnology
and the hybrid
strain
51 (1996) 287-295
BN6 (pWW60-3026)
(cells ml-‘)
growth
with 2NS
Rel. cell number
BN6 total
BN6 wild type
BN6 (pWW603026)
Ps. putida NCIB9816
2.3 x
4x
2.6 x
2.3 x
4.3 x
4.1 x
3.6 x
3x
3.8 x
4x
4.4 x
2.6 x
1.3 x lo9
3x los
1 x lo8
2.3 x 10’
<4.3x 107
<4.1 x 10’
<3.6x lo7
<3x 10’
t3.8 X lo6
<4x lo6
<4.4x lo6
<2.6x lo6
1 x 109
3.1 x lo9
2.5 x lo9
2.3 x lo9
4.3 x lo9
4x 109
3.9 x lo9
3x109
3.8 x lo9
4x lo9
4.2 x lo9
2.5 x lo9
2x lo8
2x lo8
6x lo7
5x10’
1.2x 108
6.4 x 10’
1 x los
3x 10’
1 x lo*
9.7 x 10’
1 X lo*
1.4x los
lo9
lo9
lo9
lo9
lo9
109
lo9
lo9
109
lo9
109
lo9
during
293
hybrid strains, which harbor a complete catabolic
sequence (see Fig. 2, sequence a-c + h-k).
4. Evolution of hybrid pathways
The experiences with haloaromatic compounds
have shown that new hybrid degradative capabilities for a broad spectrum of mono- and dichlorinated compounds such as chlorobenzenes (Y =
H), chlorobenzoates (Y = COOH), chlorophenols
(Y = OH), chloroanilines (Y = NH,) or chlorobiphenyls (Y = phenyl) can be evolved by natural
gene transfer (Fig. 10) (for review see Gottschalk
and Knackmuss, 1993). The basis for this evolutionary potential is, on the one hand, the
catabolic potential of bacteria that utilize aromatic and methylaromatic compounds and cooxidize the corresponding chloroaromatic structure
to chlorocatechols. On the other hand, bacteria
harboring the complementary catabolic sequence
for the assimilation of chlorocatechols are rare
but ubiquitous in natural populations and can be
enriched by chlorinated aromatics that exhibit low
toxicity. Thus, by use of 3-chlorobenzoate or 2,4dichlorophenoxyacetate,
strains were enriched
that harbor the chlorocatechol-assimilating potential on transferable genetic elements such as the
plasmids pJP4, pWR1 or pAC25. Horizontal
of hybrid
strain
(%)
43
92
99
99
>99
>99
>99
>99
>99.9
>99.9
> 99.9
> 99.9
transfer of these plasmids explains why hybrid
pathways for chlorosubstituted
aromatic compounds can be evolved in the laboratory and
during adaptation of the microflora of activated
sludge.
In the case of sulfonated naphthalenes, hybrid
strains with a complete catabolic sequence should
be generated by conjugative transfer to strain
BN6 of naturally existing plasmids that encode
salicylate degradation. This approach has not
been successful because the well known plasmids
NAH7, SAL or pWW60 were not transferred to
Sphingomonas sp. BN6. In contrast, broad host
range plasmids from the incompatibility group
Inc Pl or Inc P4 were readily transferred from
Escherichia cob to strain BN6 (10-3-10-4
per
donor cell). Therefore, the constructed plasmid
pWW60-3026 harboring the genes of salicylate
degradation plus a regulatory element from NAH
(Pseudomonas putida NCIB98 16) were successfully
transferred to strain BN6 (RUB et al., unpublished). Derivative strains, when transferred to
mineral medium with 2NS as sole source of carbon and energy, could grow with 2NS but exhibited only slightly higher catabolic performance
when compared with the mixed culture consisting
of Sphingomonas sp. BN6 and Pseudomonas
putida NCIB98 16. Neither growth characteristics
with 2NS in continuous culture nor comparison
H.-J. Knackmuss
294
! Journal of Biotechnology
of key catabolic enzymes in crude extracts of cells
of the two species and the hybrid culture could
clearly demonstrate
the advantage
of the hybrid
strain. A competition
experiment with 2NS as the
limiting
substrate
was therefore
carried out in
continuous
culture. The complementary
system of
strain BN6 and strain NCIB9816 competed with
BN6
(pWW60-3026)
harboring
the
hybrid
pathway of 2NS catabolism.
Surprisingly,
within
4-5 days, the hybrid organism clearly dominated
and completely
replaced the two-species culture
(Table 1).
5. Perspectives
These preliminary
observations
with 2NS-degrading hybrid bacteria clearly showed that genetic optimization
and construction
of hybrid
strains may increase the rate and extent of re-
7
ai
I
T
C
J
C
Fig. 11. Perspectives
for the evolution
of multifunctional
strains that degrade naphthalene
sulfonates:
mobilization
of
the genes of an uptake system for sulfonates plus those of the
upper pathway of naphthalene-2-sulfonate,
particularly
of the
desulfonating
dioxygenase,
should facilitate the evolution of
hybrid strains. Organisms
harboring
the complementary
sequences for the assimilation
of salicylates are ubiquitous
in
nature and could thus acquire the ability to mineralize substituted naphthalene
sulfonates.
51 (1996) 287-295
moval of xenobiotic
compounds
in a continuous
process.
The functionality
of Sphingomonas sp. BN6 towards a broad spectrum of substituted naphthalenes (Fig. 11) can even be expanded to suifonated
azo dyes. This, however,
requires
a two-step
anaerobic/aerobic
process in which the bacterial
2NS-grown biomass of the Sphingomonas sp. BN6
and the salicylates-degrading
strains is used for
gratuitous reduction of sulfonated azo dyes (Haug
et al., 1991). Thus, under anaerobic
conditions
and in the presence of an auxiliary electron donor,
Mordant yellow is subject to complete cleavage to
the corresponding
aromatic
amines, which are
completely mineralized
by the’subsequent
aerobic
step. Characteristically,
only cells of strain BN6
grown in the presence of naphthalene
sulfonates
can reduce azo dyes. This indicates that an uptake
system is induced that gratuitously
takes up sulfonated azo dyes, which are coreduced by an azo
reductase. This latter activity is constitutive
and
rate-limiting
in BN6, so that for practical purposes it may be necessary to improve its expression or recruit a more efficient inducible
azo
reductase.
The modular nature of the catabolic sequence
of sulfonated
aromatic compounds
and azo dyes
and its acquisition
together with an appropriate
regulatory unit by salicylates-degrading
organisms
would certainly lead to a burst of evolutionary
potential
for new and highly efficient
hybrid
routes of these xenobiotics
(Fig. 11). The mobilization of the genes of the initial catabolic
sequences,
at least for substrate
uptake
and
dioxygenolytic
desulfonation,
would create an
evolutionary
potential as described above for the
haloaromatics
(Fig. 10). Previous work has shown
that aromaticsand methylaromatics-degrading
organisms in natural populations
readily acquire
the genetic module of chlorocatechol
assimilation
and thus evolve novel biochemical
routes, not
only for single compounds
but more importantly
for mixtures of haloaromatic
xenobiotics.
In the case of sulfonated
naphthalenes,
the
genetic determinants
of the initial catabolic
sequence have to be mobilized
because these are
unique and multifunctional.
Acquisition
of this
block of genes by ubiquitous
salicylates-degrading
H.-J. Knackmuss 1 Journal of Biotechnology 51 (1996) 287-295
295
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Aerobic:
Autoxrdatlan
polymerization
precipitation
Fig. 12. Biodegradation of azo dyes could be accomplished by
bacteria harboring a highly efficient uptake and azo reductase
system that are used in a two-step anaerobic/aerobic process.
The hybrid arylsulfonates degraders (see legend of Fig. 11)
must suppress chemical misrouting of the sulfonated o- and
p-aminohydroquinones
that are generated by the initial cleavage of the azo bond.
bacteria would generate complete
catabolic
routes that allow mineralization
of a broad
spectrum of sulfonated naphthalenes. Through
appropriate process engineering which accomplishes an integrated anaerobic/aerobic treatment
(Fig. 12), a vertical expansion of the catabolic
system for sulfonated azo dyes appears to be a
realistic perspective.
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that are due to misrouting of metabolites by
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