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Applied Microbiology and Biotechnology

©

Springer
-
Verlag

2005

10.1007/s00253
-
004
-
1864
-
3

Mini
-
Review

Biodegradation of xenobiotics by anaerobic
bacteria

Chunlong

Zhang
1

and George

N.

Bennett
2


(1)


Department of Environmental Sciences, University of Houston
-
Clear Lake, Houston,
TX

77058, USA

(2)


Department of Biochemistry and Cell Biology, Rice University, 6100 Main St., Houston,
TX

77005, USA


George

N.

Bennett

Email:
gbennett@bioc.rice.edu

Phone:
+1
-
713
-
3484920

Fax:
+1
-
713
-
3485154

Received:
28

September

2004


Revised:
29

November

2004


Accepted:
30

November

2004


Published online:
26

January

2005

Abstract


Xenobiotic biodegradation under anaerobic conditions such as in groundwater,
sediment, landfill, sludge digesters and bioreactors has gain
ed increasing attention over the last
two decades. This review gives a broad overview of our current understanding of and recent
advances in anaerobic biodegradation of five selected groups of xenobiotic compounds
(petroleum hydrocarbons and fuel additives
, nitroaromatic compounds and explosives,
chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances
have been made toward the isolation of bacterial cultures, elucidation of biochemical
mechanisms, and laboratory and
field scale applications for xenobiotic removal. For certain
highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily
substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic
processes

are generally used, anaerobic biodegradation is significant under certain circumstances
(e.g., O
2
-
depleted aquifers, oil spilled in marshes). For persistent compounds including
polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for
remedial
application, but can be a significant long
-
term avenue for natural attenuation. In some cases, a
sequential anaerobic
-
aerobic strategy is needed for total destruction of xenobiotic compounds.
Several points for future research are also presented i
n this review.


Introduction

Anaerobic biodegradation of xenobiotic compounds has been a subject of extensive research
during the last two decades. Consequently, our current understanding of the dissipation
mechanisms of xenobiotics in natural anaerobic e
nvironments has considerably improved. Many
anaerobe
-
based bioreactors and remediation systems have been developed to effectively clean
-
up
contaminated media. The purpose of this review is to summarize recent advances in our
understanding and briefly descr
ibe biotechnological applications for the biodegradation of five
major groups of xenobiotic compounds: petroleum hydrocarbons and related fuel additives,
nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds,
pesticides, and
surfactants.

The review is not intended to be exhaustive, but focuses on representative anaerobes, their
biochemical mechanisms, and potential biotechnological and environmental implications.
Several excellent reviews have been published on anaerobic biode
gradation of xenobiotics, both
in general (Janke and Fritsche
1985
; Mogensen et al.
2003
a; Schink
2002
) or focused on specific
compounds including petroleum hydrocarbons (Chakraborty and Coates
2004
; Heider and Fuchs
1997
; Prince
1993
; Spormann and Widdel
2000
; Wi
ddel and Rabus
2001
), explosives (Ahmad
and Hughes
2000
; Esteve
-
Nuñez et al.
2001
; Gorontzy et al.
1994
; Marvin
-
Sikkema and de Bont
1994
; Peres and Agathos
2
000
), chlorinated compounds (Abramowicz
1990
; Bedard
2003
; Chen
2004
; El Fantrous
si et al.
1998
; Fetzner
1998
; Haggblom et al.
2000
; Ohtsubo et al.
2004
), and
pesticides (Sethunathan
1973
; Williams
1977
).

There are several reasons why
anaerobic biodegradation of xenobiotics is important to
researchers and practitioners. Aerobic processes require expensive O
2

delivery systems,
maintenance is often high due to biofouling in subsurface remedial applications (Baker and
Herson
1994
), and there are high energy costs and sludge production when bioreactors are
employed (Jewell
1987
; McCarty and Smith
1986
). In addition, anaerobic conditions naturally
prevail in most cases for contaminated groundwater, and some xenobiotic compounds [e.g.,
tetrachloroethylene, polychlorinated biphenyls (PCBs), and nitro
-
substituted aromatic
s] can be
efficiently transformed or mineralized only by anaerobic bacteria. In some cases, aerobic
degradation does not occur without a prior anaerobic process (Master et al.
2002
).


Major groups of anaerobic organisms involved in
xenobiotic biodegradation

Like their aerobic counterparts, anaerobic bacteria able to degrade xenobiotic compounds are
diverse and pr
esent in various anaerobic habitats, including sediments, water
-
laden soils,
gastrointestinal contents, reticulo
-
ruminal contents, feedlot wastes, sludge digesters, groundwater,
and landfill sites (Williams
1977
). Anaerobes use natural organics such as proteins,
carbohydrates, and many others as carbon and energy sources. Many of the so
-
called xenobiotic
compounds

of environmental concern have naturally occurring relatives (Wackett et al.
2002
).
For other xenobiotics, rep
eated exposure has resulted in the adaptation and evolution of
anaerobic bacteria capable of metabolizing these man
-
made compounds.

Table

1

lists the major groups of anaerobic microorganisms involved in biodegradation of
selected xenobiotic compounds. The pure bacterial cultures given in this table are by no means
exhaustive but are representative of each compound c
ategory. In reporting these bacteria with
compound
-
specific metabolic capability, two classical strategies are commonly employed. Some
researchers have chosen to employ pure cultures of previously isolated anaerobic strains to test
with specific compounds,

whereas others have focused on the isolation and identification of new
strains from anaerobic bacterial consortia or enrichment cultures (El Fantroussi et al.
1998
).
Without a systematic screening approach, the number of bacterial cultures successfully isolated
is limited since only a small portion of what is present in the actual microbial habitat has been
tested.

In other cases, several syntrophic bacterial strains of a bacterial consortium co
-
exist to
metabolize a specific compound (El Fantroussi et al.
1998
; Janke and Fritsche
1985
; Williams
1977
). Despite these limitations, the diversity of anaerobic microorganisms able to biodegrade
xenobiotic compounds is apparent.

Table 1


Major grou
ps of anaerobic microorganisms involved in xenobiotic biodegradation.

PAH

Polycyclic aromatic hydrocarbon,

MTBE

methyl
tert
-
butyl ether,

TNT

trinitrotoluene,

DNT

dinitrotoluene,

RDX

hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine,

HMX

octahydro
-
1,3,5,7
-
tetranitro
-
1,3,5,7
-
tetrazocine,

PCE

tetrachloroethylene,

TCE

trichloroethene,

DCE
cis
-
dichloroethene,
VC

vinyl chloride,

PCB

polychlorinated biphenyls,

PCP

pentachlorophenol,

LAS

linear alkylbenzene
sulfonate,
LAEOs

linear alcohol ethyoxylates

Compounds

Bacteria name
a
, source of isolation
b
, chemical
action

Reference

Alkane

D. oleovorans

(P): mineralizes C
12

C
20

n
-
alkane

Aeckersberg et al.
1991

Benzene

G.

spp. (P): oxidizes benzene in Fe(II)
-
reducing
conditions

Coates et al.
2001
; Rooney
-
Varga et al.
1999

Dechloromonas

spp. (S): mineralizes benzene
into CO
2

in 5

days

Toluene

G. metallireduce
ns

(S): first pure culture (Fe
3+

reducing) for toluene oxidation

Chakraborty and Coates
2004
; Lovley et al.
1989

Azoarcus

and
Thauera

spp. (S/D): facultative
toluene
-
oxidizing nitrate
-
reducers

Ethylbenzene

Thauera
-
rela
ted (S/P): denitrifying bacteria
completely mineralize ethylbenzene

Ball et al.
1996
; Rabus and
Widdel
1995

Xylene

D. acetonicum
-

and
Desulfosarcina variabilis
-
related: mineralizes
o
-

and
m
-
xylene

Harms et al.
1999
; Hess et al.
1997
; Rabus and Widdel
1995

PAHs

Acidovorax
,
Bordetella
,
Pseudomonas
,
Sphingomonas
, and
Variovorax

(S): degradation
complete for naphthalene and partial for 3

㔠物rg
P䅈猻s
P. stutzeri

and
Vibriop pelagius

related
(S): mineralizes 7

㈰2 灨瑨慬p湥

䕲楫獳潮⁥琠慬⸠
2003
; Rockne
et al.
2000

MT
BE

Pure aerobes isolated; slow under anaerobic
conditions, no pure anaerobes isolated

Finneran and Lovley
2001
;
Stocking et al.
2000

TNT, DNT

Veillonella alkalescens

(D): the earliest evidence
of anaerobic TNT degradation

C.

spp. and
Esteve
-
Nuñez et.
2001
;
Hug
hes et al.
1999
;
Compounds

Bacteria name
a
, source of isolation
b
, chemical
action

Reference

Desulfovibrio

spp. (N): most extensively studied
genera transforming TNT

McCormick et al.
1976

RDX, HMX

Desulfovibrio

spp. (S): uses RDX and HMX as
sole N source

Boopathy et al.
1998
; Kitts et
al.
1994
; Young et al.
1997
;
Zhang and Hughes
2003
;
Zhao et al.
2002

Providencia

sp., and
M. morganii

(S): transforms
into n
itroso derivatives

Serratia marcescens

(M): RDX ring cleavage
similar to McCormick
s pathway

C.

acetobutylicum

(N): transforms RDX into
NHOH and NH
2

derivatives

K. pneumoniae

(D): degrades RDX into HCHO,
CO
2

and N
2
O

PCE, TCE

A. woodii
,
C. formicoaceticum
,
Methanolobus
tindarius
,
Methanosarcina sp
.,
Methanosarcina
mazei, Methanosarcina thermphil
a
,
Sporomusa
ovata

(N): previously known strains transforming
PCE & TCE

El Fantroussi et al.
1998
;
Fathepure and

Boyd
1988
;
Jablonski and Ferry
1992
;
Terzenbach and Blaut
1994

Desulfitobac
terium

sp. (S): transforms PCE to
TCE and trace amount of DCE

De Bruin et al.
1992
; Gerritse
et al.
1996
; Gerritse et al.
1999
; Magnuson et al.
2000
;
Maymo
-
Gatell et al.
1997
;
Sung et al.
2003

Dehalobacter restrictus

(S): transforms PCE to
ethene

Desulfitobacterium frappieri

(S/D): tranforms
PCE & TCE into cis
-
DCE

Dehalococcoides ethenogenes

(N): completes
PCE & TCE degradation into ethene

Desulfu
romonas michiganensis

(S): able to grow
on free
-
phase PCE

VC

Dehalococcoides

sp. (A): able to grow with VC
and transform VC into ethene

He et al.
2003

PCBs

Desulfitobacterium dehalogenans

(S):
dehalogenates flanking Cl of OH
-
PCBs

Wiegel et al.
1999

PCP

Desulfitobacterium frappieri

(S/D): 90

99% PCP
removal forming 3
-
CP

Beaudet et al.
1998
; Bouchard
et al.
1996
; Shelton and
Tiedje
1984
; Tartakovsky et
al.
1999

Desulfitobacterium halogenans

(S),
Desulfitobacterium chlororespirans

(C),

Desulfomnile tiedje

(N): dechlorinates at
o
-

and
m
-

position

Dioxins

Dehalococcoides sp
. (S): uses dioxins as the sole
Bunge et al.
2003

Compounds

Bacteria name
a
, source of isolation
b
, chemical
action

Reference

electron acceptor

Chlorinated
pesticide

C.

sp. (N): degrades DDT as the sole C source.
Degrades other chlorinated pesticides

Ruppe et al.
2003
,
2004
;
Sethunathan
1973
; Williams
1977

Aerobacter aerogenes
,
K. pneumoniae
,
N.
vulgaris

(S): DDT
-
degrading

Dehalospirilum multivorans
: preferentially
dechlorinates technical toxaph
ene

P
-
based
pesticide

Flavobacterium

sp. (S): attacks P
-
insecticides
including diazino and parathion

Sethunathan
1973

Carbamate
pesticide

K. pneumoniae

(D): uses three chlorinated
s
-
triazines as the sole N source

Ernst and Rehm
1995
;

Dinoseb
pesticide

C. bifermentans

(D): utilizes Dinoseb as a sole C
via cometabolism

Hammill and Crawford
1996

Anionic
surfactant

Strain RZLAS (D): the only pure anaerobe using
LAS as the sole S source

Denger and Cook
1999

Nonionic
surfactant

Pelobacter propionicus

&
A.

sp. (D): LAEOs
fermented to CH
4

and CO
2

Wagener and Schink
1988

Cationic
surfactant

Unable to isolate a single bacterium using
cationic surfactant as the sole C source

Madsen et al.
2001

a
Bacteria:
A

Acetobacterium
,

C

Clostridium
,

D Desulfobacterium
,

G Geobacter
,
K

Klebsiella
,
M

Morganella
,
N

Nocardia
,

P

Pseudomonas

b
Source:

A

Aquifer materials,

C

compost;

D

sludge;

M

manure;

N

not specified;

P

petroleum
related sites;

S

soil or sediment

c
Chemicals:

CP

chlorophenol,

Dinoseb

2
-
sec
-
butyl
-
4,6
-
dinitrophenol

Pure cultures summarized in Table
1

have been isolated under strict anaerobic conditions
(sulfate
-
reducing and methanogenic). Example bacteria in this category include
Clostridia
,
Desulfobacterium
,
Desulfovibrio
,
Methanococcus
,
Methanosarcina
, and most of the newly
i
solated dehalogenating bacteria (e.g.,
Dehalococcoides
). For practical purposes, some of the
facultative denitrifying microorganisms are also included in the table such as
Flavobacterium

and
Klebsiella

to illustrate their potential role in these environmen
tal communities. Anaerobic
bacteria isolated from environmental compartments and bioreactors are preferentially illustrated
over anaerobes of pathological origin.

Attention has focused on the isolation of anaerobic bacteria that play a role in the degradat
ion of
two types of compounds due to their widespread environmental problems: the petroleum
hydrocarbons [benzene
-
toluene
-
ethylbenzene
-
xylene (BTEX); polycyclic aromatic hydrocarbons
(PAHs)] and chlorinated compounds including the pesticide DDT [1,1,1
-
tric
hloro
-
2,2
-
bis(
p
-
chlorophenyl)ethane]. In particular, extensive efforts have focused on the latter, partly because
halogenated organic compounds probably cause about half of the environmental problems
attributable to organic pollution in the world today (Ti
edje et al.
1993
), and partly because
anaerobic biodegradation is a preferred strategy. Following the discover
y of the insecticidal
properties of DDT in the late 1930s, its subsequent use and the awareness of its environmental
persistence, more than 300 bacterial strains have been shown to convert DDT into DDD [1,1
-
dichloro
-
2,2
-
bis(
p
-
chlorophenyl)ethane] (Cookson
1995
) and several novel dechlorinating strains
have been reported (Chacko et al.
1966
; Guenzi and Beard
1967
; Matsumura and Boush
1971
;
Wedemeyer
1966
) from the late 1960s to the 1970s. Research on the biodegradation of DDT
declined drastically after it was banned in the 1970s (Quensen et al.
2001
) and the focus during
the last 10

years has been directed toward chlorinated aliphatic hydrocarbons due to thei
r
worldwide prevalence. A pure culture of
Dehalococcoides ethenogenes

was able to completely
dechlorinate tetrachloroethylene (PCE) into innocuous ethene (Magnuson et al.
2000
; Maymo
-
Gatell et al.
1997
; McCarty
1997
), and
Desulfuromonas michiganensis

can even grow on free
-
phase PCE (Sung et al.
2003
). Most PCE
-
dechlorinating bacteria convert PCE into
trichloroethene (TCE) or further into cis
-
dichloroethene (DCE) (Bagley and Gossett
1990
), while
for others the more toxic vinyl chloride (VC) is produced as the end
-
product. Several recent
efforts have

therefore been made to isolate VC
-
transforming bacteria.
Dehalococcoides

sp.,
which can grow on VC and transform it into ethene in the presence of lactate and pyruvate as
electron donors (He et al.
2003
), is one such isolate.

Anaerobic degradation of the monoaromatic BTEX hydrocarbons was considered to be
negligible prior to the 1980s, partially due to the favorabl
e energetics of aerobic bacteria
(Chakraborty and Coates
2004
). These compounds have been shown to serve as carb
on and
energy sources for diverse anaerobic bacteria under nitrate
-
reducing, Fe(III)
-
reducing, sulfate
-
reducing and methanogenic conditions. Except for
p
-
xylene, isolation of pure bacterial cultures
degrading all other BTEX compounds has been successful (T
able

1
). Like BTEX, 2
-

to 4
-
ring
PAHs are quite readily biodegradable aerobically (Cerniglia
1992
), and anaerobic degradation of
PAHs was formerly thought impossible. However, naphthalene biodegradation through
denitrificati
on has been documented (Eriksson et al.
2003
; Mihelcic and Luthy
1988
), and
phenanthrene biodegradation through similar conditions was also reported (Rockne and Strand
1998
). A few PAH
-
degrading bacterial strains have been successfully isolated but none were
able to produce complete mineralization. As a concurrent contaminant with

BTEX and PAHs in
many petroleum
-
contaminated sites, methyl
tert
-
butyl ether (MTBE) is mainly susceptible to
aerobic degradation; however, anaerobic metabolism of MTBE has been reported (Finneran and
Lovley
2001
; Kolhatkar et al.
2002
; Somsamak et al.
2001
; Stocking et al.
2000
).

Anaerobic degradation of halogenated phenol, particularly pentachlorophenol (PCP), has been
the subject of several studies due to its wide use as a wood preservati
ve. Pure cultures able to
dechlorinate PCP into 3
-
chlorophenol have been isolated; some bacteria preferentially remove Cl
at the
ortho

and
meta

positions (Beaudet et al.
1998
; Tartakovsky et al.
1999
). However, no
single b
acterial culture with an ability for complete dechlorination and mineralization has yet
been isolated. For polychlorinated biphenyls (PCBs), although reductive dechlorination has been
observed frequently in many contaminated sediments and aquifers with an
array of
microorganisms (Quensen et al.
1988
), only recently have pure cultures been characterized (Wu
et al.
2002a
,
b
). A strain was isolated that could dechlorinate hydroxylated PCBs (Wiegel et al.
1999
). A pure culture that could use dioxin as the sole electron acceptor was isolated (Bunge et
al.
2003
). The isolation of dioxin
-
degrading bacteria is a good example of how bacteria have
evolved to metabolize toxic xenobiotic compounds.

The biodegradation of nitroaromatic explosives [trinitrotoluene (TNT); dinitrotoluene (DNT)]
has been studied fo
r more than two decades.
Clostridium

and
Desulfovibrio

spp. have been
extensively studied for their pathways transforming these compounds into amino
-

and
hydroxyamino
-
derivatives under anaerobic conditions. Unlike aerobic mineralization pathways
(e.g., DNT

mineralization can be readily demonstrated under aerobic conditions, Zhang et al.
2000a
,
b
), significant mineralization of TNT and DNT under anaerobic conditions has never
been achieved and anaerobic mineralizing bacter
ia never isolated. On the other hand, for non
-
aromatic explosives such as RDX (hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine) and HMX (octahyrdo
-
1,3,5,7
-
tetranitro
-
1,3,5,7
-
tetrazocine), pure bacterial cultures able to transform both agents have
been isolated (Bo
opathy et al.
1998
; Kitts et al.
1994
; Young et al.
1997
; Zhao et al.
2002
).

With a significant number of pesticides in use, dissimilar chemical structures and limited pure
bacterial isola
tes, generalizations regarding pesticide
-
degrading microorganisms are difficult to
make. For instance, in the United States alone, over 125 herbicides, 300 insecticides and 325
fungicides are registered (Cookson
1995
). The most extensively studied pesticide has been DDT
due to its persistent nature in the environment. The biodegradability of many other new synthet
ic
pesticides are of less concern due to the shorter half
-
life associated with biotic and abiotic
processes. Furthermore, studies on the biodegradation of pesticides appear to be focused mostly
on aerobic bacteria, despite some limited studies on the isola
tion of anoxic bacterial cultures (e.g.,
Ruppe et al.
2003
,
2004
).

Synthetic surfactants have created environmental problems due to the use of alkyl benzene
sulfonate (ABS) detergents that were later replaced by linear alk
ylbenzene sulfonate (LAS) in
the late 1960s. A common misconception is that surfactants are readily removed through aerobic
processes in municipal wastewater treatment plants due to sorption and aerobic biodegradation.
This is also why biodegradability dat
a of surfactants are predominantly aerobic (Swisher
1987
).
A significant percentage of surfactants escape aero
bic processes and accumulate in anaerobic
sludge digesters. A conservative estimate shows that approximately 20% of surfactants reached
the anaerobic compartment (AISE and CESIO
1999
). In addition, renewed interest in surfactant
biodegradation is based on the recent finding that many alkyl phenol polyethoxylates show
toxicity to fish and are suspected of being endocri
ne disrupters. While the importance of
anaerobic pathways is still in debate, research efforts to isolate anaerobic surfactant degrading
bacteria (Table

1
) are limited.


Biochemistry of xenobiotic biodegradation

Hydrocarbons and fuel additives

The anaerobic biochemical pathways of petroleum hydrocarbons and related fuel additives have
been the subjects of many inve
stigations during the last two decades. For hydrocarbons, the
elucidation of anaerobic BTEX (particularly toluene) degradation pathways is probably the most
advanced (Boll et al.
2002
). This is not surprising since saturated alkanes are less of a health
concern, although quantitatively they are more important than BTEX (Gieg and Suflita
2002
).

Saturated alkanes are more susceptible to aerobic bacterial attack than unsaturated aliphatic
hydrocarbons (i.e., alkene, alkyne). It
is also well established that alkanes with long carbon
chains and straight structures are more prone to aerobic biodegradation and the same is likely to
be the case for anaerobes. The most common aerobic pathway for alkane degradation is
oxidation of the t
erminal methyl group into a carboxylic acid through an alcohol intermediate,
and eventually complete mineralization through
-
oxidation (Cookson
1995
; Leahy and Colwell
1990
). Several physiologically and phylogenetically distinct anaerobes have been shown to
degrade alkanes (Aeckersberg et al.
1991
; Ehrenreich et al.
2000
; Rabus et al.
2001
; Rueter et al.
1994
). Methane can also be formed from alkanes by anaerobic organisms (Zengler et al.
1999
).
Recent data with an
n
-
hexane
-
utilizing denitrifying isolate pointed to a pathway involving initial
enzymatic attack by fumarate (

OOCCH=CHCOO

) addition in a manner similar
to that for
toluene as discussed below (Krieger et al.
2001
; Rabus et al.
2001
; Wilkes et al.
2002
). Another
pathway reported in a sulfate
-
reducing bacterium, Hxd3 (Aeckersberg et al.
1991
),

involves
carboxylation followed by removal of a terminal two
-
carbon unit to reduce the original alkane
length by one carbon as the fatty acid is formed (So et al.
2003
). Observations of a carbon
addition reaction internal to the chain were also made in studies of strain SK
-
01 (So and Young
1999a
,
b
).

Similarly, anaerobic MTBE m
etabolism is not as well understood as aerobic pathways. In the
presence of oxygen, aerobes attack MTBE with a monooxygenase. The biochemical mechanisms
of the recalcitrant ether bond cleavage have been explained in a review by Fayolle et al. (
2001
).
With anaerobic bacteria, the cleavage involves methyl transferases and tetrahydrofolate for the
degradation of lignin

(a naturally occurring ether compound) and hydroxyl group addition during
fermentation of polyethylene glycols (
-
O
-
CH
2
-
CH
2
OH). Anaerobic degradation of MTBE has
been demonstrated using compound
-
specific carbon isotope analyses in a groundwater site
(Kolha
tkar et al.
2002
), and transformation of MTBE has been observed under sulfate
-
reducing
conditions (Somsamak et a
l.
2001
).

Figure

1

delineates the major enzymes and intermediates involved in anaerobic degradation of
BETX compounds. Variations in pathways exist since various bacteria depend on different
electron acceptors correspondin
g to differing redox conditions (Chakraborty and Coates
2004
).
Complete mineralization has been reported for all

BTEX compounds except
p
-
xylene, and
research has elucidated the initial enzymatic reactions shown in Fig.

1
. A
difference from aerobic
mechanisms, which involve molecular oxygen, is the introduction of oxygen through H
2
O to
form oxygenated monoaromatic compounds that are susceptible to further ring cleavage. In some
cases, for example in the anaerobic degradation o
f
p
-
cresol, oxidation of the methyl group via
addition of oxygen derived from water occurs (Bossert et al.
1989
;

Bossert and Young
1986
).
Also shown in Fig.

1

is benzoyl coenzyme A (benzoyl
-
CoA), a common intermediate for BTEX
compounds. Benzoyl
-
CoA is formed through the addition of fumarate to the BTEX compounds
through the enzymatic

action of benzylsuccinate synthase (BSS) (for toluene) or
methylbenzylsuccinate synthase (for
o
-

and
m
-
xylene) (Biegert et al.
1996
). Studies on the
mechanism have demonstrated that these are glycyl radical enzymes (Beller and Spormann
1998
;
Krieger et al.
2001
; Leuthner et al.
1998
). After formation of benzylsuccinate, it is converted to
the CoA derivative benzylsuccinyl
-
CoA by a CoA transferase and then oxidized to benzoyl
-
CoA
and succinyl
-
CoA for further metabolism (Leutwein and Heider
1999
). The genes encoding the
benzyl succinate synthase have
been isolated (Hermuth et al.
2002
) and, in strain EbN1, are near
another operon encoding enzymes required for c
onversion of benzyl succinate to benzoyl
-
CoA
(Kube et al.
2004
). The enzyme benzylsuccinyl
-
CoA dehydrogenase i
s encoded by
bbsG

in
Thauera aromatica

(Leutwein and Heider
2002
). Benzoyl
-
CoA is transformed to 1,5
-
diene
-
1
-
c
arboxyl
-
CoA through the key enzyme, benzoyl
-
CoA reductase. After a series of hydration and
dehydrogenation steps, 3

mol acetyl
-
CoA and 1

mol CO
2

is formed from each mole of BTEX
compound (Mogensen et al.
2003a
).


Fig. 1


Anaerobic pathways for the biodegradation of petroleum hydrocarbons [benzene
-
toluene
-
ethylbenzene
-
xylene (BTEX); adapted from Chakraborty and Coates
2004
; Mogensen et al.
2003
].

A

Fumarat
e (HOOCCH=CHCOOH),

E
1

benzylsuccinate synthase (BSS),

E
2

ethylbenzylsuccinate synthase,

E
3

ethylbenzene dehydrogenase,

E
4

ethylbenzylsuccinate
synthase,

E
5

benzoyl
-
CoA reductase


The anaerobic biochemical pathways for PAHs have been studied only in the la
st few years, with
a focus on naphthalene and phenanthrene. Pure cultures of sulfate
-
reducing (Galushko et al.
19
99
)
and nitrate
-
reducing (Rockne et al.
2000
) bacteria that degrade naphthalene have been isolated.
Like mono
aromatic hydrocarbons, research has focused on the rate
-
limiting step of the initial
enzymatic attack. In contrast to earlier work that supported phenol as the major intermediate in
the fermentation of naphthalene [D. Grbic
-
Galic (1990) Microbial degradati
on of homocyclic
and heterocyclic aromatic hydrocarbons under anaerobic conditions. Unpublished report,
Department of Civil Engineering, Stanford University], recent work by several research groups
has identified 2
-
naphthoic acid (2
-
NA) as a common interme
diate (Fig.

2
) (Zhang et al.
2000a
,
b
).
This acid is formed through carboxyl
ation with the addition of a C
1

unit (Zhang and Young
1997
)
or fumarate, catalyzed by naphthyl
-
2
-
methyl
-
succin
ate synthase in the case of a substituted 2
-
methylnaphthalene (Sullivan et al.
2001
). The latter is analogous
to the benzoyl
-
CoA pathway of
monoaromatic BTEX degradation. Researchers have identified several intermediates including
two ring
-
cleaved products (Annweiler et al.
2000
,
2002
; Meckenstock et al.
2000
, Fig.

2
).


Fig. 2


Anaerobic pathways for the biodegradation of polyc
yclic aromatic hydrocarbons (PAHs)
(adapted from Annweiler et al.
2000
,
2002
).

A

Fumarate (HOOCCH=CHCOOH),

E
1

naphthyl
-
2
-
methyl
-
succinate synthase


Nitroaromatic compounds and explosives

The metabolic scheme in Fig.

3

illustrates major intermediates and end
-
products representative
of several anaerobic TNT pathways reported to date (Estev
e
-
Nuñez et al.
2001
). TNT has three
highly oxidized NO
2

groups at the 2,4,6
-
positions. Because of their electrop
hilic nature, these
external NO
2

groups are amenable to enzymatic reduction. In the meantime, since
-
electrons in
the benzene ring are shielded by four functional groups (3NO
2

and 1CH
3
) due to steric hindrance,
the aromatic structure is very stable, preventing enzymatic attack that could lead to ring cleavage.
This unique chemical structure explains, to a

large extent, why biotransformation of TNT occurs
rapidly but appreciable mineralization has never been achieved in either aerobic or anaerobic
systems even with more than two decades of intensive research effort (Hawari et al.
2000
).


Fig. 3


Anaerobic pathways for the biodegradation of nitroaromatic explosives [trinitrotoluene
(TNT)] (adapted from Esteve
-
Nuñez et al.
2001
).

A

Bamberger rearrangement,
E
1

carbon
monoxide dehydrogenase (CODH),
E
2

nitrite reductase,
E
3

the combination of enzymes
including hydrogenase, pyruvate
-
ferredoxin oxidoreductas
e, or CODH for the first step and
sulfite reductase for the final step of the reaction process (Preuss et al.
1
993
)


An advantage of anaerobic TNT biotransformation at low redox potential is to minimize
oxidative polymerization and the toxic azoxy compounds that can be readily formed in the
presence of oxygen. Among an array of end
-
products proposed or identified

(Fig.

3
), the amino
(NH
2
) and hydroxylamino (NHOH) derivatives from the reduction of NO
2

groups are frequently
reported. Results have also shown the removal of NO
2

groups as nitrite (NO
2

), and the oxidation
of CH
3

into benzoic acids (Esteve
-
Nuñez and Ramos
1998
; Esteve
-
Nuñez et al.
2000
). Boopathy
and Kulpa (
1992
) even noted the formation of NH
4
+

from the reductive elimination of NH
2

and
proposed a pathway that included toluene as the transfo
rmation end
-
product. The role of
triaminotoluene (TAT), hydroxylamino intermediates, and the resulting compounds from
subsequent hydroxyl addition
para

to NHOH (through Bamberger rearrangement) are
incompletely known under environmental conditions but have

been studied in laboratory
experiments (Hughes et al.
1998
;
1999
). TAT is considered to be a dead
-
end product that
precludes further mineralization (Hawari et al.
2000
). While hydroxylamino intermediates are
not stable, their transient toxicity could be an issue in remediation systems (Tadros et al.
2000
).
The good news, however, is that both compounds are strongly, or even irreversibly, adsorbed to
soils

a mechanism that may hold promise fo
r remediation (Daun et al.
1998
; Xue et al.
1995
),
and the chemically unstable nature of these compounds reduces long
-
term toxicity risks (Padda
et al.
2000
,
2003
). The use of cyclodextr
ins for desorption of TNT
-
related compounds has been
studied with various soils; however, the suitability of this practice over the long term is unclear
(Sheremata and Hawari
2000
).

The enzymes involved in anaerobic TNT transformation have not been fully characterized,
although several key proteins have been implicated, including ferredoxins, hydrogenases, carbon
monoxide dehydrogenase (CODH), pyruvate
-
ferredoxin oxidoreductases, and sulfite reductase
(Huang et al.
2000
; Pr
euss et al.
1993
). Perhaps more important to revitalize future research
efforts is the search for new microorg
anisms capable of TNT ring cleavage and mineralization
(Hawari et al.
2000
).

Unlike nitroaromatic TNT, the nonar
omatic cyclic nitroamines (RDX and HMX) have weak C

N bonds. Initial enzymatic attack able to change N

NO
2

or C

H bonds can readily destabilize
the cyclic structure and cause further molecular fragmentation. RDX is generally recalcitrant
under aerobic cond
itions, therefore anaerobic metabolism has been the subject of investigation.
Unfortunately, our understanding of RDX biodegradation has been limited since an early
pathway study by McCormick et al. (
1981
). In several recent studies on the examination of
approximately 24 hypothetical metabolites proposed in McCormick
s pathways, only a few were
confirmed, several intermediates were excluded, and many other new metabolites were identified
(Adrian and Chow
2001
; Hawari et al.
2000
; Zhang and Hughes
2003
). The full product analysis
of RDX biode
gradation is particularly challenging because it involves gas
-
phase mineralization
products, unstable nitroso
-

and hydroxyamino intermediates, as well as small molecules such as
formaldehyde and methanol. At the present time, enzymatic analysis is even mor
e speculative
despite the recent characterization of one enzyme (nitrate oxidoreductase) involved in RDX
biotransformation (Bhushan et al.
2002
).

Chlorinated aliphatic and aromatic hydrocarbons

The general features of anaerobic biodegradation of chlorinated compounds has been reviewed
(Haggblom et al.
2000
,
2003
). The pathways for deg
radation of chlorinated aliphatic
hydrocarbons (CAHs) such as PCE are well established (Fig.

4
). Much remains to

be understood
about the biochemical mechanisms, including the enzymes and the associated genes encoding
these metabolic enzymes in bacteria with various dechlorinating activities. A strain that has
activity on PCE and a variety of diverse halogen compound
s is
Dehalococcoides ethenogenes

195 (Fennell et al.
2004
; Maymo
-
Gatell et al.
1997
). Related Dehalococcoides
-
like organisms
have been studied (Cupples et al.
2004
; Maymo
-
Gatell et al
2001
). Aerobic bacteria can grow on
the VC intermediate of PCE degradation (Coleman et al.
2002a
,
b
). Such information is critical
so that complete PCE dechlorination can be achieved and the dechlorination rate can be
maximized
by maintaining optimal conditions such as redox, electron donors (normally H
2
), and
competing electron acceptors (e.g., nitrate, sulfate).


Fig. 4


Anaerobic pathways for the biodegradation of chlorinated aliphatic tetrachloroethylene
(PCE) (adapted from Cookson
1995
; Rittmann and McCarty
2001
).

E
1

PCE reductive
dehydrogenase (PCE
-
RDase),

E
2

trichloroethene reductive dehydrogenase (TCE
-
RDase)


PCE is one of the highly chlorinated (more oxidized) CAHs with no known microorganism
capable of aerobic biodegradation. Due to its high electron negative c
haracter, PCE can be used
as an electron acceptor (the oxidant) that is susceptible to reduction into the thermodynamically
more stable VC or ethene. Reduction is accomplished either through co
-
metabolism (fortuitous
modifications by bacteria that use othe
r primary substrates for carbon and energy) or a novel
biochemical mechanism known as dehalorespiration, where PCE is used as electron acceptor and
energy generated from exergonic dehalogenation reactions is used for bacterial growth (Cookson
1995
; El Fantroussi et al.
1998
). The electrons needed for reductive dehalogenation of PCE are
generated from the oxidation of H
2

(as electron donor, Fig.

4
), which originates from the
fermentation of other organic compounds (DiStefano et al.
1992
). Since dechlorinating bacteria
compete with H
2
-
utlilizing methanogens for H
2
, and a low H
2

concentration is favored for
dechlorinating bacteria, in practice, slow
-
release fermentation compounds such as fatty acid
s and
decaying bacterial biomass are preferred (Chen
2004
; Rittmann and McCarty
2001
).

Several enzymes and electron carriers responsible for PCE and TCE dechlorination have been
characterized. Three of the four known PCE r
eductive dehalogenases (PCE
-
RDases)
dechlorinate PCE or TCE to cis
-
DCE, but the PCE
-
RDase from
D. ethenogenes

can use PCE as
sole substrate, converting it into TCE (Magnuson et al.
1998
). Five chloroethene RDases have a
subunit molecular mass of 50

65

kDa and contain cobalamin and Fe
-
S clusters, and four
enzymes are membrane bound (Holliger et al.
1999
). TCE
-
RDase, located on the exterior of the
cytoplasmic membrane, catalyzes the dechlorination of TCE to ethene. The gene e
ncoding this
enzyme,
tceA
, was cloned and sequenced via an inverse PCR approach (Magnuson et al.
2000
).
In stu
dies on PCE respiration in
D. multivorans
, PCE dehalogenase was found in the cytoplasm
and was not tightly bound to the cell membrane (Neumann et al.
1996
).

The ability of anaerobic consortia (Kazumi et al.
1995
) and indiv
idual organisms (Song et al.
2000
,
2001
) to act on chlorinated or fluorinated aromatics (Vargas et al.
2000
) has been reported.
Little is known about the biochemical mechanisms (particularly enzymes) of the anaerobic
biodegradation of chlorinated aromatics including PCP, PCBs, and dioxins. Various anaerobic
PCP pathways have bee
n proposed, and an illustration of putative pathways is shown in Fig.

5
. It
is likely that bacteria may take sev
eral paths simultaneously for the removal of five chlorines
leading to the formation of phenol (the rate
-
limiting steps) and eventually mineralization to CH
4

and CO
2.

It is also apparent that the pathway (i.e., regiospecificity of chlorine removal) is
domi
nated by the redox potentials and whether the bacteria are acclimated prior to PCP
degradation. As can be seen from Fig.

5
, certain bacteria preferentially remove chlorines in the
order of
para

>
ortho

>
meta

(Path A, Fig.

5
) (Bryant et al.
1991
), whereas in others an
ortho

>
para

>
meta

order of chlorine removal has been reported (Pa
th B, Fig.

5
) (Mikesell and Boyd
1986
). While Fig.

5

is overly simplified, a d
etailed description of anaerobic PCP pathways is
summarized by Nicholson et al. (
1992
). Preferential chlorine
removal has practical ramifications
since some intermediates (e.g., 3,4,5
-
trichlorophenol) are more toxic than the parent compound,
while others are possible dead
-
end products.


Fig. 5


Anaerobic pathways for the biodegradation of chlorinated aromatic pentachlorophenol
(PCP) (adapted from Bryant et al.
1991
; Mikesell and Boyd
1986
). The letters
o
,
m
,
p

denote
dechlorination at the
o
,
m
, and
p

positions


PCBs and dioxins, although dissimilar in chemical structure, share some common features with
regard to their biodegradability. PCBs contain 209 di
fferent compounds (congeners) with
between 1 and 10 Cl substitutions on the backbone biphenyl structure. A typical synthetic PCB
mixture contains 60

80 different congeners.
Dioxins

have 1

8 Cl atoms s
ubstituted for H
atoms on dibenzo
-
p
-
dioxin, giving a total of 75 possible chlorinated derivatives, the most toxic
of which, i.e., 2,3,7,8
-
tetrachlorodibenzo
-
p
-
dioxin (TCDD), is commonly referred to as
dioxin.

For both PCBs and dioxins, the less chlorinated compounds are more amenable to
aerobic biodegradation. Nevertheless, reductive dechlorination is generally faster for the more
highly chlorinated compounds. Anaerobic biodegradation of both PCBs and
dioxins has been
reported (Bunge et al.
2003
) and can be enhanced by acclimation of bacteria to structurally
sim
ilar, or dissimilar yet readily biodegradable, halogenated aromatic compounds, a process
called
prim
ing

(Deweerd and Bedard
1999
; Haggblom and Young
1990
,
1995
; Wu et al.
1997
,
1998
). Early studies by Quensen et al. (
1988
) indicated that PCB dechlorination occurred
primarily from the
meta

and
para

positions, yielding less toxic and more readily degrade
d
products. A sequential anaerobic
-
aerobic treatment has recently been shown to be successful in
removing PCBs from contaminated soil (Master et al.
2002
). The degradation pattern of PCBs is
complex. Extensive
meta

and moderate
ortho

dechlorination were noted in a sediment slurry
study (Wu et al.
1998
), but a subsequent study using a sediment
-
free system indicated that
bacteria specifically removed doubly flanked chlorines (i.e., chlorines bound to C that are
flanked on b
oth sides by other Cl

C bonds) while leaving
ortho

chlorines intact (Wu et al.
2000
).
The bacterium DF
-
1 dechl
orinated several polychlorinated benzenes as well as PCB (Wu et al.
2002a
).

Like those of PCBs, the dechlorina
tion patterns of dioxins are difficult to generalize due to the
limited data available and the presence of a variety of dioxin congeners. Nevertheless, several
laboratory studies and field analysis of signature compounds have all indicated predominately
th
e initial lateral dechlorination (i.e., chlorines in the lateral 2,3,7,8 positions relative to the peri
1,4,6,9 positions), producing a characteristic 1,4
-
pattern of dioxin derivatives (Gaus et al.
2002
;
Vargas et al.
2001
). This generalization, however, contrasts with recent work by Bunge et al.
(
2003
) who proposed an initial peri
-
dechlorination pathway, demonstrating the diversity of
dechlorinating bacteria. Although no ring cleavage has been reported thus far, dechlorination is
of importance because of the reduction and even the elimination of toxicity. While the current
focus is
on dechlorination of highly chlorinated aromatic compounds, including PCP, PCBs and
dioxin, there is less awareness within the research community of the fate and effects of the less
chlorinated degradation products with higher aqueous solubility and a lowe
r octanol/water
partitioning coefficient (Mogensen et al.
2003a
). In this context, dechlorination could be a
blessing in disguise

if it yields compounds that are more readily bioavailable and mobile
(Dolfing and Beurskens
1995
; Mogensen et al.
2003a
).

Pesticides

The biochemic
al principles of pesticide biodegradation are no different from those of organic
compounds discussed earlier. Although a wealth of information is available, our current
understanding remains dispersed among a variety of pesticides and detailed biochemical
pathways are still unknown for many pesticides, even those in common use. Nevertheless, the
types of biochemical reactions are limited to a few (Alexander
1981
). Under anaerobic
conditions, the enzymatic reactions common to many pesticides include dechlorination,
hydrolysis, nitro reduction, and dealkylation (Williams
1977
). A bacterium may be partially
responsible for these metabolic activities, and in some cases the bacteria may have a metabolic
shift from one pathway to an
other (Barik et al.
1979
). To illustrate, Fig.

6

describes the anaerobic
reactions of three structurally distinct pesticides, 2,4
-
dichlorophenoxyacetic acid (2,4
-
D),
parathion (
o
,
o
-
diethyl
-
o
-
p
-
nitropheno phosphorothioate), a
nd atrazine.


Fig. 6


Anaerobic pathways for the biodegradation of three sel
ected pesticides:
a

2,4
-
dichlorophenoxyacetic acid (2,4
-
D),
b

parathion (
o
,
o
-
diethyl
-
o
-
p
-
nitropheno phosphorothioate),
and
c

atrazine (adapted from Crawford et al.
1998
; Mikesell and Boyd
1985
; Sethunathan
1973
;
Wackett et al.
2002
).

AtzA

Atrazine chlorohydrase,

AtzB

hydroxylatrazine hydrolase,
AtzC N
-
isopropylammelide amidohydrolase


Reductive dechlorination is common to all halogenated pesticides (Fig.

6
a, c), including aliphatic
(fumigants), cyclic aliphatic (lindane), aromatic (DDT; PCP, Fig.

5
), phenoxyalkanotes (2,4
-
D),
aniline
-
based (alachlor), and cyclodiene (aldrin) (Cookson
1995
). While lightly halogenated
pesticides are more biodegradable under aerobic conditions, it is commonly believed that highly
halogenated pesticides often biodegrade more rapidly unde
r anaerobic conditions.

Hydrolysis of phosphate esters, catalyzed by esterase, is an important mechanism for
organophosphate pesticides. For example, an esterase hydrolyzed the P

O

C linkage in
parathion subsequent to a nitro reduction, which leads to the
formation of
p
-
aminophenol
(Fig.

6
b). Various other esterases catalyze degradation of aliphatic and aromatic est
er pesticides
(e.g., carbamates; Sethunathan
1973
). The degradation of the nitrogen
-
containing pesticide
atraz
ine shown in Fig.

6
c (partially aerobic processes) involves hydrolytic dechlorination,
dealkylation, and the cle
avage of C

N in the cyclic ring, yielding ultimate mineralization to CO
2

and NH
3
. Anaerobic degradation of atrazine by mixed consortium and in wetlands by a co
-
metabolic process has been reported (Ghosh and Philip
2004
; Kao et al.
2001
; Seybold et al.
2001
). Although N
-
dealkylated intermediates were not confirmed under denitrifying
conditions,
evidence of a hydroxyatrazine intermediate and ring cleavage was provided by Crawford et al.
(
1998
)
with the bacterial isolate M91
-
3. Three enzymes have been characterized in
Pseudomonas

sp. ADP, including atrazine chlorohydrase (AtzA), hydroxylatrazine hydrolase (AtzB), and
N
-
iso
-
pylammelide amidohydrolase (AtzC) (Wackett et al.
2002
).

Surfactants

For each of the three major surfactants (anionic, nonionic and cationic), current understanding of
the anaerobic bi
ochemical pathways is based on a few limited studies. Even with recent advances
in sensitive analytical instrumentation, such as high resolution GC
-
MS, LC
-
MS and tandem MS,
many of the putative pathways are based on a few tentatively identified intermediat
es. Other
added challenges include cumbersome derivatization procedures, effects of sorption, difficulty in
obtaining pure surfactant homologues, and the requirement for a consortium of bacteria to
completely degrade a surfactant with various moieties. In
this context, any detailed discussion on
anaerobic surfactant degradation pathways must be speculative. Described briefly below are
likely bacterial strategies in attacking nonionic and anionic surfactant moieties based on several
recent studies using anae
robic microorganisms.

The nonionic linear alcohol ethyoxylates (LAE) have the common structural formula
CH
3
(CH
2
)
m
O(CH
2
CH
2
O)
n
H (
m
=7

17,
n
=1

25). Initial bacterial attack proposed by Steber and
Wierich (
1987
) included the central scission at the center ether bond linking the alkyl chain with
the ethoxy (EO) chain

a strategy well
-
known for aerobic bacteria. Wagener a
nd Schink (
1988
)
suggested that the initial step is a hydroxyl group exchange reaction, followed by a shorteni
ng of
the EO chain by stepwise cleavage of acetaldehyde. Huber et al. (
2000
) recently concluded that
central sci
ssion is unlikely and that the first step of microbial attack is cleavage of the terminal
EO unit, releasing acetaldehyde stepwise and shortening the EO until the lipophilic moiety is
reached. Another major nonionic surfactant, nonylphenol ethoxylates (NPE
O), has a benzene
ring with an EO chain
para

to the C
9
H
19

functional group. Although rapid mineralization has
been reported, a recent study by Ferguson and Brownawell (
2003
) concluded that aromatic ring
mineralization was not a major pathway for NPEO biodegradation.

Anionic linear alkylbenzene sulfonate (LAS) is a mixture of related isomers and homologues
consisting

of a
para
-
sulfonated benzene molecule with an alkyl chain attached to any position
except the terminal one. This structural uniqueness requires the alteration of an alkyl chain, a
benzene ring, and a sulfonate linkage for complete mineralization (Mogensen

et al.
2003b
).
Under aerobic conditions, LAS biodegradation is initiated with an
-
oxidation of the terminal
methyl group of the alkyl chain to form a carboxylic acid. Further degradation proceeds by a
stepwise
shortening of the alkyl chain by
-
oxidation, leaving a short
-
chain sulfophenyl
carboxylic acid. The a
romatic ring hydrolyzes to form a dihydroxy
-
benzene structure that is
opened before desulfonation of the formed sulfonated dicarboxylic acid (Madsen et al.
2001
).
Such needed information is lacking for LAS biodegradation under various anaerobic conditions.
C
12

3
LAS was desulfonated under sulphur
-
limited anoxic conditions (Denger and Cook
1999
),
suggesting that LAS may not be entirely persistent. Current data on anaerobic biodegradability
does not allow an accurate survey of

anaerobic biodegradation pathways of surfactants.


Practical applications of anaerobic processes in
xenobiotic biodegradation

Conventional anaerobic processes have been used for the treatment of concentrated municipal
and industrial wastewaters for over
a century as they enjoy energy savings from methane and
lower sludge production than aerobic activated sludge processes (Jewell
1987
; McCarty and
Smith
1986
). The rapidly growing knowledge of chemical
-
specific bacteria and

biochemical
pathways suggests that the treatment of xenobiotics, commonly at very low concentration, is
technically feasible and, in many instances, also economically viable. Evidence for xenobiotic
biodegradability under various anaerobic environments ha
s stemmed predominately from lab
-
scale studies using serum bottles, microcosms, columns, and small
-
sized bioreactors. These lab
-
scale studies, along with many field and large
-
scale demonstrations are based mostly on
indigenous bacteria or enriched cultures
. Biodegradation tests using pure bacterial cultures or
studies aimed at isolation of pure cultures (Table

1
) ha
ve been almost exclusively lab
-
scale,
although there are a few reported uses of pure bacterial inocula in pilot and field tests (e.g.,
Dybas et al.
1998
; El Fantroussi et al.
1999
). A summary in Table

2

focuses on practical
applications of anaerobic bacterial consortia in field or large
-
scale studies and, whenever
applicable, document
ed sources from peer reviewed journals are selected although many studies
of this type are reported frequently in conference proceedings and industrial notices.

Table 2


Selected large and field scale anaerobic processes in xenobiotic degradation
a
.

BTEX

P
etroleum hydrocarbons (benzene
-
toluene
-
ethylbenzene
-
xylene),

CT

carbon tetrachloride,

CF

chloroform,

UASB

upflow anaerobic sludge bioreactor,

CSTR

continuous stirred tank reactor,

APEO
, alkylphenylethoxylate,

AE

alcohol ethyoxylate,

3
-
MCP

3
-
monochloropheno
l,

HCH

hexachlorocyclobenzene,

MCB

monochlorobenzene

Compounds

Type and scale

Performance results

Reference

Alkane

Microcosm

44%
n
-
alkanes removed in
12

months

Salminen et al.
2004

BTEX

8



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ㄲ〠摡ys

Ba瑴e牭a渠
1983

Aquifer

NO
2


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牥浯癡m

Ba牫e爠r琠a氮l
1987

Fuel
spill site

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2


e湨n湣e搠
m
-

and
p
-
xylene
removal

Hutchins et al.
1991

PAHs

Microcosm

2

㔠物湧⁐䅈猠摥杲gde搠畮u
e爠

4
2

Ⱐ乏
3

-
牥摵d楮i co湤楴楯湳

R潣歮k⁡湤⁓瑲t湤n
1998
; Rothermich et al.
2002

MTBE

Microcosm;
groundwater

Stimulated by humic substances;
field evidence in groundwater using
13
C of MTBE

Finneran and Lovley
2001
; Kolhatkar et al.
2002

TNT, DNT,
RDX, HMX

Full
-
scale reactor

TNT to mine
ralizable and
nonaromatic products by
Clostridium

Funk et al.
1995

5×1.8×2

m Sludge
reactor

Providing sucrose
and NH
4
Cl, 98%
TNT, DNT and RDX removal in
30

weeks

Lenke et al.
1998

PCE, TCE, CT

Shallow aquifer

Nitrate an
d acetate injection
Semprini et al.
1992

Compounds

Type and scale

Performance results

Reference

transformed 95

97% CT to CF

Industrial site

Significant ethene and
CH
4

in a
TCE
-
contaminated aquifer

McCarty and Wilson
1992

4.5
-
Acre chemical
plant

Strong correlation between

PCE,
ethene and electron donor

Major et al.
1991

Aquifer

Nutrients, enrichment culture
injection converted
TCE to ethene

Ellis et al.
2000

Pilot
-
scale

Pseudomonas stutzeri

KC removed
CT

Dybas et al.
1998

PCBs

6
-
l Batch
bioreactor

11% and 23% of total Cl/biphenyl
was reduced after 13

weeks

Pagano et al.
1995

PCP

488
-
m Long
stream

PCP disappeared: O
2
-
rich > O
2
-
poor
anaerobic > sorption

Pignatello et al.
1985

Pilot digester

>97.5% removal; 95% converted to
3
-
MCP

Chen and Berthouex
2001

UASB, biofi
lm
and CSTR

95% Removal with C source
provided, dechlorinated at the
o
-

then
m
-
, but not
p
-

chlorines;
reactor optimization studies

Hendriksen et al.
1992
;
Ribarova et al.
2002
;
Woods et al.
1989

Dioxins

Microcosm

Dechlorinated under methanogenic
conditions

Vargas et al.
2001

Chlorinated
pesticide

In situ anaerobic
bioscreen

HCH converted to MCB and
benzene which was mineralized in
an aerobic treatment plant

Langenhoff
2003

P
-
Based
pesticide

Field studies

Nonpersistent, degraded under P
-
limited conditions

Ternan et al.
1998

Carbamate
pesticide

500
-
l Fermentor;
35 yard
3

(=26.8

m
3
)
bioreactor

Large
-
scale aerobic systems using
Pseudomonas

sp. and recomb
inant
Escherichia coli
; no large
-
scale
anaerobic processes

Newcombe and
Crowley
1999
; Strong
et al.
2000

Dinoseb
pesticide

2,600
-
l Static
reactors

Undetectable by 15

days after
addition of C and acclimated culture

Rober
t et al.
1993

Anionic
surfactant

3.5
-
l Digester;
field data

14

25% LAS12 was transformed in
a CSTR reactor. F
ield data support
anaerobic biodegradation in
sediments, landfill and soils

Federle and Pastwa
1988
; Haggensen e
t al.
2002
; Mogensen et al.
2003b

Nonionic
surfactant

Batch to
microcosm

Partially (APEO) to well (AE)
biodegradable

Ferguson and
Brownawell
2003
;
Huber et al.
2000
;

Compounds

Type and scale

Performance results

Reference

Cationic
surfactant

Batch

Strongly adsorbed, toxic, scarce
anaerobic biodegradation data

Madsen et al.
2001

a
Laboratory microcos
m studies were included for certain xenobiotic compounds

Compounds particularly suited to anoxic/anaerobic processes have included highly halogenated
compounds such as carbon tetrachloride (CT), PCE, PCBs and some of the organochlorine
pesticides that pers
ist under aerobic conditions. Nonhalogenated compounds such as
nitroaromatic and aminoaromatic compounds, including herbicides and hazardous energetic
organonitro compounds, persist under aerobic conditions and decompose only under
anoxic/anaerobic conditi
ons (Baker and Herson
1994
). Morgan and Watkinson (
1989
) indicated
that the persistent nature of compounds such as DDT and PCBs is evidence of microbial
fallibility, and therefore biological cleanup of sites contaminated w
ith this type of compound is
unlikely to be generally feasible unless an extremely long treatment period is acceptable. The
debate continues over whether persistent organic pollutants (POP) can be remediated by any
biological means. Studies demonstrated th
at DDE [1,1
-
bis
(chlorophenyl)
-
ethylene], a toxic
byproduct of DDT, can be biodegraded into DDMU [1,1
-
bis
(
p
-
chlorophenyl)
-
2
-
chloro
-
ethylene]
under methanogenic and sulfidogenic conditions (Quensen et al.
1998
). DDMU has one less Cl
atom and does not bioaccumulate as readily as its parent, and is also subject to dechlorination.
This finding, however, was discounted
by others who believe that the rate was insignificant in
the field and the dechlorinating bacteria are often less favorable in competing with other bacteria
(Renner
1998
). Despite much success in lab studies, in practice timely remediation of POPs such
as PCBs and DDT still relies heavily on non
-
biological means such as sediment dredging and
natural capping.

Chlor
inated aliphatic hydrocarbons provide perhaps the most successful example of anaerobic
biodegradation in anoxic aquifer environments. Under proper conditions, deliveries of electron
donors and nutrients significantly stimulated the activities of reductive
dechlorination in many
field studies (Major et al.
1991
; McCarty and Wilson
1992
; Semprini et al.
1992
). Field success,
however, often entails expensive monitoring of the contaminant plume and the end
-
products
including methane and ethene. In several cases where indigenous bacteria were unable to
dechlorinate, bioaugmentation wi
th pure dechlorinating bacteria has been shown to be successful
(Dybas et al.
1998
; Ellis et al.
2000
).

Field experience in remediating hydrocarbons using aerobic bacteria and pathways dates back to
the early 1970s. For inst
ance, Raymond (
1974
) received a patent on a process designed to
remove hydrocarbon contaminants from groundwat
er by stimulating indigenous aerobic bacteria
with nutrients and oxygen. Anaerobic processes, however, have received little attention and have
had limited success in the field even with monoaromatic hydrocarbons (BTEX). Recently, field
data have suggested
anaerobic biodegradation could be a significant process in contaminated
aquifers depleted of oxygen (Table

2
). F
ield evidence regarding the exclusive role of anaerobes
are sometimes equivocal since groundwater normally considered to be anoxic can sometimes
contain dissolved oxygen (DO) as high as 1

mg/l (Batterman
1983
; Hutchins et al.
1991
;
Steinbach et al.
2004
). Future research and field demonstrations with hydrocarbons, both in
terrestria
l and marine environments, are likely to increase. For the terrestrial environment,
research is motivated largely by the clean
-
up of gasoline spills in leaking underground storage
tanks and the increased recognition of natural attenuation as part of the re
medial strategy. For the
marine environment, work is largely driven by oil spills, particularly of crude oil. Prince (
1993
)
stated that there is room to extend current applications to oiled marshes and other anaerobic
sediments as these are the frequent recipients of spill incidents. Thirty percent of gasoline sold in
the United States contains 11% by volume MTBE
and crude oils are composed of more than
75% aliphatic and aromatic hydrocarbons (Stocking et al.
2000
). Anaer
obic MTBE
biodegradation is still considered to be a rare occurrence, therefore remedial applications for
MTBE and other fuel oxygenates are almost exclusively aerobic processes (Fayolle et al.
2001
;
Stocking et al.
2000
).

Anaerobic processes for the degradation of explosive compounds have been employed in both in
situ and ex situ reactor systems (Funk et al.
1995
; Lenke et al.
1998
). Processes such as land
farming, composting and slurry re
actors have been very successful in transforming or
detoxifying explosives and, in some cases, result in complete mineralization. Since
mineralization of explosives is very unlikely in anaerobic processes, remediation is often
achieved by two strategies, i
.e., transformation into innocuous products or irreversible binding
with soil components. Recently, increasing evidence has pointed toward the use of sequential
anaerobic
-
aerobic processes to destroy nitroaromatic explosives (Esteve
-
Nuñez et al.
2001
;
Hawari et al.
2000
).

The anaerobic biodegradation of pesticides and surfactants has witnessed limited in situ and ex
situ applications relative to their extensive usage and disposal. Most pesticide biodegradation
studies stem fr
om the need to minimize dispersion outside of the agriculture environment, and
remedial applications are limited to some contaminated pesticide manufacturing sites and
accidental spills as shown in Table

2

(Langenhoff
2003
; Newcombe and Crowley
1999
; Roberts
et al.
1993
; Strong et al.
2000
; Tern
an et al.
1998
). There is a paucity of data regarding the
anaerobic biodegradation of surfactants, and surfact
ants commonly in use are considered as not
persistent in the environment as implied from the extensive aerobic biodegradation database
currently available. Surfactants are in fact the most abundant organic species in domestic sewage
sludge, where concentra
tions exceeding g/kg levels are frequently observed (Mogensen et al.
2003a
). Field monitoring data support evi
dence of anaerobic biodegradation in sediment below
sewage treatment plant (STP) outfalls, domestic septic systems, landfill sites receiving sludge,
and subsurface soils beneath laundromat wastewater discharge (Federle and Pastwa
1988
). One
area of needed research is the anaerobic biodegradation in sludge digesters of municipal STPs.
Such anaerobic digesters are gen
erally not designed for the removal of surfactants, hence
improved designs and optimization of various anaerobic reactor systems has been the subject of
several studies (Haggensen et al.
2002
; Mogensen et al.
2003a
). Furth
er research is needed with
regard to surfactants of current environmental concern, particularly LAS and NPEO (Ferguson
and Brownawell
2003
; Huber et al.
2000
).

Conclusions and future prospects

The mounting evidence accumulat
ed during the last two decades supports the argument that
anaerobic biodegradation, once considered to be negligible, could be significant for a variety of
xenobiotic compounds in anaerobic environments such as groundwater, sediment, landfill,
sludge diges
ters and bioreactor systems. The elucidation of biochemical mechanisms using
isolated bacteria strains, and laboratory feasibility studies using mainly enrichment cultures has
enabled successful large
-

and field
-
scale in situ and ex situ remediation applic
ations (Tables

1
,
2
).
For certain highly chlorinated hydrocarbons (e.g., PCE), anaerobic processes cannot easily be
substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic
processes are gener
ally used, anaerobic biodegradation could become significant, and an
economically viable option under certain circumstances (e.g., oxygen
-
depleted aquifer, oil
-
spilled marsh). For persistent compounds including PCBs, dioxins, and DDT, anaerobic
processes a
re slow for remedial applications, but can represent a significant avenue if natural
attenuation is an option. For many xenobiotic compounds, particularly PCBs and explosives,
anaerobic processes could be complementary to aerobic processes for complete con
taminant
destruction.

With the increasing appreciation of anaerobic processes, along with recent advances in
biochemical, molecular technology and analytical instrumentation, new strains will continue to
be isolated and novel enzymes and biochemical pathwa
ys will be characterized. Further research
will be needed to characterize genes encoding the enzymes that bacteria have evolved to degrade
such xenobiotics. Recombinant strains, although still a debated issue in practice, have been
explored in the case of
aerobic microorganisms and show some success in outdoing the
performance of indigenous bacteria (Shimazu et al.
2001
; Wackett et al.
2002
). Genetically
engineered microorganisms capable of multiple pathways are likely to

offer solutions to some of
the most recalcitrant xenobiotic compounds, most likely at contained wastestreams associated
with industrial facilities. An ignored area of research is the characterization of enrichment
cultures. This is particularly important
for recalcitrant compounds that require a consortium of
syntrophic bacteria. Elucidating the ecology of these bacterial consortia is critical, but such
information is almost nonexistent. A related approach involving the sequential use of anaerobic
and aero
bic bacteria (Esteve
-
Nuñez et al.
2001
; Lenke et al.
1998
; Master et al.
2002
) may also
allow advances in treatment to be attained.

Other knowledge gaps include the understanding and manipulation of bacterial strategies in
utilizing compounds with various functional moieties. Not only the initial enzymatic attack but
also the comp
lete mineralization potential needs to be characterized. Not discussed in this review
are the optimization of anaerobic processes and the provision of optimal electron donors and
acceptors.

Acknowledgements


Research in authors

laboratories has been supported by the Welch
Foundation (C
-
1268) and BC
-
0022, DSWA, EIH and SERDP. This material is also based o
n
work supported in part by the United States Army Research Laboratory and the United States
Army Research Office (Grant DOD Army W911NF
-
04
-
1
-
0179)


References

Abramowicz DA (1990) Aerobic and anaerobic biodegradation of PCBs: a review. Crit Rev
Biotechno
l 10:241

251




Adrian NR, Chow T (2001) Identification of hydroxylamino
-
dinitroso
-
1,3,5
-
triazine as a
transient intermediate formed during the anaerobic

biodegradation of hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine. Environ Toxicol Chem 20:1874

1877



Aeckersberg F, Bak F, Widdel F (1991) Anaerobic oxidation of saturated hydrocarbons to CO
2

by a new type of sulfate
-
reducing bacterium. Arch Microbiol 156:5

1
4




Ahmad F, Hughes JB (2000) Anaerobic transformation of TNT by
Clostridium
. In: Spain JC,
Hughes JB, Knackmuss H
-
J (eds) Biodegradation of nitroaromat
ic compounds and explosives,
Lewis, Boca Raton, Fla., pp

185

212



AISE and CESIO (1999) Environmental relevance of anaerobic biodegradability of surfactants.
http://www.aise
-
net.org/PDF/ana
erobicBiopub1.pdf
, p

6



Alexander M (1981) Biodegradation of chemicals of environmental concern. Science 211:132

138



Annweiler E, Materna A, Safinowski S, Kappler A, Richnow HH, Michaelis W, Meckensrock
RU (2000) Anaerobic degradation of 2
-
methylna
phthalene by a sulfate reducing enrichment
culture. Appl Environ Microbiol 66:5329

5333



Annweiler E, Michaelis W, Meckenstock RU (2002) Identical ring cleavage products during
anaerobic degradation of naphthalene, 2
-
methylnaphthalene, and tetralin indi
cate a new
metabolic pathway. Appl Environ Microbiol 68:852

858



Bagley DM, Gossett JM (1990) Tetrachloroethene transformation to trichloroethene and cis
-
1,2
-
dichloroethene by sulfate
-
reducing enrichment cultures. Appl Environ Microbiol 56:2511

2516



Baker KH, Herson DS (1994) Bioremediation. McGraw Hill, New York, NY



Ball HA, Johnson HA, Reinhard M, Spormann AM (1996) Initial reactions in anaerobic
ethylbenzene oxidation by a denitrifying bacterium, strain EB1. J Bacteriol 178:5755

5761



Barik

S, Wahid PA, Ramakrishna C, Sethunathan N (1979) A change in the degradation pathway
of parathion after repeated applications to flooded soil. J Agric Food Chem 27:1391

1392



Barker JF, Patrick GC, Major DW (1987) Natural attenuation of aromatic hydroc
arbons in a
shallow sand aquifer. Ground Water Monit Rev 7:64

71



Batterman G (1983) A large scale experiment on in situ biodegradation of hydrocarbon in the
subsurface. In: Ground water in water resources planning, vol II. Proc Int Symp. IASA
Publicati
on 142. International Association of Hydrological Sciences, London, p

93



Beaudet R, Levesque MJ, Villemur R, Lanthier M, Chenier M, Lepine F, Bisaillon JG (1998)
Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a
met
hanogenic consortium or with
Desulfitobacterium frappieri

strain PCP
-
1. Appl Microbiol
Biotechnol 50:135

141



Bedard DL (2003) Polychlorinated biphenyls in aquatic sediments: environmental fate and
outlook for biological treatment. In: Bossert ID, Haggb
lom MM (eds) Dehalogenation. Kluwer,
Norwell, Mass., pp

443

465



Beller HR, Spormann AM (1998) Analysis of the novel benzylsuccinate synthase reaction for
anaerobic toluene activation based on structural studies of the product. J Bacteriol 180:5454

5457



Bhushan B, Halasz A, Spain J, Thiboutot S, Ampleman G, Hawari J (2002) Biotransformation of
hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine catalyzed by a NAD(P)H: nitrate oxidoreductase from
Aspergillus niger
. Environ Sci Technol 36:3104

3108



Biegert T,
Fuchs G, Heider J (1996) Evidence that anaerobic oxidation of toluene in the
denitrifying bacterium
Thauera aromatica

is initiated by formation of benzylsuccinate from
toluene and fumarate. Eur J Biochem 238:661

668



Boll M, Fuchs G, Heider J (2002) Ana
erobic oxidation of aromatic compounds and
hydrocarbons. Curr Opin Chem Biol 6:604

611




Boopathy R, Gurgas M, Ullian J, Manning JF (1998) M
etabolism of explosive compounds by
sulfate
-
reducing bacteria. Curr Microbiol 37:127

131



Boopathy R, Kulpa CF (1992) Trinitrotoluene as a sole nitrogen source for a sulfate
-
reducing
bacterium
Desulfovibrio

sp. (B strain) isolated from an anaerobic dige
ster. Curr Microbiol
25:235

241



Bossert ID, Young LY (1986) Anaerobic oxidation of
p
-
cresol by a denitrifying bacterium. Appl
Environ Microbiol 52:1117

1122



Bossert ID, Whited G, Gibson DT, Young LY (1989) Anaerobic oxidation of
p
-
cresol mediated
b
y a partially purified methylhydroxylase denitrifying bacterium. J Bacteriol 171:2956

2962



Bouchard B, Beaudet R, Villemur R, McSween G, Lepine F, Bisaillon JG (1996) Isolation and
characterization of
Desulfitobacterium frappieri

sp. nov., an anaerobic

bacterium which
reductively dechlorinates pentachlorophenol to 3
-
chlorophenol. Int J Syst Bacteriol 46:1010

1015



Bryant FO, Hale DD, Rogers JE (1991) Regiospecific dechlorination of pentachlorophenol by
dichlorophenol
-
adapted microorganisms in freshwa
ter, anaerobic sediment slurries. Appl
Environ Microbiol 57:2293

2301



Bunge M, Adrian L, Kraus A, Lorenz WG, Andreesen JR, Gorisch H, Lechner U (2003)
Reductive dehalogenation of chlorinated dioxins by the anaerobic bacterium
Dehalococcoides
ethenogene
s sp
. strain CBDB1. Nature 421:357

360




Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation
3:351

368




Chacko CI, Lockwood JL, Zabik M (1966) Chlorinated hydrocarbon pesticides: degradation by
microbes. Science 154:893

895



Chakraborty R, Coates JD (2004) An
aerobic degradation of monoaromatic hydrocarbons. Appl
Microbiol Biotechnol 64:437

446



Chen G (2004) Reductive dehalogenation of tetrachloroethylene by microorganisms: current
knowledge and application strategies. Appl Microbiol Biotechnol 63:373

377



Chen ST, Berthouex PM (2001) Treating an aged pentachlorophenol
-

(PCP
-
) contaminated soil
through three sludge handling processes, anaerobic sludge digestion, post
-
sludge digestion and
sludge land application. Water Sci Technol 44:149

56



Coates JD,
Chakraborty R, Lack JG, O
Connor SM, Cole KA, Bender KS, Achenbach LA
(2001) Anaerobic benzene oxidati
on coupled to nitrate reduction in pure culture by two strains of
Dechloromonas
. Nature 411:1039

1043



Coleman NV, Mattes TE, Gossett JM, Spain JC (2002a) Biodegradation of cis
-
dichloroethene as
the sole carbon source by a beta
-
proteobacterium. Appl Env
iron Microbiol 68:2726

2730



Coleman NV, Mattes TE, Gossett JM, Spain JC (2002b) Phylogenetic and kinetic diversity of
aerobic vinyl chloride
-
assimilating bacteria from contaminated sites. Appl Environ Microbiol
68:6162

6171



Cookson JT Jr (1995) Bio
remediation engineering: design and application. McGraw
-
Hill, New
York, NY



Crawford JJ, Sims GK, Mulvaney RL, Radosevich M (1998) Biodegradation of atrazine under
denitrifying conditions. Appl Microbiol Biotechnol 49:618

623



Cupples AM, Spormann AM
, McCarty PL (2004) Comparative evaluation of chloroethene
dechlorination to ethene by
Dehalococcoides
-
like microorganisms. Environ Sci Technol
38:4768

4774



Daun G, Lenke H, Reuss M, Knackmuss H
-
J (1998) Biological treatment of TNT
-
contaminated
soil. 1
. Anaerobic cometabolic reduction and interaction of TNT and metabolites with soil
components. Environ Sci Technol 32:1956

1963



De Bruin WP, Koterman MJJ, Posthumus MA, Schraa G, Zehnder AJB (1992) Complete
biological reductive transformation of tetrac
hloroethene to ethane. Appl Environ Microbiol
58:1996

2000



Denger K, Cook AM (1999) Linear alkylbenzenesulphonate (LAS) bioavailable to anaerobic
bacteria as a source of sulphur. J Appl Microbiol 86:165

168



Deweerd KA, Bedard DL (1999) Use of halog
enated benzoates and other halogenated aromatic
compounds to stimulate the microbial dechlorination of PCBs. Environ Sci Technol 33:2057

2063



DiStefano TD, Gossett JM, Zinder SH (1992) Hydrogen as an electron donor for dechlorination
of tetrachloroethe
ne by an anaerobic mixed culture. Appl Environ Microbiol 58:3622

3629



Dolfing J, Beurskens JEM (1995) The microbial logic and environmental significance of
reductive dehalogenation. Adv Microbial Ecol 14:143

206



Dybas MJ, Barcelona M, Bezborodnikov

S, Davies S, Forney L, Heuer H, Kawka O, Mayotte T,
Sepulveda
-
Torres L, Smalla K, Sneathen M, Tiedje J, Voice T, Wiggert DC, Witt ME, Criddle
CS (1998) Pilot
-
scale evaluation of bioaugmentation for in
-
situ remediation of a carbon
tetrachloride
-
contaminate
d aquifer. Environ Sci Technol 32:3598

3611



Ehrenreich P, Behrends A, Harder J, Widdel F (2000) Anaerobic oxidation of alkanes by newly
isolated denitrifying bacteria. Arch Microbiol 173:58

64



El Fantroussi S, Naveau H, Agathos SN (1998) Anaerobic
dechlorinating bacteria. Biotechnol
Prog 14:167

188




ElFantroussi S, Belkacemi M, Top EM, Mahillon J, Vaveau H, Agathos SN (1999)
Bioaugmentation of a s
oil bioreactor designed for pilot
-
scale anaerobic bioremediation studies.
Environ Sci Technol 33:2992

3001



Ellis DE, Lutz EJ, Odom JM, Buchanan RJ Jr, Bartlett CL, Lee MD, Harkness MR, Deweerd
KA (2000) Bioaugmentation for accelerated in situ anaerobic

bioremediation. Environ Sci
Technol 34:2254

2260



Eriksson M, Sodersten E, Yu Z, Dalhammar G, Mohn WW (2003) Degradation of polycyclic
aromatic hydrocarbons at low temperature under aerobic and nitrate
-
reducing conditions in
enrichment cultures from no
rthern soils. Appl Environ Microbiol 69:275

84



Ernst C, Rehm HJ (1995) Utilization of chlorinated
s
-
triazines by a new strain of
Klebsiella
pneumoniae
. Appl Microbiol Biotechnol 42:763

768




Esteve
-
Nuñez A, Ramos JL (1998) Metabolism of 2,4,6
-
trinitrotoluene (TNT) by
Pseudomonas

sp. JLR11. Environ Sci Technol 32:3802

3808



Esteve
-
Nuñez A, Luchessi, Phillipps B, Schink B, Ramos JL (2000) Respiration
of 2,4,6
-
trinitrotoluene by
Pseudomonas

sp. strain JLR11. J Bacteriol 182:1352

1355



Esteve
-
Nuñez A, Caballero A, Ramos JL (2001) Biological degradation of 2,4,6
-
trinitrotoluene.
Microbiol Mol Biol Rev 65:335

352




Fathepure BZ, Boyd SA (1988) Dependence of tetrachloroethylene dechlorination on
methanogenic substrate consumption by
Methanosarcina

sp. strain DCM. Appl Environ
Microbiol 54:2
976

2980



Fayolle F, Vandecasteele J
-
P, Monot F (2001) Microbial degradation and fate in the environment
of methyl
tert
-
butyl ether and related fuel oxygenates. Appl Microbiol Biotechnol 56:339

349



Federle TW, Pastwa GM (1988) Biodegradation of surf
actants in saturated subsurface sediments:
a field study. Ground Water 26:761

770



Fennell DE, Nijenhuis I, Wilson SF, Zinder SH, Haggblom MM (2004)
Dehalococcoides
ethenogenes

strain 195 reductively dechlorinates diverse chlorinated aromatic pollutants
.
Environ Sci Technol 38:2075

2081



Ferguson PL, Brownawell BJ (2003) Degradation of nonylphenol ethoxylates in estuarine
sediment under aerobic and anaerobic conditions. Environ Toxicol Chem 22:1189

1199



Fetzner S (1998) Bacterial dehalogenation. A
ppl Microbiol Biotechnol 50:633

657




Finneran KT, Lovley DR (2001) Anaerobic degradation of methyl
tert
-
butyl ether (MTBE) and
tert
-
butyl alcohol (T
BA). Environ Sci Technol 35:1785

1790




Funk SB, Crawford DL, Crawford RL, Mead G, Davis
-
Hooker W (1995) Full
-
scale anaerobic
bioremediation of trinitro
toluene contaminated soils. Appl Biochem Biotechnol 51:625

633



Galushko A, Minz D, Schink B, Widdel F (1999) Anaerobic degradation of naphthalene by a
pure culture of a novel type of marine sulphate
-
reducing bacterium. Environ Microbiol 1:415

420



G
aus C, Brunskill GJ, Connell DW, Prange J, Muller JF, Papke O, Webber R (2002)
Transformation processes, pathways, and possible sources of distinctive polychlorinated
dibenzo
-
p
-
dioxin signatures in sink environments. Environ Sci Technol 36:3452

3549



Ge
rritse J, Renard V, Gomes TMP, Lawson PA, Collins MD, Gottschal J (1996)
Desulfitobacterium

sp. strain, an anaerobic bacterium that can grow by reductive dechlorination
of tetrachloroethene or
ortho
-
chlorinated phenols. Arch Microbiol 165:132

140



Gerri
tse J, Drzyzga O, Kloetstra G, Keijmel M, Wiersum LP, Hutson R, Collins MD, Gottschal
JC (1999) Influence of different electron donors and acceptors on dehalorespiration of
tetrachloroethene by
Desulfitobacterium frappieri

TCE1. Appl Environ Microbiol 65:5
212

5221



Ghosh PK, Philip L (2004) Atrazine degradation in anaerobic environment by a mixed microbial
consortium. Water Res 38:2276

2283



Gieg LM, Suflita JM (2002) Detection of anaerobic metabolites of saturated and aromatic
hydrocarbons in petrole
um
-
contaminated aquifers. Environ Sci Technol 36:3755

3762



Gorontzy T, Drzyzga O, Kahl MW, Bruns
-
Nagel D, Breitung J, von Loew E, Blotevogel KH
(1994) Microbial degradation of explosives and related compounds. Crit Rev Microbiol 20:265

284



Guenzi W
D, Beard WE (1967) Anaerobic biodegradation of DDT to DDD in soil. Science
156:1116

1117



Haggblom MM, Young LY (1990) Chlorophenol degradation coupled to sulfate reduction. Appl
Environ Microbiol 56:3255

3260



Haggblom MM, Young LY (1995) Anaerobic
degradation of halogenated phenols by sulfate
-
reducing consortia. Appl Environ Microbiol 61:1546

1550



Haggblom MM, Knight VK, Kerkhof LJ (2000) Anaerobic decomposition of halogenated
aromatic compounds. Environ Pollut 107:199

207



Haggblom MM, Ahn Y
B, Fennell DE, Kerkhof LJ, Rhee SK (2003) Anaerobic dehalogenation
of organohalide contaminants in the marine environment. Adv Appl Microbiol 53:61

84



Haggensen F, Mogensen AS, Angelidaki I, Ahring BK (2002) Anaerobic treatment of sludge:
focusing on r
eduction of LAS concentration in sludge. Water Sci Technol 46:159

165



Hammill TB, Crawford RL (1996) Degradation of 2
-
sec
-
butyl
-
4,6
-
dinitrophenol (Dinoseb) by
Clostridium bifermentans

KMR
-
1. Appl Environ Microbiol 62:1842

1846



Harms G, Zengler K, R
abus R, Aeckersberg F, Minz D, Rosselló
-
Mora R, Widdel F (1999)
Anaerobic oxidation of
o
-
xylene,
m
-
xylene, and homologous alkylbenzenes by new types of
sulfate
-
reducing bacteria. Appl Environ Microbiol 65:999

1004



Hawari J, Beaudet S, Halasz A, Thibout
ots S, Ampleman G (2000) Microbial degradation of
explosives: biotransformation versus mineralization. Appl Microbiol Biotechnol 54:605

618



He J, Ritalahti KM, Yang K
-
L, Koenigsberg SS, Löffler FE (2003) Detoxification of vinyl
chloride to ethene coupl
ed to an anaerobic bacterium. Nature 424:62

65



Heider J, Fuchs G (1997) Anaerobic metabolism of aromatic compounds. Eur J Biochem
243:577

96




Hendriksen HV, Larsen S, Ahring BK (1992) Influence of a supplemental carbon source on
anaerobic dechlorination of pentachlorophenol in granular sludge. Appl Environ Microbiol
58:365

370



Hermuth K, Leuthner B, Heider J (2002) Operon structure and
expression of the genes for
benzylsuccinate synthase in
Thauera aromatica

strain K172. Arch Microbiol 177:132

138



Hess A, Zarda B, Hahn D, Haner A, Stax D, Hohener P, Zeyer J (1997) In situ analysis of
denitrifying toluene
-

and
m
-
xylene degrading bacte
ria in a diesel fuel
-
contaminated laboratory
aquifer column. Appl Environ Microbiol 65:2136

2141



Holliger C, Wohlfarth G, Diekert G (1999) Reductive dechlorination in the energy metabolism
of anaerobic bacteria. FEM Microbiol Rev 22:383

398



Huang S
, Lindahl PA, Wang C, Bennett GN, Rudolph FB, Hughes JB (2000) 2,4,6
-
Trinitrotoluene reduction by carbon monoxide dehydrogenase from
Clostridium thermoaceticum
.
Appl Environ Microbiol 66:1474

1478



Huber M, Meyer U, Rys P (2000) Biodegradation mechanism
s of linear alcohol ethoxylates
under anaerobic conditions. Environ Sci Technol 34:1737

1741



Hutchins SR, Down WC, Wilson JT, Smith GB, Kovacs DA, Fine DD, Douglass RH, Hendrix
DJ (1991) Effect of nitrate addition on bioremediation of fuel
-
contaminated

aquifer: field
demonstration. Ground Water 29:571

580



Hughes JB, Wang C, Yesland K, Richardson A, Bhadra R, Bennett G, Rudolph F (1998)
Bamberger rearrangement during TNT metabolism by
Clostridium acetobutylicum
. Environ Sci
Technol 32:494

500




Hughes JB, Wang CY, Zhang C (1999) Anaerobic Biotransformation of 2,4
-
dinitrotoluene and
2,6
-
dinitrotoluene by
Clostridium acetobutylicum
: a pathway through dihydroxylamino
intermediates.

Environ Sci Technol 33:1065

1070



Jablonski PE, Ferry JG (1992) Reductive dechlorination of trichloroethylene by the CO
-
reduced
CO dehydrogenase enzyme complex from
Methanosarcina thermophila
. FEMS Microbiol Lett
96:55

60



Janke D, Fritsche W (1985)

Nature and significance of microbial cometabolism of xenobiotics. J
Basic Microbiol 25:603

619



Jewell WJ (1987) Anaerobic sewage treatment, part 6. Environ Sci Technol 21:14

21



Kao CM, Wang JY, Wu MJ (2001) Evaluation of atrazine removal processes

in a wetland. Water
Sci Technol 44:539

544



Kazumi J, Haggblom MM, Young LY (1995) Diversity of anaerobic microbial processes in
chlorobenzoate degradation: nitrate, iron, sulfate and carbonate as electron acceptors. Appl
Microbiol Biotechnol 43:929

93
6



Kitts CL, Cunningham DP, Unkefer PJ (1994) Isolation of three hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine degrading species of the family Enterobacteriaceae from nitramine explosive
-
contaminated soil. Appl Environ Microbiol 60:4608

4711



Kolhatkar R,
Kuder T, Philp P, Allen J, Wilson JT (2002) Use of compound
-
specific stable
carbon isotope analyses to demonstrate anaerobic biodegradation of MTBE in groundwater at a
gasoline release site. Environ Sci Technol 36:5139

5146



Krieger J, Roseboom W, Albra
cht SP, Spormann AM (2001) A stable organic free radical in
anaerobic benzylsuccinate synthase of
Azoarcus

sp. strain T. J Biol Chem 276:12924

12927



Kube M, Heider J, Amann J, Hufnagel P, Kuhner S, Beck A, Reinhardt R, Rabus R (2004)
Genes involved in
the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1.
Arch Microbiol 181:182

194




Langenhoff A (2003) In
-
situ bioremed
iation of pesticides.
(
http://www.mep.tno.nl/Informatiebladen_eng/304e.pdf
)



Leahy JG, Colwell RR (1990) Microbial degradation of hydrocarbons in the environment.
Microbiol Rev 54:305

3
15




Lenke H, Warrelmann J, Daun G, Hund K, Sieglen U, Knackmuss H
-
J (1998) Biological
treatment of TNT
-
contaminated soil.
2. Biologically induced immobilization of the contaminants
and full
-
scale application. Environ Sci Technol 32:1964

1971



Leutwein C, Heider J (1999) Anaerobic toluene
-
catabolic pathway in denitrifying
Thauera
aromatica
: activation and beta
-
oxidation of
the first intermediate, (R)
-
(+)
-
benzylsuccinate.
Microbiology 145:3265

3271



Leutwein C, Heider J (2002) (R)
-
Benzylsuccinyl
-
CoA dehydrogenase of
Thauera aromatica
, an
enzyme of the anaerobic toluene catabolic pathway. Arch Microbiol 178:517

524



Leut
hner B, Leutwein C, Schulz H, Horth P, Haehnel W, Schiltz E, Schagger H, Heider J (1998)
Biochemical and genetic characterization of benzylsuccinate synthase from
Thauera aromatica
:
a new glycyl radical enzyme catalysing the first step in anaerobic toluene

metabolism. Mol
Microbiol 28:615

628



Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Phillips EJP, Siegel DI (1989)
Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339:297

299



Madsen T, Boyd HB, Nylén D, Pederse
n AR, Petersen GI, Simonsen F (2001). Environmental
and health assessment of substances in household detergents and cosmetic detergent products.
Environmental Project No. 615.
http://www.mst.dk/udgiv
/publications/2001



Magnuson JK, Stern RV, Gossett JM, Zinder SH, Burris DR (1998) Reductive dechlorination of
tetrachloroethene to ethene by a two
-
component enzyme pathway. Appl Environ Microbiol
64:1270

1275



Magnuson JK, Romine MF, Burris DR, Kin
gsley MT (2000) Trichloroethene reductive
dehalogenase from
Dehalococcoides ethenogenes
: Sequence of
tceA

and substrate range
characterization. Appl Environ Microbiol 66:5141

5147



Major DW, Hodgins WW, Butler BJ (1991) Field and laboratory evidence of
in situ
biotransformation of tetrachloroethene to ethene and ethane at a chemical transfer facility in
North Toronto. In: Hinchee RE, Olfenbuttel (eds) On site bioremediation: processes for
xenobiotic and hydrocarbon treatment. Butterworth
-
Heinemann, Stone
ham, Mass. pp

141

171



Marvin
-
Sikkema FD, de Bont JA (1994) Degradation of nitroaromatic compounds by
microorganisms. Appl Microbiol Biotechnol 42:499

507



Master ER, Lai VW
-
M, Kuipers B, Cullen WR, Mohn WW (2002) Sequential anaerobic
-
aerobic
treatme
nt of soil contaminated with weathered Aroclor 1260. Environ Sci Technol 36:100

103



Matsumura F, Boush GM (1971) DDT metabolized by microorganisms from Lake Michigan.
Nature 230:325

326



Maymo
-
Gatell X, Chien Y, Gossett JM, Zinder SH (1997) Isolatio
n of a bacterium that
reductively dechlorinates tetrachloroethene to ethene. Science 276:1568

1571




Maymo
-
Gatell X, Nijenhuis I, Zinder SH (2001
) Reductive dechlorination of cis
-
1,2
-
dichloroethene and vinyl chloride by
Dehalococcoides ethenogene
s
. Environ Sci Technol
35:516

521



McCarty PL (1997) Breathing with chlorinated solvents. Science
276:1521

1522




McCarty PL, Smith DP (1986) Anaerobic wastewater treatment. Environ Sci Technol 20:1200

1206



McCarty PL, Wilson JT (1992) Nat
ural anaerobic treatment of a TCE plume St. Joseph,
Michigan, NPL site. In: United States Environmental Protection Agency (ed) Bioremediation of
hazardous wastes. EPA /600/R
-
92/126. US EPA, Washington, D.C., pp

57

50



McCormick NG, Cornell JH, Kaplan AM

(1981) Biodegradation of hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
trazine. Appl Environ Microbiol 42:817

823



McCormick NG, Feeherry FE, Levinson HS (1976) Microbial transformation of 2,4,6
-
TNT and
other nitroaromatic compounds. Appl Environ Microbiol 31:949

958



Meckenstock RU, Annweiler E, Michaelis W, Richnow HH, Schink B (2000) Anaerobic
naphthalene degradation by a sulfate
-
reducing enrichment culture. Appl Environ Microbiol
66:2743

2747



Mihelcic JR, Luthy RG (1988) Microbial
-
degradation of acenaphthen
e and naphthalene under
denitrification conditions in soil
-
water systems. Appl Environ Microbiol 54:1188

1198



Mikesell MD, Boyd S (1985) Reductive dechlorination of the pesticides 2,4
-
D and 2,4,5
-
T, and
pentachlorophenol in anaerobic sludges. J Environ

Qual 14:337

340



Mikesell MD, Boyd SA (1986) Complete reductive dechlorination and mineralization of
pentachlorophenol by anaerobic microorganisms. Appl Environ Microbiol 52:861

865



Mogensen AS, Dolfing J, Haagensen F, Ahring BK (2003a) Potential f
or anaerobic conversion
of xenobiotics. Adv Biochem Eng Biotechnol 82:69

134



Mogensen AS, Haagensen F, Ahring BK (2003b) Anaerobic degradation of linear alkylbenzene
sulfonate. Environ Toxicol Chem 22:706

711



Morgan P, Watkinson RJ (1989) Microbiol
ogical methods for the clean up of soil and
groundwater contaminated with halogenated organic compounds. FEMS Microbiol Rev 63:277

300



Neumann A, Wohlfarth G, Diekert G (1996) Purification and characterization of
tetrachloroethene reductive dehalogenas
e from
Dehalospirillum multivorans
. J Biol Chem
271:16515

16519



Newcombe D, Crowley DE (1999) Bioremediation of atrazine
-
contaminated soil by repeated
applications of atrazine
-
degrading bacteria. Appl Microbiol Biotechnol 51:877

882



Nicholson DK, W
oods SL, Istok JD, Peek DC (1992) Reductive dechlorination of chlorophenols
by a pentachlorophenol
-
acclimated methanogenic consortium. Appl Environ Microbiol 58:2280

2286



Ohtsubo Y, Kudo T, Tsuda M, Nagata Y (2004) Strategies for bioremediation of poly
chlorinated
biphenyls. Appl Microbiol Biotechnol 65:250

258



Padda RS, Wang CY, Hughes JB, Bennett GN (2000) Mutagenicity of trinitrotoluene and its
metabolites formed during anaerobic degradation by
Clostridium acetobutylicum

ATCC 824.
Environ Toxicol
Chem 19:2871

2875



Padda RS, Wang C, Hughes JB, Bennett GN (2003) Mutagenicity of nitroaromatic explosives
during anaerobic transformation by
Clostridium acetobutylicum
. Environ Toxicol Chem
22:2293

2297



Pagano JJ, Scrudato RJ, Roberts RN, Bemis JC
(1995) Reductive dechlorination of PCB
-
contaminated sediments in an anaerobic bioreactor system. Environ Sci Technol 29:2584

2589



Peres CM, Agathos SN (2000) Biodegradation of nitroaromatic pollutants: from pathways to
remediation. Biotechnol Annu Rev
6:197

220



Pignatello JJ, Johnson LK, Martinson MM, Carlson RE, Crawford RL (1985) Response of the
microflora in outdoor experimental streams to pentachlorophenol: compartmental contributions.
Appl Environ Microbiol 50:127

132



Preuss A, Fimpel J, Di
ekert G (1993) Anaerobic transformation of 2,4,6
-
trinitrotoluene (TNT).
Arch Microbiol 159:345

353



Prince RC (1993) Petroleum spill bioremediation in marine environments. Crit Rev Microbiol
19:217

242



Quensen JF III, Tiedje JM, Boyd SA (1988) Reduc
tive dechlorination of polychlorinated
biphenyls by anaerobic microorganisms from sediments. Science 242:752

754



Quensen JF III, Mueller SA, Jain MK, Tiedje JM (1998) Reductive dechlorination of DDE to
DDMU in marine sediment microcosms. Science 280:72
2

724



Quensen JF III , Tiedje JM, Jain MK, Mueller SA (2001) Factors controlling the rate of DDE
dechlorination to DDMU in Palos Verdes margin sediments under anaerobic conditions. Environ
Sci Technol 35:286

291



Rabus R, Widdel F (1995) Anaerobic d
egradation of ethylbenzene and other aromatic
hydrocarbons by new denitrifying bacteria. Arch Microbiol 163:96

103




Rabus
R, Wilkes H, Behrends A, Armstroff A, Fischer T, Pierik AJ, Widdel F (2001) Anaerobic
initial reaction of
n
-
alkanes in a denitrifying bacterium: evidence for (1
-
methylpentyl)succicate
as initial product and for involvement of an organic radical in
n
-
hexane

metabolism. J Bacteriol
183:1707

1715



Raymond RL (1974) Reclamation of hydrocarbon contaminated ground water. US Patent
3

846

290, 5 November 1974



Renner R (1998)
Natural

remediation of DDT, PC
Bs debated. Environ Sci Technol 32:360

363A



Ribarova I, Topalova J, Ivanov I, Kozuharov D, Dimkov R, Cheng C (2002) Anaerobic
sequencing batch reactor as initiating stage in complete pentachlorophenol biodegradation.
Water Sci Technol 46:565

569



Ri
ttmann BE, McCarty PL (2001) Environmental biotechnology: principles and applications.
McGraw
-
Hill, New York



Roberts DJ, Kaake RH, Funk SB, Crawford DL, Crawford RL (1993) Anaerobic remediation of
dinoseb from contaminated soil. An on
-
site demonstratio
n. Appl Biochem Biotechnol 39

40:781

789



Rockne KJ, Strand SE (1998) Biodegradation of bicyclic and polycyclic aromatic hydrocarbons
in anaerobic enrichments. Environ Sci Technol 32:3962

3967



Rockne KJ, Chee
-
Sanford JC, Sanford RA, Hedlund BP, Stal
ey JT, Strand SE (2000) Anaerobic
naphthalene degradation by microbial pure culture under nitrate
-
reducing conditions. Appl
Environ Microbiol 66:1595

1601



Rooney
-
Varga JN, Anderson RT, Fraga JL, Ringelberg D, Loveley DR (1999) Microbial
communities ass
ociated with anaerobic benzene degradation in a petroleum
-
contaminated
aquifer. Appl Environ Microbiol 65:3056

3063




Roth
ermich MM, Hayes LA, Lovley DR (2002) Anaerobic, sulfate
-
dependent degradation of
polycyclic aromatic hydrocarbons in petroleum
-
contaminated harbor sediment. Environ Sci
Technol 36:4811

4817



Rueter P, Rabus R, Wilkes H, Aeckersberg F, Rainey FA, Jannas
ch HW, Widdel F (1994)
Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate
-
reducing bacteria.
Nature 372:455

458



Ruppe S, Neumann A, Vetter W (2003) Anaerobic transformation of compounds of technical
toxaphene. I. Regiospecific re
action of chlorobornanes with geminal chlorine atoms. Environ
Toxicol Chem 22:2614

2621



Ruppe S, Neumann A, Vetter W (2004) Anaerobic transformation of compounds of technical
toxaphene. II. Fate of compounds lacking geminal chlorine atoms. Environ Toxi
col Chem
23:591

598



Salminen JM, Tuomi PM, Suortti A
-
M, Jørgensen KS (2004) Potential for aerobic and anaerobic
biodegradation of petroleum hydrocarbons in boreal subsurface. Biodegradation 15:29

39




Schink B (2002) Anaerobic digestion: concepts, limits and perspectives. Water Sci Technol
45:1

8



Semprini L, Hopkins GD, McCarty PL, Roberts PV (1992) In situ biotransformation of carbon

tetrachloride and other halogenated compounds resulting from biostimulation under anoxic
conditions. Environ Sci Technol 26:2454

2461



Sethunathan N (1973) Microbial degradation of insecticides in flooded soil and in anaerobic
cultures. Residue Rev 47:
143

165



Seybold CA, Mersie W, McNamee C (2001) Anaerobic degradation of atrazine and metolachlor
and metabolite formation in wetland soil and water microcosms. J Environ Qual 30:1271

1277



Shelton DR, Tiedje JM (1984) Isolation and partial character
ization of bacteria in an anaerobic
consortium that mineralizes 3
-
chlorobenzoic acid. Appl Environ Microbiol 48:840

848



Sheremata TW, Hawari J (2000) Cyclodextrins for desorption and solubilization of 2,4,6
-
trinitrotoluene and its metabolites from soil
. Environ Sci Technol 36:3462

3468



Shimazu M, Mulchandani A, Chen W (2001) Simultaneous degradation of organophosphorus
pesticides and
p
-
nitrophenol by a genetically engineered
Moraxell

sp. with surface
-
expressed
organophosphorus hydrolase. Biotechnol
Bioeng 76:318

324



So CM, Young LY (1999a) Initial reactions in anaerobic alkane degradation by a sulfate reducer,
strain AK
-
01. Appl Environ Microbiol 65:5532

5540



So CM, Young LY (1999b) Isolation and characterization of a sulfate
-
reducing bacteri
um that
anaerobically degrades alkanes. Appl Environ Microbiol 65:2969

2976



So CM, Phelps CD, Young LY (2003) Anaerobic transformation of alkanes to fatty acids by a
sulfate
-
reducing bacterium, strain Hxd3. Appl Environ Microbiol 69:3892

3900



Somsa
mak P, Cowan RM, Haggblom MM (2001) Anaerobic biotransformation of fuel
oxygenates under sulfate
-
reducing conditions. FEMS Microbiol Ecol 37:259

264



Song B, Palleroni NJ, Haggblom MM (2000) Isolation and characterization of diverse
halobenzoate
-
degradi
ng denitrifying bacteria from soils and sediments. Appl Environ Microbiol
66:3446

3453



Song B, Palleroni NJ, Kerkhof LJ, Haggblom MM (2001) Characterization of halobenzoate
-
degrading, denitrifying
Azoarcus

and
Thauera

isolates and description of

Thauer
a
chlorobenzoica

sp. nov. Int J Syst Evol Microbiol 51:589

602



Spormann AM, Widdel F (2000) Metabolism of alkylbenzenes, alkanes, and other hydrocarbons
in anaerobic bacteria. Biodegradation 11:85

105




Steber J, Wierich P (1987) The anaerobic degradation of detergent range fatty alcohol
ethoxylates. Studies with
14
C
-
labbelled model surfactant. Water Res 21:661

667



Steinbach A, Seifert R, An
nweiler E, Michaelis W (2004) Hydrogen and carbon isotope
fractionation during anaerobic biodegradation of aromatic hydrocarbons
-
a field study. Environ
Sci Technol 38:609

616



Stocking AJ, Deeb RA, Flores AE, Stringfellow W, Talley J, Brownell R, Kavana
ugh MC
(2000) Bioremediation of MTBE: a review from a practical perspective. Biodegradation 11:187

201



Strong LC, McTavish H, Sadowsky MJ, Wackett LP (2000) Field
-
scale remediation of atrazine
-
contaminated soil using recombinant
Escherichia coli

expres
sing atrazine chlorohydrolase.
Environ Microbiol 2:91

98



Sullivan ER, Zhang X, Phelps C, Young LY (2001) Anaerobic mineralization of stable
-
isotope
-
labeled 2
-
methylnaphthalene. Appl Environ Microbiol 67:4353

4357



Sung Y, Ritalahti KM, Sanford RA, U
rbance JW, Flynn SJ, Tiedje JM, Löffler FE (2003)
Characterization of two tetrachloroethene
-
reducing, acetate
-
oxidizing anaerobic bacteria and
their description as
Desulfuromonas michiganesis

sp. nov. Appl Environ Microbiol 69:2964

2974



Swisher RD (198
7) Surfactant biodegradation, 2nd edn. Dekker, New York, N.Y.



Tadros MG, Crawford A, Mateo
-
Sullivan A, Zhang C, Hughes JB (2000) Toxic effects of
hydroxylamino intermediates on algae
Selenastrum capricornutum
. Bull Environ Contam
Toxicol 64:579

585



Tartakovsky B, Levesque M, Dumortier R, Beaudet R, Guiot SR (1999) Biodegradation of
pentachlorophenol in a continuous anaerobic reactor augmented with
Desulfitobacterium
frappieri

PCP
-
1. Appl Environ Microbiol 65:4357

4362



Ternan NG, McGrath JW, McMu
llan G, Quinn JP (1998) Organophosphonates: occurrence,
synthesis and biodegradation by microorganisms. World J Microbiol Biotechnol 14:635

647



Terzenbach DP, Blaut M (1994) Transformation of tetrachloroethylene to trichloroethylene by
homoacetogenic b
acteria. FEMS Microbiol Lett 123:213

218



Tiedje JM, Quensen JF III, Chee
-
Sanford J, Schimel JP, Boyd SA (1993) Microbial reductive
dechlorination of PCBs. Biodegradation 4:231

240




Vargas C, Song B, Camps M, Haggblom MM (2000) Anaerobic degradation of fluorinated
aromatic compounds. Appl Microbiol Biotechnol 53:342

347



Vargas C, Fennell DE, Häggblom MM (2001) Anaerobi
c reductive dechlorination of chlorinated
dioxins in estuarine sediments. Appl Microbiol Biotechnol 57:786

790



Wackett LP, Sadosky MJ, Martinez B, Shapir N (2002) Biodegradation of atrazine and related
s
-
triazine compounds: from enzymes to field studie
s. Appl Microbiol Biotechnol 58:39

45




Wagener S, Schink B (1988) Fermentative degradation of nonionic surfactants and polyethylene
glycol by e
nrichment cultures and by pure cultures of homoacetogenic and propionate
-
forming
bacteria. Appl Environ Microbiol 54:561

565



Wedemeyer G (1966) Dechlorination of DDT by
Aerobacter aerogenes
. Science 152:647



Widdel F, Rabus R (2001) Anaerobic biodeg
radation of saturated and aromatic hydrocarbons.
Curr Opin Biotechnol 12:259

276



Wiegel J, Zhang X, Wu Q (1999) Anaerobic dehalogenation of hydroxylated polychlorinated
biphenyls by
Desulfitobacterium dehalogenans
. Appl Environ Microbiol 65:2217

2221



Wilkes H, Rabus R, Fischer T, Armstroff A, Behrends A, Widdel F (2002) Anaerobic
degradation of
n
-
hexane in a denitrifying bacterium: further degradation of the initial
intermediate (1
-
methylphentyl)succinate via C
-
skeleton rearrangement. Arch Microbiol

177:235

243



Williams PP (1977) Metabolism of synthetic organic pesticides by anaerobic microorganisms.
Residue Rev 66:63

135



Woods SL, Ferguson JF, Benjamin MM (1989) Characterization of chlorophenol and
chloromethoxybenzene biodegradation during
anaerobic treatment. Environ Sci Technol 23:62

68



Wu Q, Bedard DL, Wiegel J (1997) Temperature determines pattern of anaerobic microbial
dechlorination of Aroclor 1260 primed by 2,3,4,6
-
tetrachlorobiphenyl in Woods Pond sediments.
Appl Environ Microbio
l 63:4818

4825



Wu Q, Sowers KR, May HD (1998) Microbial reductive dechlorination of Aroclor 1260 in
anaerobic slurries of estuarine sediments. Appl Environ Microbiol 64:1052

1058



Wu Q, Sowers KR, May HD (2000) Establishment of a polychlorinated bip
henyl
-
dechlorinating
microbial consortium, specific for doubly flanked chlorines, in a defined, sediment
-
free medium.
Appl Environ Microbiol 66:49

53



Wu Q, Milliken CE, Meier GP, Watts JE, Sowers KR, May HD (2002a) Dechlorination of
chlorobenzenes by a

culture containing bacterium DF
-
1, a PCB dechlorinating microorganism.
Environ Sci Technol 36:3290

3294




Wu Q, Watts JE, Sowers KR, May HD (2002b) Ide
ntification of a bacterium that specifically
catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked
chlorines. Appl Environ Microbiol 68:807

812



Xue SK, Iskandar IK, Selim HM (1995) Adsorption
-
desorption of 2,4,6
-
trinit
rotoluene and
hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine in soils. Soil Sci 160:317

327



Young DM, Unkefer PJ, Ogden KL (1997) Biotransformation of hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine (RDX) by a prospective consortium and its most effective isolate
Ser
ratia marcescens
.
Biotechnol Bioeng 53:515

522



Zengler K, Richnow HH, Rosselló
-
Mora R, Michaelis W, Widdel F (1999) Methane formation
from long
-
chain alkanes by anaerobic microorganisms. Nature 401:266

269



Zhang C, Hughes JB (2003) Biodegradation p
athways of hexahydro
-
1,3,5
-
trinitro
-
1,3,5
-
triazine
(RDX) by
Clostridium acetobutylicum

cell
-
free extract. Chemosphere 50:665

671



Zhang C, Hughes JB, Nishino SF, Spain J (2000a) Slurry
-
phase biological treatment of 2,4
-
dinitrotoluene and 2,6
-
dinitrotolu
ene: Role of bioaugmentation and effects of high dinitrotoluene
concentrations. Environ Sci Technol 34:2810

2816



Zhang X, Young LY (1997) Carboxylation as an initial reaction in the anaerobic metabolism of
naphthalene and phenanthrene by sulfidogenic c
onsortia. Appl Environ Microbiol 63:4759

4764



Zhang X, Sullivan ER, Young LY (2000b) Evidence for aromatic ring reduction in the
biodegradation pathway of carboxylated naphthalene by a sulfate
-
reducing consortium.
Biodegradation 11:117

124



Zhao J
-
S
, Halasz A, Paquet L, Beaulieu C, Hawari J (2002) Biotransformation of hexahydro
-
1,3,5
-
trnitro
-
1,3,5
-
triazine and its mononitroso derivative hexahydro
-
1
-
nitroso
-
3,5
-
dinitro
-
1,3,5
-
triazine by
Klebsiella pneumoniae

strain SCZ
-
1 isolated from an anaerobic slu
dge. Appl Environ
Microbiol 68:5336

5341