FEMS Microbiology Letters 119 (1994) 199-208
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
199
FEMSLE 05992
Anaerobic degradation of halogenated benzoic acids
by photoheterotrophic bacteria
B.J. van der Woude
and J.C. Gottschal
*, M . d e B o e r , N . M . J . v a n d e r P u t , F . M . v a n d e r G e l d , R . A . P r i n s
Department of Microbiology, University of Groningen, P.O. Box 14, 9750 AA Haren, the Netherlands
(Received 2 March 1994; revision received and accepted 29 March 1994)
Abstract: From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was
obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in
the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a
Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph
capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if
grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.
Key words: Photoheterotrophy; Anaerobic dehalogenation; Rhodopseudomonas palustris; Benzoic acid; Bromobenzoic acid;
Chlorobenzoic acid
Introduction
I n d u s t r i a l a n d a g r i c u l t u r a l activities a r e t h e
main source of a great variety of halogenated
c o m p o u n d s , m a n y o f which have a p p e a r e d to b e
r e c a l c i t r a n t in m o s t e n v i r o n m e n t s . In spite o f t h e
fact t h a t since t h e e a r l y seventies investigations
have focussed on t h e c a p a c i t i e s o f a e r o b i c a n d
a n a e r o b i c b a c t e r i a to d e g r a d e x e n o b i o t i c c o m p o u n d s [1,2], it is only r e c e n t l y t h a t p h o t o t r o p h i c
m i n e r a l i z a t i o n o f s u b s t i t u t e d a r o m a t i c s has g a i n e d
some attention.
P h o t o h e t e r o t r o p h i c b a c t e r i a a r e k n o w n for
* Corresponding author. Tel: (050) 632191; Fax: (050) 632154.
SSDI 03 78-1097(94)00138-H
t h e i r use o f a w i d e r a n g e o f substrates, e s p e c i a l l y
if grown a n a e r o b i c a l l y in t h e light [3-8]. T h e
m o s t obvious a d v a n t a g e s o f the t r a n s f o r m a t i o n o f
x e n o b i o t i c c o m p o u n d s for p h o t o t r o p h i c b a c t e r i a
w o u l d be: (1) detoxification; (2) t h e use as a
s o u r c e o f C, S o r N; a n d (3) use as a sink for
excess r e d u c i n g power. T h e p a r t i a l d e h a l o g e n a tion o f PCBs in t h e p r e s e n c e o f light a n d a c e t a t e
was c l a i m e d to b e t h e result o f r e d u c t i v e d e h a l o g e n a t i o n by p h o t o t r o p h i c b a c t e r i a [9]. D i n i t r o p h e n o l e s c a n b e r e d u c t i v e l y t r a n s f o r m e d to
m o n o n i t r o p h e n o l e s by p h o t o t r o p h i c b a c t e r i a [10].
T h e c o m p l e t e use o f 3 - c h l o r o b e n z o a t e ( 3 - C B A )
by Rhodopseudomonas palustris W S 1 7 [11] is dep e n d e n t on t h e p r e s e n c e o f b e n z o a t e . B e n z o a t e g r o w n cells o f t h e p h o t o t r o p h i c b a c t e r i u m H 4 5 - 2 ,
200
similar to Rhodopseudomonas palustris, were able
to photometabolize 3-CBA, but again did not
utilize it as a sole growth substrate [12].
In this study we describe the enrichment and
some properties of a phototrophic bacterium able
to grow on 3-CBA as the sole carbon source
anaerobically in light. Its characteristics are compared with those of Rhodopseudomonas palustrisa
strain WS17.
Materials and Methods
Organisms
For enrichments with halogenated benzoic
acids, a mixture of samples collected from a ditch
along a railway, a ditch near a polluted paint-factory site and De Biesbosch marsh sediment, all
sites in the Netherlands, was used as an inoculum
(50 g wet material/l). Sampling was done in
sterile serum bottles which were completely filled
with surface sediment and water.
Rhodopseudomonas palustris strain WS17, able
to metabolise 3-chlorobenzoate (3-CBA) in the
presence of benzoate (BA) [11], was kindly supplied by Dr. Wyndham (Carleton University, Ottawa, Ontario, Canada).
amide, 0.4 mg; pyridoxamine, 1.0 mg; pantothenic
acid, 0.2 mg; cobalamin, 0.2 mg; biotin, 0.04 mg.
Non-halogenated growth substrates and halogenated benzoic acids were added from separately autoclaved stock solutions.
Enrichment cultures, 10 ml in closed 16-ml
glass tubes, contained a mixture of mono- and
dichlorinated benzoates (2-CBA, 3-CBA, 4-CBA,
2,3-CBA, 2,4-CBA, 2,5-CBA, 3,4-CBA and 3,5CBA: 0.2 mM each) or monobromobenzoates (2BrBA, 3-BrBA and 4-BrBA: 0.2 mM each). They
were incubated without or in the presence of
additional non-halogenated substrates: benzoate
(1 mM), ethanol (3 mM) or decanoate (1 mM). In
successive transfers of enrichment cultures, inhibition of algal growth was accomplished by the
addition of 5 /xM 3(3,4-dichlorophenyl)l,l-dimethylurea (DCMU) from an ethanolic filtersterile 1 mM solution [13]. Cultures of R.palustris
WS17 were kept viable by transfers in media
containing BA (1 mM) and 3-CBA (1 mM).
Cultures were checked for purity by streaking
on Nutrient Broth (BBL, Cockeysville, USA) agar
plates, which were incubated aerobically in the
dark and anaerobically in the light, and by microscopic observation.
Analytical procedures
Growth media and enrichment
Batch cultures were routinely incubated in the
light at 30°C, pH = 7.0, in closed tubes or serum
bottles, containing anaerobically prepared liquid
medium and a N 2 gas phase. The low-chloride
mineral salts medium contained the following
components (per 1): MgSOa.7H20, 0.1 g;
Ca(NOa)2.4H20 , 0.04 g; yeast extract, 0.1 g;
EDTA, 1 mg; FeSO4.7H20, 2 mg; ZnSO4.7H20,
0.1 mg; MnC12.4H20, 0.03 mg; HaBO3, 0.3 mg;
COC12.6H20, 0.2 mg; CUC1E.2H20, 0.01 mg;
NiC1E.6H20 , 0.02 mg; Na2MoO4.2H20, 0.03 mg;
Na2SeO3.5H20 0.026 mg; NaEWO4.2H20 0.033
mg. After autoclaving, the medium was completed by the addition of a K(NH4)PO 4 buffer,
pH = 7.0, to a final concentration of 25 mM, and
by adding a filter-sterile vitamin solution (values
give final concentrations): para-aminobenzoic
acid, 0.2 mg; folic acid, 0.1 mg; fatty acid, 0.1 mg;
riboflavin, 0.2 mg; thiamin, 0.4 mg; nicotinic acid
Chloride was measured colorimetrically according to the method of Bergman and Sanic [14]
with NaCI as a standard. Benzoates were analyzed as described previously by Gerritse and
Gottschal [15], with a lower detection limit of
0.01 mM. Growth was determined by measuring
optical density at 660 nm. Dissolved or cell-carbon
were analyzed with a total carbon analyzer as
described by Gerritse et al. [16]. Absorption spectra of whole cells and of methanol-extracted pigments were made after the method by Yurkov
and van Gemerden [17].
Chemicals
All chemicals were of analytical grade. BA,
2-CBA, 3-CBA, 4-CBA and 2,4-CBA were purchased from Merck (Darmstadt, FRG). 2,3-CBA,
2,5-CBA, 3,4-CBA, 2-BrBA, 3-BrBA and 4-BrBA
were from Aldrich (Gillingham, UK). 3,5-CBA
was from Fluka (Buchs, Switzerland)
201
Results
Enrichment of anaerobic phototrophic bacteria on
chlorobenzoate and bromobenzoate
Enrichment cultures were set up in the presence of a mixture of chloro- or bromobenzoates
(see Material and Methods) either without an
additional substrate or with benzoate, decanoate,
or ethanol. After 2 months of anaerobic incubation in the light, tubes with BA or decanoate had
turned reddish-brown. In BA-containing tubes,
concentrations of 2-CBA and 3-CBA or 4-BrBA
had decreased, and 4-BrBA and 3-BrBA had
partly disappeared in tubes containing decanoate.
Initial transfers from these cultures in fresh
medium containing the CBA-mixture showed a
decrease in concentration of 3-CBA only if BA (1
mM) was added. A decrease in 2-BrBA and 3BrBA was observed in subcultures containing the
BrBA-mixture and BA. Subsequently, all CBAcontaining enrichment cultures were pooled and
used to inoculate (10%) tubes containing a mixture of CBAs, with and without additional benzoate (BA, 1 mM). The same procedure was
followed for BrBA cultures.
In the subcultures, in the absence of BA, only
poor growth of pigmented bacteria occurred, presumably at the expense of some yeast extract
components, and no degradation of halogenated
substrates was observed. However, if BA was
present, CBAs containing subcultures turned red
and showed complete disappearance of 3-CBA
within 14 days. Disappearance was also observed
for 3-BrBA in subcultures containing a mixture of
BrBAs and BA. In anaerobic controls, autoclaved
or incubated in the absence of light, no decrease
of halogenated substrate concentrations was observed over a period of one month.
After 3 successive transfers (1:10) in fresh
medium containing 3-CBA (0.5 m M ) + BA (1
mM) the 3-CBA degrading culture appeared able
to degrade 3-CBA or 3-BrBA without the addition of BA. The same was true for the 3-BrBA
degrading culture. By anaerobic plating on 3-CBA
in the light, a pure culture of a phototrophic
bacterium, able to degrade 3-CBA as well as
3-BrBA, was obtained from the 3-CBA degrading
mixed culture. The organism will be referred to
as isolate DCP3.
Identification of isolate DCP3
Isolate DCP3 is a motile, irregularly shaped
rod (3 × 0.5 /~m) showing a budding type of cell
division. Spectral analysis of whole cells cultured
anaerobically in the light showed absorption maxima at 865, 806, 591, 384 nm (Bchla) and at 531,
499, 476 nm (carotenoids) [18]. In absorption
spectra of methanol extracts these first two Bchla
peaks shifted together to 770 nm, the third peak
to 609 nm and the last peak to 364 nm. The three
carotenoid peaks shifted to 509, 476 and 455 nm,
respectively [17]. The red coloured cells contained lamellar arrangements of m e m b r a n e s (Fig.
1). Isolate DCP3 grew with acetate (/-/'max = 0.147
h - l ) , formate + CO2, thiosulphate + CO2, citrate, benzoate (/.t.max=0.066 h -1, Fig. 2A) or
4-hydroxybenzoate as sole carbon and electron
sources anaerobically in the light. U n d e r aerobic
conditions in the dark, yeast extract, acetate,
pyruvate, citrate and 4-hydroxybenzoate can be
used as sole carbon and energy sources, but not
benzoate. On the basis of these characteristics,
A
Fig. 1. Transmission electron micrograph of a longitudinal section of isolate DCP3. It was embedded in Epon after fixation with 3%
glutaraldehyde (1 h on ice) and post-fixation with osmium tetroxide and potassium bichromate.
202
8
1[
Ai"
o,~,~
1
to +,
i/\
1° ' ~
t/\
1 +°
o.01 ~
0.o
1.6
-I-, , , , , , B
:""""'N
\
.-o-o--o
,,
0
(D
ID
---
%%
oO.,<~
/so".:
1.0
:S
E
C
0
a
L
0.1
o-
\
÷* ~~
,4"
o/
8
~.
f/
,,+'"
@
O.E
0
0
0
~
0.0
0.01
C
1.8
///0-0
1
A
I
C
~'.%
%
-+~Oao.1 /
/
U~
0
g
I
l
I
I
l
l
I
i
0.01
0
1.0
l
I
l
1
50
:E
vE
0.8
100
c
e
c
o
0,0
160
time (h)
Fig. 2. Growth of isolate DCP3 on (A) benzoate (BA), (B) 3-chlorobenzoate (3-CBA) or (C) 3-CBA added after growth with BA.
OD660, ©; BA, e; 3-CBA, • CI-, +.
203
T h e d e c r e a s e in c o n c e n t r a t i o n s o f 2 - C B A o r
3 , 5 - D B A in m i x e d s u b s t r a t e c u l t u r e s in the prese n c e o f 3 - C B A c o n t i n u e d , slowly, after the d e p l e tion o f 3 - C B A (Fig. 3). This was a c c o m p a n i e d by
s o m e i n c r e a s e in cell density. I n such m i x e d subs t r a t e cultures, 4 - C B A was n o t d e g r a d e d completely. U p o n r e n e w e d a d d i t i o n of 2 - C B A o r
3 , 5 - C B A a f t e r t h e i r c o m p l e t e d i s a p p e a r a n c e , furt h e r g r o w t h at t h e e x p e n s e o f t h e s e c o m p o u n d s
was o b s e r v e d with t h e s a m e cell-yield as o b s e r v e d
with 3-CBA alone. Yet, if fresh m e d i a c o n t a i n i n g
2- o r 3 , 5 - C B A w e r e i n o c u l a t e d ( 1 : 2 0 ) with thus
p r e - g r o w n m a t e r i a l , no g r o w t h o c c u r r e d in t h e
a b s e n c e o f 3-CBA.
isolate D C P 3 c a n b e t e n t a t i v e l y i d e n t i f i e d as a
Rhodopseudomonas palustris strain.
Use of halogenated substrates by isolate DCP3
I s o l a t e D C P 3 was a b l e to grow o n meta substit u t e d c h l o r o - o r b r o m o b e n z o a t e as t h e sole carb o n s o u r c e with m a x i m u m specific g r o w t h r a t e s
of 0.032 h - 1 a n d 0.023 h - 1 , r e s p e c t i v e l y (Fig. 2B,
T a b l e 1). C u l t u r e s g r o w n on B A d e g r a d e d 3 - C B A
within 2 h a f t e r its a d d i t i o n (Fig. 2C). N o g r o w t h
was o b s e r v e d with any o f t h e o t h e r m o n o - or
d i h a l o g e n a t e d b e n z o a t e s t e s t e d as sole substrates: 2 - C B A , 2 - B r B A , 4 - C B A , 4 - B r B A , 2,3C B A , 2,4-CBA, 2,5-CBA, 3,4-CBA, 3,5-CBA.
If D C P 3 was g r o w n o n 3 - C B A in t h e p r e s e n c e
o f o n e o f t h e s e h a l o g e n a t e d b e n z o a t e s only 2C B A , 4 - C B A o r 3 , 5 - d i c h l o r o b e n z o a t e (3,5-CBA)
w e r e u s e d to s o m e extent. T h e c h l o r i n e r e c o v e r ies w e r e similar to t h o s e with 3 - C B A a l o n e (approx. 100%). A l s o t h e cell-yields ( c o r r e c t e d for
growth on y e a s t extract a l o n e ) p e r s u b s t r a t e carb o n u s e d w e r e t h e s a m e as on 3 - C B A alone. This
i n d i c a t e d t h a t 2 - C B A , 4 - C B A a n d 3,5-CBA, alt h o u g h n o t c o n s u m e d as sole s u b s t r a t e s by D C P 3 ,
w e r e d e h a l o g e n a t e d a n d c o m p l e t e l y u s e d for cell
c a r b o n in t h e p r e s e n c e o f 3 - C B A ( T a b l e 1). A l s o
c u l t u r e s grown on B A c o u l d use 2-CBA, b u t
c u l t u r e s grown on a c e t a t e did not.
Use of halogenated substrates by Rhodopseudomonas palustris strain WS17
S t r a i n W S 1 7 d e h a l o g e n a t e d 3 - C B A if grown
on B A with s t o i c h i o m e t r i c c h l o r i n e r e l e a s e ( T a b l e
1), b u t n o t if grown on a c e t a t e (3 mM). C u l t u r e s
g r o w n with 3 - C B A a n d B A s h o w e d i n c r e a s e d
b i o m a s s a n d a similar cell-yield if c o m p a r e d with
c u l t u r e s g r o w n on B A a l o n e ( T a b l e 1).
A p a r t from 3 - C B A also 2-CBA, 2 - B r B A o r
3 - B r B A d i s a p p e a r e d in c u l t u r e s of W S - 1 7 grown
on B A ( T a b l e 1). N o o t h e r m o n o - o r d i h a l o g e n a t e d b e n z o a t e s t e s t e d (see above) d e c r e a s e d
in c o n c e n t r a t i o n within 14 days of i n c u b a t i o n .
Table 1
Growth parameters of isolate DCP3 and Rhodopseudomonaspalustris WS17 with several substituted benzoates. Data are means of
duplicates. BA, benzoate; CBA, chlorobenzoate; BrBA, bromobenzoate
Substrate
Isolate DCP3
p~
(h- 1)
BA
2-CBA
3-CBA
0.066
0.032
4-CBA
-
3,5-CBA
2-BrBA
3-BrBA
4-BrBA
0.023
-
Strain WS 17
CIrecovery 1
(%)
92
99
106
96
Yield 2
(g/mol)
Relative
consumption
(mM/mM)
79
75
79
n.a.
0.5
0.6
0.06
0
0.3
0.2
0
82
3
/z
(h - I)
0.065
-
CI
recovery
(%)
101
96
Yield
(g/mol)
82
82
74
85
77
n.a.: not applicable.
1 C I - produced/substrate-bound chlorine disappeared.
2 mg cell-carbon produced/primary+ additional substrate (mM) disappeared.
3 CBA or BrBA consumption relative to BA consumption after 4 days of incubation in the presence of BA (1 mM).
204
oI ° - ' ° ' - ° ' - °
0 1
"'~"
0.01
"
~t.O
i
'"
"--
"~
"
-"
0.0
B
~
0...0--0---0~0
2.6
2.0
A
E
I:
1.6
0
@
@
:S
E
v
0
a
4~
0.1
1.0
0
0
0.6
.++
b
c
@
o
c
0
o
\
0.01
,
0.0
i
C
2.6
~0.---0--0
0 O''O--O''v, .'"
Cj/
|
,+'''If"
/
k
0
@
@
v
~
1.8
£
0
1.0
C
@
O
C
0
0
II
0.1
0
0
0.8
0.01
0
100
time
200
0.0
800
(h)
Fig. 3. Use of (A) 2-chlorobenzoate (2-CBA) (B) 4-chlorobenzoate (4-CBA) or (C) 3,5-dichlorobenzoate (3,5-CBA) by isolate DCP3
during and after growth on 3-CBA. Legends as in Fig. 2; 2-CBA, . 4-CBA, • 3,5-CBA, T.
205
1.0
0.8
00.0/0~0~0~0--0~0
A
E
c
o
(D
cD
X
0.1
I
¢3
O
.'
0.4
c
C
0.01
100
"+
c
0
'.~
0
?---- ..,;%_.,. --.,..
0
""
0.6
200
0.2
o
o
0.0
3OO
time (h)
Fig. 4. Use of 3-CBA and 2-CBA by Rhodopseudomonas
palustris strain WS17 during and after depletion of BA. The
culture was inoculated with cells from an identical culture
sampled after benzoate had been used completely.Legends as
in Fig. 2; 2-CBA, *.
In a culture of strain WS17 containing 2-CBA
and 3-CBA, a continued slow decrease of 2-CBA
and 3-CBA was observed after complete consumption of BA. This was even more apparent in
a second transfer (Fig. 4), in which both 2-CBA
and 3-CBA disappeared completely.
Discussion
From light-exposed enrichments with mixtures
of chlorinated (CBA) or brominated (BrBA) benzoates, a phototrophic bacterium was isolated,
able to use 3-chlorobenzoic acid (3-CBA) or 3bromobenzoic acid (3-BrBA) as a sole substrate
for growth, anaerobically in the light. Based on its
physiological and morphological properties, this
isolate DCP3 is tentatively characterized as a
strain of Rhodopseudomonas palustris [3,18]. This
organism resembles Rhodopseudomonas palustris
WS17, with the main difference being that strain
WS17, in pure culture, is dependent on benzoate
(BA) for growth on 3-CBA [11]. For several aerobic organisms it is known that xenobiotic substrates often fail to induce pathways leading to
successful degradation and growth [19]. The initial dependence on BA of the subcultures from
which DCP3 was isolated disappeared completely, either due to a shift in composition of the
population or as a result of genetic changes in the
initially enriched organism.
Interestingly, the use of other chlorinated benzoic acids was not observed in the initial enrichment transfers. However, pure cultures of DCP3,
grown on 3-CBA, also degraded 2-, 4-and 3,5CBA. Therefore the phototrophic isolate DCP3
appears to possess a dehalogenation mechanism
relatively unspecific to the position of the substituent at the aromatic ring. This is in contrast
with the capacities of most dehalogenating anaerobes isolated thus far [20,21].
As is the case with denitrifying organisms degrading halogenated BAs [22], the location of the
dehalogenating step in the degradation route of
DCP3 is unclear. Removal of the halogen substituent does not necessarily take place by initial
dehalogenation, as observed in, for example,
methanogenic systems [23] and during PCB dehalogenation by a phototrophic culture [9], but
may also occur somewhere downstream the benzoate pathway. Reductive removal of a substituent in such a reductive ring-fission pathway
has been shown for deamination of 4-aminobenzoyl-CoA to benzoyl-CoA in Desulfobacteriurn anilini [24].
The following observations reinforce the suggestion [11] that anaerobic dehalogenating phototrophs use a BA-inducible reductive ring-fission
pathway [25]: (1) after the disappearance of BA,
3-CBA or 2-CBA were still slowly used by strain
WS17; (2) isolate DCP3 can use 2-CBA not only
if grown on 3-CBA but also if grown on BA; and
(3) cultures of isolate DCP3 degraded 3-CBA
without delay if pre-grown with BA.
The lower growth rates of DCP3 with 3-CBA
and 3-BrBA compared with BA may indicate that
the halogen substituent impedes transport over
the cell-membrane or perhaps one or more steps
in this benzoate pathway proceed more slowly
with the chlorine-substituted molecules than with
the unsubstituted benzoate intermediates. So far
the benzoate CoA-ligase is the only enzyme of
the reductive ring-fission pathway studied. The
purified enzyme of a R. palustris strain, not previously grown in the presence of chlorinated ben-
206
zoates, expressed only little activity for 3 - C B A
(1%) a n d 2 - c h l o r o b e n z o a t e (10%) relative to B A
[26]. Strains possessing d e h a l o g e n a t i n g capacities
thus seem to have developed e n z y m e s with a
wider substrate r a n g e for m e t a b o l i s m of halog e n a t e d b e n z o i c acids a n d higher specific activities for such c o m p o u n d s t h a n their n o n - d e h a l o g e n a t i n g relatives.
T h e ecological i m p o r t a n c e of the d e c h l o r i n a t ing capacities of p h o t o t r o p h s is obscure. Estim a t e s c o n c e r n i n g the role of p h o t o t r o p h s in the
t u r n o v e r of organic substrates in n a t u r a l systems
are lacking [27]. T h e fact that the 3 - C B A m e t a b o lizing R. palustris WS17 has b e e n isolated from
a n e n v i r o n m e n t with n o k n o w n history of pollution [11] suggests that, in m a n y a q u e o u s systems,
the capacity exists for r a p i d acclimation to the
use of h a l o g e n a t e d aromatics if a n a e r o b i c a n d
light c o n d i t i o n s occur together. I n c o m b i n a t i o n
with the results p r e s e n t e d above, this observation
suggests that the role of p h o t o h e t e r o t r o p h i c bacteria in the removal of c h l o r i n a t e d aromatic comp o u n d s from c o n t a m i n a t e d e n v i r o n m e n t s may be
m o r e i m p o r t a n t t h a n hitherto assumed.
Acknowledgements
W e t h a n k K.A. Sjollema for the p r e p a r a t i o n of
the e l e c t r o n m i c r o g r a p h s a n d B.E.M. Schaub for
technical assistance with p r e p a r a t i o n of the spectra. W e are grateful to Dr. T.A. H a n s e n for
v a l u a b l e discussions, a n d to Dr. R.C. W y n d h a m
for his kind gift of Rhodopseudomonas palustris
WS17.
References
1 Reineke, W. and Knackmuss, H.J. (1988) Microbial degradation of haloaromatics. Ann. Rev. Microbiol. 42, 263-287.
2 Mohn, W.W. and Tiedje, J.M. (1982) Microbial reductive
dehalogenation. Microbiol. Rev. 56, 482-507.
3 Imhoff, J.F. and Triiper, H.G. (1992) The genus Rhodospirillum and related genera. In: The Prokaryotes, Vol.
II1 (Balows, A., Triiper, H.G., Dworkin, M., Harder, W.
and Schleifer K.H., Eds.), pp. 2141-2155. Springer Verlag,
New York.
4 Harwood, C.S. and Gibson, J. (1988) Anaerobic and aero-
bic metabolism of diverse aromatic compounds by the
photosynthetic bacterium Rhodopseudomonas palustris.
Appl. Environ. Microbiol. 54, 712-717.
Wright, G.E. and Madigan, M.T. (1991) Photocatabolism
of aromatic compounds by the phototrophic purple bacterium Rhodomicrobium vannielii. Appl. Environ. Microbiol. 57, 2069-2073.
Elder, D.J.E., Morgan, P. and Kelly, D.J. (1992) Anaerobic degradation of trans-cinnamate and w-phenylalkane
carboxylic acids by the photosynthetic bacterium
Rhodopseudomonas palustris: evidence for a B-oxidation
mechanism. Arch. Microbiol. 157, 148-154.
Rahalkar, S.B., Joshi, S.R. and Shivaraman, N. (1993)
Photometabolism of aromatic compounds by Rhodopseudomonas palustris. Curr. Microbiol. 26, 1-9.
Khanna, P., Rajkumar, B. and Jothikumar, N. (1992)
Anoxygenicdegradation of aromatic substances by Rhodopseudomonas palustris. Curr. Microbiol. 25, 63-67.
Montgomery, L. and Vogel, T.M. (1992) Dechlorination of
2,3,5,6-tetrachlorobiphenyl by a phototrophic enrichment
culture. FEMS Microbiol. Lett. 94, 247-250.
10 Blasco, R. and Castillo, F. (1992) Light-dependent degradation of nitrophenols by the phototrophic bacterium
Rhodobacter capsulatus EIF1. Appl. Environ. Microbiol.
58, 690-695.
11 Kamal, V.S. and Wyndham, R.C. (1990) Anaerobic phototrophie metabolism of 3-chlorobenzoate by Rhodopseudomonas palustris WS17. Appl. Environ. Microbiol. 56,
3871-3873.
12 Tanaka, H., Maeda, H., Suzuki, H., Kamibayashi, A. and
Tonomura, K. (1981) Metabolism of thiophene-2-carboxylate by a photosynthetic bacterium. Agric. Biol. Chem. 46,
1429-1438.
13 de Wit, R., van Boekel, W.H.M. and van Gemerden, H.
(1988). Growth of the cyanobacterium Microcoleus chtonoplastes on sulfide. FEMS Microbiol. Ecol. 53, 203-209.
14 Bergman, J.G. and Sanik, J. (1957) Determination of trace
amounts of chlorine in naphtha. Anal. Chem. 2, 241-243.
15 Gerritse, J. and Gottschal, LC. (1992) Mineralization of
the herbicide 2,3,6-trichlorobenzoicacid by a co-culture of
anaerobic and aerobic bacteria. FEMS Microbiol. Ecol.
101, 89-98.
16 Gerritse, J., Schut, F. and Gottschal, J.C. (1990) Mixed
chemostat cultures of obligately aerobic and fermentative
or methanogenic bacteria grown under oxygen-limiting
conditions. FEMS Microbiol. Len. 66, 87-94.
17 Yurkov, V.V. and van Gemerden, H. (1993) Abundance
and salt tolerance of obligately aerobic, phototrophic bacteria in a marine microbial mat. Neth. J. Sea Res. 31:
57-62.
18 Uffen, R.L. and Wolfe, R.S. (1970) Anaerobic growth of
purple nonsulfur bacteria under dark conditions. J. Appl.
Bacteriol 104, 462-472.
19 Haigler, B.E., Pettigrew, C.A. and Spain, J.C. (1992)
Biodegradation of mixtures of substituted benzenes by
Pseudomonas sp. strain JS150. Appl. Environ. Microbiol.
58, 2237-2244.
207
20 DeWeerd, K. and Suflita, J.M. (1990) Anaerobic aryl reductive dehalogenation of halobenzoates by cell extracts of
"Desulfomonile tiedjei". Appl. Environ. Microbiol. 56,
2999-3005.
21 Madsen, T. and Licht, D. (1992) Isolation and characterization of an anaerobic chlorophenol-transforming baterium. Appl. Environ. Microbiol. 58, 2874-2878.
22 Schennen, U., Braun, K. and Knackmuss, H.J. (1985)
Anaerobic degradation of 2-fluorobenzoate by benzoatedegrading, denitrifying bacteria. J. Bacteriol. 161: 321-325.
23 Suflita, J.M., Horowitz, A., Shelton, D. and Tiedje, J.M.
(1982) Dehalogenation: a novel pathway for the anaerobic
biodegradation of haloaromatic compounds. Science 218,
1115-1117
24 Schnell, S. and Schink, B. (1991) Anaerobic aniline degra-
dation via reductive deamination of 4-aminobenzoyl-CoA
in Desulfobacterium anilini. Arch. Microbiol. 155: 511-556.
25 Dispensa, M., Thomas, C.T., Kim, M.K., Perrotta, J.A.,
Gibson, J. and Harwood, C. (1992) Anaerobic growth of
Rhodopseudomonas palustris on 4-hydroxybenzoate is dependent on AadR, a member of the cyclic AMP receptor
protein family of transcriptional regulators. J. Bacteriol.
174, 5803-5813.
26 Geissler, J.F., Harwood, C.S. and Gibson, J. (1988) Purification and properties of benzoate-coenzyme A ligase, a
Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate. J. Bacteriol. 170, 1709-1714.
27 Sleat, R. and Robinson, J.P. (1984) The bacteriology of
anaerobic degradation of aromatic compounds. J. Appl.
Bacteriol. 57, 381-394.