FEMS Microbiology Reviews 22 (1999) 459^473
Review
Anaerobic bacterial metabolism of hydrocarbons
Johann Heider a; *, Alfred M. Spormann
a
b;c
, Harry R. Beller b , Friedrich Widdel
d
Mikrobiologie, Institut fuër Biologie II, Universitaët Freiburg, Schaënzlestr. 1, D-79104 Freiburg, Germany
Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305, USA
c
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
d
Max Planck Institut fuër marine Mikrobiologie, Celsiusstrasse 1, D-28359 Bremen, Germany
b
Received 1 September 1998 ; accepted 23 September 1998
Abstract
The capacity of some bacteria to metabolize hydrocarbons in the absence of molecular oxygen was first recognized only
about ten years ago. Since then, the number of hydrocarbon compounds shown to be catabolized anaerobically by pure
bacterial cultures has been steadily increasing. This review summarises the current knowledge of the bacterial isolates capable
of anaerobic mineralization of hydrocarbons, and of the biochemistry and molecular biology of enzymes involved in the
catabolic pathways of some of these substrates. Several alkylbenzenes, alkanes or alkenes are anaerobically utilized as
substrates by several species of denitrifying, ferric iron-reducing and sulfate-reducing bacteria. Another group of anaerobic
hydrocarbon degrading bacteria are `proton reducers' that depend on syntrophic associations with methanogens. For two
alkylbenzenes, toluene and ethylbenzene, details of the biochemical pathways involved in anaerobic mineralization are known.
These hydrocarbons are initially attacked by novel, formerly unknown reactions and oxidized further to benzoyl-CoA, a
common intermediate in anaerobic catabolism of many aromatic compounds. Toluene degradation is initiated by an unusual
addition reaction of the toluene methyl group to the double bond of fumarate to form benzylsuccinate. The enzyme catalyzing
this first step has been characterized at both the biochemical and molecular level. It is a unique type of glycyl-radical enzyme,
an enzyme family previously represented only by pyruvate-formate lyases and anaerobic ribonucleotide reductases. Based on
the nature of benzylsuccinate synthase as a radical enzyme, a hypothetical reaction mechanism for the addition of toluene to
fumarate is proposed. The further catabolism of benzylsuccinate to benzoyl-CoA and succinyl-CoA appears to occur via
reactions of a modified L-oxidation pathway. Ethylbenzene is first oxidized at the methylene carbon to 1-phenylethanol and
subsequently to acetophenone, which is then carboxylated to 3-oxophenylpropionate and converted to benzoyl-CoA and
acetyl-CoA. Anaerobic mineralization of alkanes involves an oxygen-independent oxidation to fatty acids, followed by Loxidation. In one strain of an alkane-mineralizing sulfate-reducing bacterium, the activation appears to proceed via a chainelongation, possibly by addition of a C1 -group at the terminal methyl group of the alkane. Finally, aspects concerned with the
regulation and ecological significance of anaerobic hydrocarbon catabolic pathways are discussed. z 1999 Federation of
European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Anaerobic bacterial catabolism; Hydrocarbon; Toluene; Ethylbenzene ; Alkane
* Corresponding author. Tel.: +49 (761) 203-2774; Fax: +49 (761) 203-2626; E-mail [email protected]
0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 5 - 4
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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Bacteria capable of metabolizing hydrocarbons under anoxic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Anaerobic catabolism of toluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Pathway of anaerobic toluene conversion to benzoate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Benzylsuccinate synthase, the enzyme catalyzing the initial reaction in anaerobic toluene mineralization
3.2.1. Properties of benzylsuccinate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. Proposed reaction mechanism of benzylsuccinate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Genetics of anaerobic toluene mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Anaerobic catabolism of ethylbenzene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Anaerobic metabolism of alkanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Regulation of anaerobic hydrocarbon metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Ecological aspects of anaerobic hydrocarbon degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Conclusion/outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Hydrocarbons can be classi¢ed into saturated
compounds (aliphatic and alicyclic alkanes), compounds containing C^C double-bonds (alkenes),
compounds with C^C triple-bonds (alkynes), and
the mono- and polycyclic aromatic hydrocarbons.
Details regarding the chemistry of these compounds
can be found in most organic chemistry textbooks
(e.g. [1^4]). Most hydrocarbon compounds exhibit
high homolytic and heterolytic dissociation energies
of their C^H and C^C bonds and weak chemical
reactivity. Therefore, hydrocarbons do not participate in acid-base reactions in aqueous systems, as
indicated by their extremely high theoretical pKa values. Protonation of alkanes (yielding carbenium ions
and molecular hydrogen) requires the use of super
acids. Addition reactions to unsaturated C^C bonds
occur at the double- and triple-bonds of alkenes and
alkynes, but not at aromatic rings.
Certain redox reactions of alkenes occur under
relatively mild conditions; for example, their reduction to alkanes with hydrogen by catalytic hydrogenation. Similarly, methyl or methylene groups directly attached to aromatic rings can be
catalytically oxidized to the corresponding carboxylor carbonyl-groups, respectively. The most common
reaction of hydrocarbons is their combustion as fuel
with oxygen to CO2 and water. These reactions,
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which proceed via radical intermediates, are initiated
by oxygenation reactions, involving molecular oxygen as a direct reactant. Enzyme-catalyzed oxygenations, which also occur via radical intermediates,
have until recently been the only known initial reactions for degradation of alkanes and aromatic hydrocarbons in biological systems (see below).
Radical mechanisms are also involved in some
known oxygen-independent chemical reactions of hydrocarbons. Alkanes and alkyl side chains of aromatic hydrocarbons can be chemically `cracked' to
smaller alkanes and alkenes by pyrolysis, or halogenated with elemental halogens in the presence of light.
Both reactions involve free radical intermediates.
These reactions are technically performed under conditions which are not compatible with biological systems. Yet, it was recently elucidated that some bacteria actually employ oxygen-independent radical
reactions to make hydrocarbons available as substrates (see below).
Aromatic hydrocarbons can be chemically derived
through electrophilic substitution. Well known examples of substitution reactions at the aromatic
ring are halogenation, nitration, sulfonation, diazonium coupling and reactions with carbon electrophiles, e.g. carbocations generated in Friedel^Crafts
reactions. These reactions normally require conditions which probably can not exist within living organisms.
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Hydrocarbons are widespread in the environment.
Their major industrial source is petroleum and its
associated natural gases, formed geochemically
from biomass under conditions of high pressure
and temperature. However, signi¢cant amounts of
hydrocarbons are also formed by biological processes. For example, methane is produced as a metabolic end product by methanogenic bacteria. Biosynthesis of high-molecular alkanes by decarbonylation
of the corresponding (n+1) aldehydes has been reported for a marine alga [5]. The simplest alkene
compound, ethylene, is synthesized and released by
higher plants as a ripening hormone; in addition,
some ethylene-producing bacteria and fungi are
known [6]. High-molecular mass alkenes are found
as constituents of the cuticulas of insects and higher
plants and serve as protection against loss of water
as well as sex pheromones [7]. They are formed either by decarboxylation of unsaturated fatty acids [8]
or by monoxygenase-catalyzed conversion of unsaturated aldehyde precursors to alkenes and CO2 [9].
Another well known class of natural hydrocarbons,
which often contain double bonds, are the isoprenoids, e.g. carotenoids and terpenes of many plants,
insects and microorganisms [10]. Anaerobic catabolism of isoprenoids is reviewed by Hylemon and
Harder in this issue. Certain aromatic hydrocarbons
are also formed biologically. Low concentrations of
toluene have been detected in pristine environments,
such as the anaerobic hypolimnia of lakes [11]; it
originates from phenylalanine degradation by several
species of anaerobic bacteria. These bacteria ¢rst oxidize phenylalanine to phenylacetate, which is then
decarboxylated [12,13]. Even biological formation of
naphthalene has recently been reported in some
plant and animal species [14].
Catabolism of hydrocarbons has long been considered as a strictly oxygen-dependent process. Common aerobic hydrocarbon-utilizing organisms are
found among fungi and bacteria. These microorganisms are capable of metabolizing virtually all naturally formed and a wide range of industrially produced hydrocarbons. In aerobic organisms, the
initial attack of hydrocarbons always requires molecular oxygen as a co-substrate. The ¢rst enzymes in
the metabolic pathways of alkanes are monooxygenases, while aromatic hydrocarbons are attacked by
either monooxygenases or dioxygenases. These en-
461
zymes incorporate hydroxyl groups, derived from
molecular oxygen, into the aliphatic chain or the
aromatic ring. The alcohols formed from aliphatic
hydrocarbons are then oxidized to the corresponding
acids; the phenolic compounds generated by ring
hydroxylation of aromatic hydrocarbons are direct
precursors for oxidative ring cleavage (last reviewed
in [15]).
As demonstrated throughout the last decade of
microbiological research, particular microorganisms
are also able to catabolize hydrocarbon compounds
under anaerobic conditions. Hydrocarbons that can
be degraded anaerobically include aliphatic alkenes
and alkanes with chain lengths of 6-20 carbon atoms,
monocyclic alkylbenzenes, such as toluene, ethylbenzene, propylbenzene, p-cymene, xylene- and ethyltoluene-isomers, as well as benzene and naphthalene.
Some hydrocarbons that are degraded anaerobically
by pure bacterial cultures are shown in Fig. 1. Obviously, bacteria capable of this metabolic capacity
must have developed alternative, oxygen-independent reactions for the initial attack of their hydrocarbon substrates. No organism which mineralises hydrocarbons containing less than six C-atoms
anaerobically has yet been discovered.
Fig. 1. Structures of some hydrocarbons which are metabolized
anaerobically by pure bacterial cultures.
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2. Bacteria capable of metabolizing hydrocarbons
under anoxic conditions
Hydrocarbons are highly reduced organic molecules. In chemotrophic organisms, the reducing
equivalents generated during transformation of hydrocarbons to metabolic intermediates need to be
transferred to an electron acceptor with a more positive redox potential to allow energy conservation for
growth. Based on our present biochemical knowledge, energy conservation from hydrocarbon metabolism by a chemotrophic organism in pure culture is
not conceivable in the absence of an external electron acceptor. In the absence of oxygen as terminal
electron acceptor, energy conservation may be accomplished by anaerobic respiration with nitrate, ferric iron or sulfate (Table 1). Accordingly, all anaerTable 1
Stoichiometric equations of anaerobic bacterial toluene oxidation
coupled to the reduction of di¡erent electron acceptors
(1) Denitrifying bacteria :
‡
C7 H8 +7.2 NO3
3 ‡ 0:2 H
C 7 HCO3
3 ‡ 3:6 N2 +0.6 H2 O
vG³P = 33554 kJ
(mol toluene)31
(2) Iron(III) reducing bacteria:
C7 H8 +94 Fe(OH)3
C 7 FeCO3 +29 Fe3 O4 +145 H2 O
vG³P = 33398 kJ
(mol toluene)31
(3) Sulfate reducing bacteria:
‡
3
C7 H8 +4.5 SO23
C 7 HCO3
4 ‡ 3 H2 O
3 ‡ 2:5 H +4.5 HS
vG³P = 3205 kJ
(mol toluene)31
(4) Methanogenic consortia: reactions catalyzed by `protonreducing' bacteria (a) and methanogens (b, c).
3
(a) C7 H8 +9 H2 O
C HCO3
3 ‡ 3 H3 C-COO
+4 H‡ +6 H2
vG³P = +166 kJ
(mol toluene)31
‡
(b) 6 H2 +1.5 HCO3
3 ‡ 1:5 H C 1.5 CH4 +4.5 H2 O
vG³P = 3203 kJ (6 mol H2 )31
(c) 3 H3 C-COO3 +3 H2 O
C 3 CH4 +3 HCO3
3
vG³P = 393 kJ
(3 mol acetate)31
Sum: C7 H8 +7.5 H2 O
‡
C 4.5 CH4 +2.5 HCO3
3 ‡ 2:5 H
vG³P = 3131 kJ
(mol toluene)31
obic hydrocarbon degrading strains, which are available as pure cultures, are either denitrifying, ferric
iron-reducing or sulfate-reducing bacteria (Table 2).
In addition, some bacteria may dispose of the reducing equivalents recovered from hydrocarbon oxidation by reducing protons to hydrogen, but this is
thermodynamically feasible only in syntrophic association with hydrogen-consuming microorganisms,
such as methanogens (see below). None of the `proton-reducing' hydrocarbon-degrading bacteria is
available in a de¢ned coculture or in pure culture.
A last group of anaerobic bacteria, which may principally use hydrocarbons as carbon- and electron
sources, are the anoxygenic photosynthetic bacteria.
However, although these bacteria are long known to
metabolize polar aromatic compounds, no hydrocarbon-metabolizing phototrophic bacteria are yet reported.
The denitrifying species described mineralize a variety of alkylbenzenes, including toluene, m-xylene,
ethylbenzene, propylbenzene, p-ethyltoluene and pcymene (for structures see Fig. 1) [16^23]. Some of
these strains were formerly classi¢ed as Pseudomonas
sp.; based on 16S rDNA sequence comparison, they
are now a¤liated with the genera Thauera and
Azoarcus within the L-subclass of the Proteobacteria
(Table 2). The known strains exhibit a wide substrate
spectrum for polar aromatic compounds, but are restricted to few aromatic hydrocarbons. Only a few of
the isolated strains use more than one aromatic hydrocarbon compound as substrate (Table 2). Denitrifying bacteria grow relatively fast on alkylbenzenes: the maximum growth rates on toluene reach
0.12 h31 (doubling time 6 h), which represents 70%
of the growth rate obtained when benzoate serves as
substrate [24]. The reaction balance for toluene oxidation under denitrifying conditions is given in Table
1. Hydrocarbon oxidation at the expense of nitrate
yields a high amount of free energy (Table 1).
Only one species of a ferric iron-reducing bacterium which degrades an aromatic hydrocarbon, Geobacter metallireducens (Table 2), has been reported.
Toluene is the only metabolisable hydrocarbon for
this bacterium, which is also capable of anaerobic
degradation of several other polar aromatic substrates [25]. The stoichiometry of toluene oxidation
by G. metallireducens is shown in Table 1. The theoretical free energy yield of hydrocarbon utilization
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Table 2
Overview of bacterial strains available in pure culture, which are capable of anaerobic hydrocarbon degradation
Species/strain [reference]
Hydrocarbons metabolized
Other key substrates (other than benzoate)
I. Denitrifying bacteria (L-subclass of proteobacteria)
Thauera aromatica K172 [42]
Toluene
Thauera aromatica T1 [73]
Toluene
Azoarcus sp. strain T [16] and
Toluene, m-xylene
unpublisheda
Azoarcus tolulyticus Tol4 (and
Toluene
other strains) [74]
Azoarcus tolulyticus Td15 [74]
Toluene, m-xylene
Strain ToN1 [21]
Toluene
Strain EbN1 [21]
Toluene, ethylbenzene
Strain PbN1 [21]
Ethylbenzene, propylbenzene
Strain EB1 [22]
Ethylbenzene
Strain mXyN1 [21]
Toluene, m-xylene
Strain T3 [75]
Toluene
Strain M3 [75]
Toluene, m-xylene
Strain mCyN1 [23]
Toluene, p-ethyltoluene, p-cymene
Strain mCyN2b [23]
p-Cymene
II. Ferric iron reducing bacterium (N-subclass of proteobacteria)
Geobacter metallireducens GS15 [25]
Toluene
b
Not reported
Not reported
Phenol, p-cresol, phenylacetate
Acetophenone, phenylalanine
Acetophenone, propiophenone, phenylacetate, phenol
Acetophenone, phenylacetate, phenol
3-Methylbenzoate, p-cresol
Not reported
Not reported
p-Cresol, phenylalanine, p-ethylbenzoate,
p-isopropylbenzoate
p-Ethylbenzoate, p-isopropylbenzoate
Phenol, p-cresol, phenylacetate
III. Sulfate reducing bacteria (N-subclass of proteobacteria)
Desulfobacula toluolica Tol2 [32]
Toluene
Strain PRTOL1 [33]
Toluene
Desulfobacterium cetonicum [34]
Toluene
Strain oXyS1 [34]
Toluene, o-xylene, o-ethyltoluene
Strain mXyS1 [34]
Toluene, m-xylene, m-ethyltoluene,
m-cymene
Strain Hxd3 [29]
Alkanes (C12 ^C20 ), 1-hexadecene
Strain Pnd3 [30]
Alkanes (C14 ^C17 ), 1-hexadecene
Strain TD3 [31]
Alkanes (C6 ^C16 )
a
Phenol, p-cresol, anthranilate, phenylalanine
p-Cresol, 3-methylbenzoate
p-Cresol, cyclohexanecarboxylate
Phenylacetate
p-Cresol, phenylacetate
Not reported
o-Methylbenzoate, benzylsuccinate
m-Methylbenzoate
1-Hexadecanol, 2-hexadecanol, fatty acids (C4 ^C18 )
1-Hexadecanol, fatty acids (C3 ^C18 )
Fatty acids (C4 ^C18 )
C.J. Krieger, M. Reinhard and A.M. Spormann, unpublished results.
Does not grow with benzoate.
by ferric iron-reducing bacteria is relatively high (Table 1); growth of these bacteria is probably limited
by the availability of the insoluble Fe(OH)3 . Reports
on degradation of benzene under ferric iron reducing
conditions indicate that the metabolic potential of
ferric iron reducing bacteria is probably wider than
demonstrated so far [26,27]. Other oxidized metals,
e.g. Mn(IV), may also serve as electron acceptors for
toluene-metabolizing bacteria, as suggested by experiments in sediments [28].
As shown in Table 1, several pure cultures of sulfate-reducing bacteria were isolated which were capable of utilizing alkanes and alkenes (Table 2; [29^
31]). In addition, four strains of alkylbenzene-metabolizing sulfate-reducing bacteria have been described
(Table 2). Two of those were restricted to toluene as
the only hydrocarbon substrate [32,33]. Two new
strains were recently isolated on o-xylene and m-xylene, respectively ; the o-xylene-degrading strain also
metabolises toluene and o-ethyltoluene, the m-xylene-degrading strain toluene, m-ethyltoluene and mcymene (for structures see Fig. 1) [34]. The reaction
equation for toluene oxidation by sulfate-reducing
bacteria is given in Table 1. This process yields
only small amounts of free energy (Table 1) and
relatively low growth rates of the bacteria are observed [30,32,33]. The metabolic potential of sulfate-reducing bacteria for degradation of alkylbenzenes is probably much wider than presently
known; even benzene and naphthalene appear to
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be degraded in anaerobic sediments under sulfatereducing conditions [35,36].
Finally, a fourth group of anaerobic bacteria utilizing various alkylbenzenes have been found in syntrophic association with methanogenic archaea
[37,38]. Hydrocarbons are converted to CO2 , H2 ,
and acetate. This disproportionation is thermodynamically feasible only if the steady state concentration of hydrogen (and possibly also of acetate) is
kept at a low level. This is achieved by hydrogen
and acetate consumption by methanogens. The low
free energy available from conversion of alkylbenzenes to CO2 and methane (Table 1) must sustain
all organisms involved in the syntrophic association.
The hydrogen partial pressure, which just allows
growth of both organisms of the consortium, is in
the range of 1 Pa (1035 atm). As a result, the syntrophic consortia disproportionate aromatic hydrocarbons to methane and bicarbonate (see Table 1).
3. Anaerobic catabolism of toluene
Interest in anaerobic toluene mineralization resulted initially from the observation that toluene
was readily degraded in sewage sludge and in anaerobic hydrocarbon contaminated sediments [37,39,40].
In an anaerobic enrichment culture with crude oil
under conditions of sulfate reduction, toluene was
the most rapidly consumed of the utilisable alkylbenzenes [31,41]. Since 1990, pure cultures of bacteria
mineralizing toluene anaerobically have been isolated. These include denitrifying, sulfate-reducing,
as well as iron-reducing bacteria that belong to the
L- and N-subclasses of the Proteobacteria (e.g. [18^
20,25,32,33,42]).
Benzoate (or its CoA-thioester) has been recognized as a central intermediate in anaerobic mineralization of numerous aromatic compounds. In many
anaerobic toluene-mineralizing cultures, benzoate
has been detected as a transiently excreted product
[17,43^46]. Thus, the initial series of reactions in
anaerobic toluene degradation apparently involves
the conversion of toluene to benzoate (or benzoylCoA). The reactions leading to de-aromatization of
benzoyl-CoA and further degradation of the alicyclic
intermediates are discussed by Harwood et al. in this
volume.
3.1. Pathway of anaerobic toluene conversion to
benzoate
Based on in vitro studies with two denitrifying
bacteria, Thauera aromatica and Azoarcus sp. strain
T [24,47], a pathway of toluene oxidation to benzoylCoA was proposed (Fig. 2).
The ¢rst reaction is the addition of toluene to
fumarate to form benzylsuccinate (Fig. 2). Free benzylsuccinate was identi¢ed as a transient intermediate
of anaerobic toluene oxidation by both in vitro experiments and in vivo isotope trapping experiments
in T. aromatica [24]. Kinetic studies of benzylsuccinate formation from toluene and fumarate in Azoarcus sp. strain T demonstrated that the in vitro rate of
benzylsuccinate formation was about 30% of the in
vivo rate of toluene consumption [47], suggesting
that this reaction actually represents the ¢rst step
in anaerobic toluene mineralization. Accumulation
Fig. 2. Proposed pathway of anaerobic oxidation of toluene to benzoyl-CoA. The initial reaction is the addition of fumarate to the methyl
group of toluene, catalyzed by benzylsuccinate synthase (enzyme 1). The hypothetical further steps are analogous to L-oxidation of Kmethyl-branched fatty acids, with a CoA-transferase (enzyme 2) initiating the pathway. The proposed enzymes catalyzing these further reactions are given below (3^6).
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of benzylsuccinate in culture media was observed in
earlier studies of anaerobic toluene mineralization,
but was interpreted as the result of dead-end metabolism [33,43^46].
Oxidation of benzylsuccinate to benzoyl-CoA was
proposed to proceed via a modi¢ed L-oxidation
pathway (Fig. 2). The existence of this pathway is
supported by several experimental ¢ndings: (1)
CoA-dependent conversion of benzylsuccinate to
benzoyl-CoA did not require ATP [24]; (2) E-phenylitaconate (or the CoA thioester) was identi¢ed as an
oxidation product of benzylsuccinate [47]; (3) benzylsuccinate oxidation to E-phenylitaconate and benzoyl-CoA was signi¢cantly increased when succinylCoA served as the source of CoA [48]; and (4) benzylsuccinate:succinyl-CoA CoA-transferase activity
was detected in toluene-grown cells of T. aromatica
(C. Leutwein and J. Heider, unpublished). Therefore,
activation of benzylsuccinate to the CoA-thioester is
apparently catalyzed by a CoA-transferase rather
than an ATP-dependent CoA ligase. Benzylsuccinyl-CoA is thought to be subsequently oxidized to
E-phenylitaconyl-CoA by a benzylsuccinyl-CoA dehydrogenase. The next three postulated enzymatic
reactions are hydration to 2-carboxymethyl-3-hydroxy-phenylpropionyl-CoA, oxidation to benzoylsuccinyl-CoA, and thiolytic cleavage to benzoylCoA and succinyl-CoA (Fig. 2). None of these
postulated intermediates or enzyme activities have
yet been detected in cell-free extracts. Considering
the expected redox potentials in analogy to L-oxidation, the reducing equivalents generated from oxidation of benzylsuccinyl-CoA to E-phenylitaconyl-CoA
(Fig. 2, enzyme 3) are probably transferred to the
quinone pool, whereas the 3-hydroxyacyl-CoA dehydrogenase (Fig. 2, enzyme 5) probably reduces
NAD‡ or NADP‡ . The third two-electron oxidation
step required for toluene oxidation to benzoyl-CoA
is accomplished by utilizing the oxidized co-substrate
fumarate in the ¢rst step, which is released in the
reduced form as succinate (Fig. 2). Regeneration of
fumarate from succinate by succinate dehydrogenase
involves another transfer of reducing equivalents to
the quinone pool.
Elucidation of the unusual pathway of anaerobic
toluene degradation described above in T. aromatica
and Azoarcus strain T raises the question of whether
this pathway is unique to these species or a general
465
mode for anaerobic toluene mineralization. Benzylsuccinate formation from toluene and fumarate was
also found in two toluene-mineralizing sulfate-reducing strains [49,50]. The sulfate-reducing strains represent a phylogenetically distant group of bacteria
(N-subclass of proteobacteria), suggesting that anaerobic toluene metabolism generally proceeds via benzylsuccinate.
3.2. Benzylsuccinate synthase, the enzyme catalyzing
the initial reaction in anaerobic toluene
mineralization
Benzylsuccinate formation from toluene and fumarate appears to be the initial reaction in anaerobic
toluene oxidation [24,47]. This enzymatic reaction,
catalyzed by benzylsuccinate synthase, has several
novel features. Firstly, enzymatic toluene addition
to fumarate does not involve a net redox reaction.
This is in contrast to all known toluene-transforming
oxygenases, which catalyze an oxidation of the hydrocarbon substrate [51,52]. Secondly, benzylsuccinate formation represents a unique biochemical reaction of forming a carbon^carbon bond, which di¡ers
from carboxylations, aldolase-type and oxo-acid
lyase-type reactions [53].
3.2.1. Properties of benzylsuccinate synthase
The novel enzyme benzylsuccinate synthase catalyzes the addition of toluene to fumarate (Fig. 2).
Benzylsuccinate synthase activity was extremely sensitive to inactivation by air, exhibiting a half life time
of only 20^30 s. The enzyme was reversibly inhibited
by the substrate analogs benzyl alcohol, benzaldehyde or phenylhydrazine [24,54]. Benzylsuccinate
synthase was puri¢ed under anoxic conditions from
toluene-grown cells of T. aromatica. The pure enzyme was extremely oxygen-sensitive and did not
require further co-substrates. The enzyme contained
a redox-active £avin cofactor, but no iron^sulfur
clusters. It had a native mass of 220 kDa and consisted of three subunits of apparent masses of 98
kDa (K), 8.5 kDa (L) and 6.4 kDa (Q). Based on
the native mass, an K2 L2 Q2 composition of benzylsuccinate synthase is assumed. Half of the K-subunits
showed a C-terminal truncation of 4 kDa, producing
an KP-fragment of 94 kDa with identical N-terminal
amino acid sequences.
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The amino acid sequences of the subunits of benzylsuccinate synthase were derived from the corresponding genes (see below). The K-subunit showed
strong similarity to enzymes containing glycyl radicals, which have so far been represented only by
pyruvate-formate lyase and anaerobic ribonucleotide
reductase (Sawers, this issue). Based on the catalyzed
reactions and the recorded similarity scores, benzylsuccinate synthase represents a new subclass of the
glycyl-radical containing enzymes. The presence of a
glycyl radical in benzylsuccinate synthase was inferred from characterization of the observed truncation product (KP) of the large subunit, which is
thought to be generated by oxygenolytic cleavage
of the polypeptide backbone at the site of the glycyl
radical [54]. The observed pattern of nearly equal
amounts of intact and truncated K-subunits suggests
that only one of the two large subunits of the benzylsuccinate synthase holoenzyme carries a glycylradical. The same type of oxygenolytic cleavage is
known to exist for the other glycyl-radical enzymes
([55,56]; Sawers, this issue).
3.2.2. Proposed reaction mechanism of
benzylsuccinate synthase
A reaction mechanism of benzylsuccinate synthase
involving radical intermediates is suggested by a
number of experimental observations: ¢rstly, benzylsuccinate synthase was identi¢ed as a possible new
glycyl-radical enzyme by sequence similarity [54,57].
Secondly, the predicted glycyl radical site was identical with the determined site of oxygenolysis [54].
Thirdly, the proposed radical-carrying glycine and
a conserved cysteine of benzylsuccinate synthase,
which are also part of the active center of pyruvate
formate-lyase [58,59], were essential for growth on
toluene, as shown by mutagenesis and genetic complementation studies [57] (see below). Finally, GC/
MS analysis of benzylsuccinate formed from [methyl-D3 ]toluene showed that the deuterium atom abstracted from the methyl group of toluene is retained
in the succinyl moiety of benzylsuccinate [47]. Considering these observations, a radical reaction mechanism is proposed as shown (Fig. 3).
The proposed reaction mechanism of benzylsuccinate synthase begins with the radical-containing, activated form of the enzyme (Fig. 3). It appears plaus-
Fig. 3. Proposed reaction mechanism of benzylsuccinate synthase.
The radical-containing enzyme produces a benzyl radical from
toluene, which adds to fumarate to form a benzylsuccinyl radical.
The enzyme converts the product radical to benzylsuccinate, and
the radical form of benzylsuccinate synthase is regenerated. The
H-atoms originating from the methyl group of toluene are highlighted to indicate their retention in the benzylsuccinate formed.
ible that an enzyme-based radical of active benzylsuccinate synthase ¢rst abstracts a hydrogen atom
from toluene to yield a benzyl radical at the active
site. This radical would then add to the double bond
of fumarate, forming a benzylsuccinyl radical. This
type of radical addition to a C^C double bond is well
known in organic chemistry and is employed in freeradical polymerization reactions [4]. Finally, the enzyme would donate the abstracted hydrogen atom
back to the benzylsuccinyl radical, thus forming benzylsuccinate and at the same time regenerating the
enzyme radical (Fig. 3). The strong homology to
pyruvate formate-lyase suggests that the glycyl radical may react with the conserved cysteine residue to
form an intermediate thiyl radical, which actually
abstracts the hydrogen atom.
Formation of the radical form of pyruvate formate-lyase in Escherchia coli is catalyzed by pyruvate
formate-lyase activase with S-adenosyl-methionine
and reduced £avodoxin as co-substrates [56]. The
discovery that the benzylsuccinate synthase operon
contains an essential open reading frame which exhibits signi¢cant similarity to pyruvate formate-lyase
activase, suggests that the protein encoded by this
open reading frame may well generate the radical
(active) form of benzylsuccinate synthase by a similar mechanism [54,57].
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3.3. Genetics of anaerobic toluene mineralization
The genes coding for the subunits of benzylsuccinate synthase (bssA, B and C) were cloned and sequenced from T. aromatica. A single gene codes for
both forms of the large subunit (K and KP), whereas
the small L and Q subunits are encoded by separate
genes [54]. Together with another gene, bssD (Fig. 4),
these genes form a toluene-inducible operon (Fig. 4).
The predicted bssD gene product shows strong similarity with activating enzymes required for radical
generation in other glycyl radical enzymes. The
bssD gene is cotranscribed with the structural genes
of benzylsuccinate synthase and is probably regulated at the translational level [54].
An independent genetic analysis of anaerobic toluene metabolism was conducted in Thauera sp.
strain T1. Screening of 10 000 chemically induced
mutants for loss of the ability to grow anaerobically
with toluene resulted in the identi¢cation of four
mutants (named tut for toluene utilization), which
were unable to grow with toluene anaerobically,
but still metabolized benzoate. Three of these mutants have so far been characterized. They are affected in three di¡erent genes, which are closely clustered on one cosmid clone [57,60]. One of these
genes, tutB, appears to be involved in regulation of
gene expression [60]. The other two genes are virtually identical to the genes coding for the large subunit of benzylsuccinate synthase (tutD and bssA;
80% identity) and the activating enzyme (tutE and
bssD; 62% identity) from T. aromatica. In addition,
equally close homologs of the genes for the two
small subunits of benzylsuccinate synthase (bssB
and bssC) are linked to tutD and tutE in Thauera
sp. strain T1, and the operon organization is identical in these two strains. Substitutions of the predicted active site residues glycine 828 and cysteine
Fig. 4. Organization of the bss operon, coding for benzylsuccinate synthase. The gene products encoded by the di¡erent genes
are indicated below the genes.
467
492 by alanine in TutD/BssA of Thauera sp. strain
T1 failed to complement the mutant phenotype [57].
Thus, a biochemical and a genetic approach have
identi¢ed independently the same genes for benzylsuccinate synthase and its putative activase.
The gene products of the bssABC genes of T. aromatica have been shown to be the subunits of benzylsuccinate synthase; the bssD gene probably codes
for the activating enzyme required for converting
benzylsuccinate synthase into the active (radical-containing) state. Although it has not yet been demonstrated, it appears likely that Thauera sp. strain T1
utilises a pathway of anaerobic toluene catabolism
identical to the benzylsuccinate pathway shown in
Fig. 2. This, along with the above mentioned identities of the tut and bss genes, leads us to suggest the
adoption of the bss nomenclature for the structural
genes involved in toluene conversion to benzylsuccinate also for strain T1.
4. Anaerobic catabolism of ethylbenzene
Anaerobic mineralization of ethylbenzene has been
reported in three denitrifying bacteria, strains EbN1,
PbN1 and EB1 [21,22]. The ¢rst two strains were
isolated from freshwater mud, while the latter strain
originated from an oil re¢nery treatment pond. All
three isolates are closely related to each other, and
are a¤liated with the genus Azoarcus in the L-subclass of Proteobacteria, as indicated by 16S rDNA
sequence analysis.
In ethylbenzene-mineralizing cultures, benzoate
was detected as a transient intermediate [22]. This
suggested that benzoate or benzoyl-CoA was an intermediate in the catabolism of ethylbenzene, as
found in the degradation of many other aromatic
compounds. The pathways employed for anaerobic
oxidation of the two alkylbenzenes toluene and ethylbenzene to the oxidation level of benzoate, however, appear to be quite di¡erent.
A pathway for the anaerobic oxidation of ethylbenzene to benzoyl-CoA has been proposed, based
on growth experiments [21] and cell suspension studies ([22]; K. Zengler, C. Champion, R. Rabus and F.
Widdel, unpublished data). Strains EbN1 and EB1
were able to grow with 1-phenylethanol and acetophenone as sole carbon and electron sources under
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Fig. 5. Proposed pathway of anaerobic ethylbenzene degradation to the level of benzoyl-CoA. The enzymes needed for catalyzing the reactions of the proposed pathway are given (1^5). Note that input of energy is required for carboxylation of acetophenone and for CoAthioester formation of benzoylacetate. The co-substrate requirements of these reactions are not known.
denitrifying conditions [21,22]. Formation of both
compounds from ethylbenzene was demonstrated in
cell suspensions and cell extracts ([22,50]; H.A.
Johnson and A.M. Spormann, unpublished data).
The proposed initial reaction for this pathway is
the oxidation of ethylbenzene to 1-phenylethanol.
The oxygen atom of the hydroxyl group of 1-phenylethanol is derived from water, as shown by labeling
studies with 18 O-water and GC/MS analysis, con¢rming that the reaction de¢nitely occurred under
strict exclusion of molecular oxygen [22]. 1-Phenylethanol is further oxidized to acetophenone. As inferred from the calculated redox potentials of analogous compounds (e.g. isopropanol/propane,
E³P = 328 mV; acetone/isopropanol, E³P = 3323
mV), the redox equivalents generated from oxidation
of ethylbenzene to 1-phenylethanol (enzyme 1, Fig.
5) are probably transferred to the quinone pool;
those generated from oxidation of 1-phenylethanol
to acetophenone may have a su¤ciently negative potential to be transferred to NAD‡ or NADP‡ .
Only indirect evidence is available for the further
reactions involved in acetophenone conversion to
benzoyl-CoA. It is proposed that acetophenone is
carboxylated to benzoylacetate (3-oxophenylpropionate) in a reaction analogous to reactions found in
aerobic and anaerobic degradation of aliphatic ketones [61,62]. This is supported by several experimental ¢ndings. Firstly, growth of both organisms
on ethylbenzene or acetophenone occurred only in
the presence of CO2 ([22]; K. Zengler, C. Champion,
R. Rabus and F. Widdel, unpublished data). Furthermore, an unknown compound, which exhibited
a similar UV-visible spectrum to acetophenone and
may be the proposed carboxylation product benzoylacetate, was found to be transiently formed during
acetophenone metabolism [22]. Benzoylacetate is
proposed to be activated to the CoA thioester and
to be cleaved thiolytically to acetyl-CoA and benzoyl-CoA (Fig. 5).
Strain EbN1 is able to metabolize two di¡erent
hydrocarbon substrates, toluene and ethylbenzene
[21]. Cells grown on either of the two substrates exhibited the enzyme activities necessary for metabolism of the respective growth substrate, but did not
metabolize the other hydrocarbon compound. This is
consistent with the use of completely di¡erent initial
metabolic routes for ethylbenzene and toluene
([21,22,50]; H.A. Johnson and A.M. Spormann, unpublished data; K. Zengler, C. Champion, R. Rabus
and F. Widdel, unpublished data).
5. Anaerobic metabolism of alkanes
The ¢rst pure culture of an alkane-degrading bacterium was reported in 1991. This bacterium, sulfatereducing strain Hxd3, grows on hexadecane and other long chain alkanes under strictly anaerobic conditions. The degradation balance showed that hexadecane was completely oxidized to CO2 at the
expense of sulfate [29]. Since then, several further
sulfate-reducing alkane-degrading isolates have
been obtained which grow on alkanes with chains
of six and more carbon atoms [30,31]. The known
alkane-degrading sulfate-reducing bacteria are nutri-
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tionally and phylogenetically unrelated to Desulfovibrio species [30]. The biochemical basis of hydrocarbon metabolism in these organisms is still poorly
understood. Experiments with cell suspensions have
indicated that anaerobic alkane degradation probably does not occur via dehydrogenation to a 1-alkene and hydration to an alcohol [30], a hypothetical
mechanism presented in former literature. Most interestingly, one of the isolated alkane-degrading sulfate reducers, strain Hxd3 (Table 2), was found to
produce membrane lipids containing odd numbers of
C-atoms from alkanes with an even number of Catoms (and vice versa). In contrast, control cells
grown on 1-alkenes or fatty acids did not show
this shift in the carbon chain length of cellular fatty
acids. This indicates that alkanes and alkenes have
di¡erent metabolic routes in this strain, and that
addition or removal of a metabolite containing an
odd number of C-atoms is involved in anaerobic
alkane catabolism. The most plausible explanation
for this would involve an initial carboxylation or
carbonylation of the activated alkane to produce
the Cn‡1 -fatty acid or aldehyde [30]; this would formally correspond to a reversal of the assumed biosynthetic reactions involved in alkane biosynthesis
[5]. However, the described alteration of the chain
length of fatty acids upon growth on alkanes has
not been observed in any of the other known strains
capable of growing anaerobically on alkanes (see
Table 2). This suggests that there are di¡erent mechanisms for initiating anaerobic alkane metabolism,
and that the mechanism employed depends on the
bacterial strain [30].
6. Regulation of anaerobic hydrocarbon metabolism
In all cases studied, the enzymes of anaerobic hydrocarbon metabolism are clearly substrate-induced.
Catabolic enzymes for toluene are only present in
cells grown on toluene, not in cells grown on other
substrates, as demonstrated for T. aromatica [63],
strain EbN1 and the sulfate-reducing D. toluolica
[50]. Genes coding for two-component regulatory
systems possibly involved in induction of gene expression by toluene have been described in Thauera
strain T1 [60] and T. aromatica [64]. The exact regulatory mechanisms remain unknown.
469
Although ethylbenzene is chemically very similar
to toluene, no cross-induction of catabolic enzymes
for the two alkylbenzenes was observed in cells of
strain EbN1 grown either on toluene or on ethylbenzene [50]. Details of the regulatory systems are not
known.
Anaerobic alkane metabolism is induced only in
cells grown on alkanes. It has been shown that synthesis of new proteins is necessary in fatty acidgrown cells in order to gain the ability to metabolize
alkanes [30]. All known anaerobic alkane-degrading
bacteria use a limited and clearly de¢ned range of
chain lengths; this range di¡ers between the various
strains [29^31].
7. Ecological aspects of anaerobic hydrocarbon
degradation
Hydrocarbon compounds as substrates for aerobic
and anaerobic bacteria have probably always been
available near natural petroleum deposits or petroleum formation sites, e.g. the Guaymas Basin [65].
Since one can assume continuous spreading of hydrocarbons into anaerobic environments over geological periods, the existence of bacteria capable of
anaerobic hydrocarbon degradation is understandable from an ecological standpoint. In modern environments, polluted by human activity, these organisms are probably enriched, together with the longknown aerobic hydrocarbon degraders thriving in
the oxic zones.
Hydrocarbon catabolism by anaerobic bacteria
may cause serious problems in the petroleum industry. Secondary extraction of petroleum deposits is
often performed by injection of sea water, which
may introduce a bacterial inoculum, along with sulfate as an electron acceptor, into the sulfate-depleted
reservoir. The extracted oil^water mixtures are suitable growth media for sulfate-reducing bacteria. Several constituents of petroleum serve directly as electron donors for sulfate reduction to sul¢de [31],
which causes `souring' of the petroleum and gas.
Furthermore, precipitation of insoluble metal sul¢des may interfere with extraction of the oil and its
separation from water, and free sul¢de corrodes
pipelines and storage tanks even in the absence of
oxygen. Hydrogen sul¢de escaping into the air
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presents a hazard for the workers in oil production
plants.
Anaerobic bacterial hydrocarbon degradation may
be technically exploited in the bioremediation of
some hydrocarbon-polluted sites. Because the anaerobic processes are usually slower and less e¤cient
than degradation under aerobic conditions, this application is only appropriate at sites with limited
access of air, or which can not be aerated easily.
Examples are contaminated groundwater aquifers,
for which treatment with nitrate as anaerobic electron acceptor has been shown to favor the rate and
the extent of bioremediation [66,67].
8. Conclusion/outlook
The ¢eld of anaerobic microbial degradation of
hydrocarbon compounds has developed only recently, but we are already beginning to understand
some of the underlying mechanisms by which these
rather inert substrates are attacked without the aid
of molecular oxygen. The few anaerobic initiation
reactions known are surprisingly diverse, compared
to aerobic pathways, which are always initiated by
an oxygenation reaction. Only in the case of hydrocarbons with similar structure and reactivity can we
expect bacteria to make use of similar anaerobic degradation pathways. For example, propylbenzene catabolism is probably analogous to that of ethylbenzene [21], and m-xylene is apparently degraded by
certain bacterial strains via a pathway analogous to
that outlined for toluene (C.J. Krieger, M. Reinhard
and A.M. Spormann, unpublished data). The o- and
p-xylene isomers can also be co-metabolized with
toluene and converted to dead-end products by
some bacterial strains [33,44,47,68]. So far, only
one pure culture growing on o-xylene has been obtained [34], and no pure cultures growing on p-xylene
have yet been reported. The metabolic pathways
used for the mineralization of these xylene isomers
remain unknown. Anaerobic degradation of some
other hydrocarbons probably proceeds via unique
and novel pathways. Examples include benzene and
naphthalene, which have been considered to be very
recalcitrant to anaerobic degradation in the laboratory in the past, but which have recently been shown
to be degraded in anaerobic environments [35^
37,69]. The initial attack of these compounds under
anoxic conditions is thought to be accomplished by
novel mechanisms, for example by direct oxidation
or carboxylation of the aromatic ring, as suggested
by metabolic studies of mixed cultures [37,70]. Finally, there have been reports for about 20 years
that there is signi¢cant anaerobic oxidation of the
most abundant alkane in nature, methane, in anoxic
sediments and sewage sludge [71]. Some reports suggest that methanogenic archaea participate in this
reaction, formally via a reversal of methanogenesis
[71,72], but there is no de¢nitive information available on the organisms and the biochemical reactions
involved in anaerobic methane oxidation in natural
environments.
Acknowledgments
J.H. acknowledges Professor G. Fuchs (Mikrobiologie, Universitaët Freiburg) for his constant support
and encouragement, as well as the ¢nancial support
of the Deutsche Forschungsgemeinschaft. Research
in the laboratory of A.M.S. was supported by NSF
Grants MCB 9723312 and MCB 9733535, by Grant
R-815738 of the O¤ce of Research and Development, U.S. Environmental Protection Agency
through the Western Region Hazardous Research
Center, and by a Terman Award to A.M.S. The
work of F.W. was supported by the Deutsche Forschungsgemeinschaft, the Max-Planck-Gesellschaft
and the Fonds der chemischen Industrie.
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