Preliminary characterization of four 2-chlorobenzoate-degrading anaerobic bacterial consortia

Article abstract of DOI:10.1023/A:1008348123672

Dechlorination was the initial step of 2CB biodegradation in four 2-chlorobenzoate-degrading methanogenic consortia. Selected characteristics of ortho reductive dehalogenation were examined in consortia developed from the highest actively dechlorinating dilutions of the original 2CB consortia, designated consortia M34^-9, P20^-9, P21^-9 and M50^-7. In addition to 2-chlorobenzoate, all four dilution consortia dehalogenated 4 of 32 additional halogenated aromatic substrates tested, including 2-bromobenzoate; 2,6-dichlorobenzoate; 2,4-dichlorobenzoate; and 2-chloro-5-hydroxybenzoate. Dehalogenation occurred exclusively at the ortho position. Both ortho chlorines were removed from 2,6-dichlorobenzoate. Benzoate was detected from 2-bromobenzoate and 2,6-dichlorobenzoate. 4-Chlorobenzoate and 3-hydroxybenzoate were formed from 2,4-dichlorobenzoate and 2-chloro-5-hydroxybenzoate, respectively. Only benzoate was further degraded. Slightly altering the structure of the parent "benzoate molecule" resulted in observing reductive biotransformations other than dehalogenation. 2-Chlorobenzaldehyde was reduced to 2-chlorobenzyl alcohol by all four consortia. 2-chloroanisole was O-demethoxylated by three of the four consortia forming 2-chlorophenol. GC-MS analysis indicated reduction of the double bond in the propenoic side chain of 2-chlorocinnamate forming 2-chlorohydrocinnamate. None of the reduction products was dechlorinated. The following were not dehalogenated: 3- and 4-bromobenzoate; 3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate; 2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-chlorophenol; 2-chloroaniline; 2-chloro-5-methylbenzoate; 2,3-dichlorobenzoate; 2,5-dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid; and 2,4-dichlorophenoxyacetic acid. Consortia M34^-9, P20^-9, P21^-9, and M50^-7 dechlorinated 2-chlorobenzoate at <= 4 mm. Dechlorination rates were highest for consortia P20^-9 followed by those of M50^-7 with rates declining above 2 and 3 mm 2CB, respectively. The major physiological types of microorganisms in consortia M34^-9, P20^-9, P21^-9, and M50^-7 were sulfate-reducing and hydrogen-utilizing anaerobes.

Full text of DOI:10.1023/A:1008348123672

Biodegradation 10: 27–33, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
27
Preliminary characterization of four 2-chlorobenzoate-degrading
anaerobic bacterial consortia
Barbara R. Sharak Genthner
University of West Florida Center for Environmental Diagnostics and Bioremediation, Pensacola, FL 32514, USA
(e-mail: bgenthne@uwf.edu)
Accepted 5 October 1998
Key words: anaerobic biodegradation/biotransformation, 2-chlorobenzoate, reductive dehalogenation.
Abstract
Dechlorination was the initial step of 2CB biodegradation in four 2-chlorobenzoate-degrading methanogenic con-
sortia. Selected characteristics of ortho reductive dehalogenation were examined in consortia developed from the
highest actively dechlorinating dilutions of the original 2CB consortia, designated consortia M34−9, P20−9, P21−9
and M50−7. In addition to 2-chlorobenzoate, all four dilution consortia dehalogenated 4 of 32 additional halo-
genated aromatic substrates tested, including 2-bromobenzoate; 2,6-dichlorobenzoate; 2,4-dichlorobenzoate; and
2-chloro-5-hydroxybenzoate. Dehalogenation occurred exclusively at the ortho position. Both ortho chlorines were
removed from 2,6-dichlorobenzoate. Benzoate was detected from 2-bromobenzoate and 2,6-dichlorobenzoate. 4-
Chlorobenzoate and 3-hydroxybenzoatewere formed from 2,4-dichlorobenzoateand 2-chloro-5-hydroxybenzoate,
respectively. Only benzoate was further degraded. Slightly altering the structure of the parent “benzoate molecule”
resulted in observing reductive biotransformations other than dehalogenation. 2-Chlorobenzaldehyde was reduced
to 2-chlorobenzyl alcohol by all four consortia. 2-chloroanisole was O-demethoxylated by three of the four con-
sortia forming 2-chlorophenol. GC-MS analysis indicated reduction of the double bond in the propenoic side chain
of 2-chlorocinnamate forming 2-chlorohydrocinnamate. None of the reduction products was dechlorinated. The
following were not dehalogenated: 3- and 4-bromobenzoate; 3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate;
2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-chlorophenol; 2-chloroaniline; 2-chloro-5-methylbenzoate; 2,3-
dichlorobenzoate; 2,5-dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid; and 2,4-dichlorophenoxyacetic acid.
Consortia M34−9, P20−9, P21−9, and M50−7 dechlorinated 2-chlorobenzoate at ≤4 mm. Dechlorination rates
were highest for consortia P20−9 followed by those of M50−7with rates declining above 2 and 3 mm 2CB,
respectively. The major physiological types of microorganisms in consortia M34−9, P20−9, P21−9, and M50−7
were sulfate-reducing and hydrogen-utilizing anaerobes.
Abbreviations: 2CB = 2-chlorobenzoate; 2CHC = 2-chlorohydrocinnamate; 2CPA = 2-chlorophenylacetic acid;
BESA = bromoethane sulfonic acid; HPLC = High performance liquid chromatography; MPN = Most probable
Number.
Introduction
tium was dechlorination at the ortho position to form
benzoate that was then degraded to carbon dioxide
and methane. Reductive dechlorination at the ortho
position of multi-chlorinated benzoic acids has been
observed in anaerobic enrichments (Gerritse et al.
1992; Van der Woude et al. 1996) and with aerobic
bacterial strains (van den Tweel et al. 1987; Ro-
manov & Hausinger 1994; 1996). Aerobic biodegra-
We previously reported anaerobic biodegradation of
2-chlorobenzoate (2CB) in freshwater sediment en-
richments (Sharak Genthner et al. 1989a), and in a
methanogenic bacterial consortium derived from one
of these enrichments (Sharak Genthner et al. 1989b).
The initial step of 2CB biodegradation in this consor-

28
dation of 2CB by Pseudomonas sp. via oxygenases
with subsequent release of chlorine has also been re-
ported (Engesser & Schulte 1989; Fetzner et al. 1989;
Romanov & Hausinger 1994). However, anaerobic
biodegradation and/or reductive dechlorination of the
mono-chlorinated compound, 2CB, is not usually ob-
served in anaerobic enrichments (Horowitz et al. 1982;
Horowitz et al. 1983; Häggblom et al. 1993; Van
der Woude et al. 1996; Maloney et al. 1997). If
2CB loss was observed, it had not been accompanied
by the detection of benzoate, the product of reduc-
tive dehalogenation of 2CB, and stable 2CB-degrading
bacterial consortia were not obtained for further study
(Häggblom et al. 1993; 1996; Van der Woude et al.
1996).
Thus, our previous studies (Sharak Genthner et
al. 1989a; 1989b) in which we detected benzoate
as an intermediate of 2CB anaerobic biodegradation
have been the only substantiated reports of anaerobic
2CB reductive dechlorination. In the current inves-
tigation, we examine characteristics of four stable
2CB-dechlorinating/2CB-degrading anaerobic bacte-
rial consortia obtained during our earlier study, and
of four additional consortia derived from the highest
actively-dechlorinating dilutions of the original 2CB
consortia.
and 10 mm, respectively, of bromoethane sulfonic
acid (BESA), a potent inhibitor of methanogenesis
(Gunsalus, et al. 1978). Since BESA (1 mm) did not
enhance biodegradation of 2CB in the first laboratory
transfer of M50, but 10 mm BESA inhibited biodegra-
dation of 2CB in P21 (Sharak Genthner et al. 1989a),
subsequent laboratory transfers were prepared without
BESA. In both cases consortia were methanogenic.
Media were routinely inoculated with 10% of a stable
consortium.
MPN analyses
MPN analyses were done in serum stopper tubes (18
× 150 mm) by preparing triplicate dilutions (10−2
through 10−9) of a stable consortia in a medium
appropriate for the particular MPN. The MPN sets
were incubated up to 6 months. Enumeration of 2CB
dechlorinators was performed in anaerobic medium
containing 800 µm 2CB. Loss of the dechlorination
product, benzoate, was the criterion for enumerating
benzoate-degrading bacteria. The highest dechlorinat-
ing dilution, i.e. 1 × 10−7 or 1 × 10−9, of each
consortium was propagated as described for the sta-
ble consortia and were designated M50−7, M34−9
,
P20−9, and P21−9
.
The MPN anaerobic medium was modified to
enumerate other physiological types of bacteria in
consortia M50−7, M34−9, P20−9, and P21−9. Ben-
zoate and acetate-utilizing anaerobes were enumer-
ated in medium containing 10 mm sodium benzoate
and 50 mm sodium acetate, respectively. Nitrate-
and sulfate-reducing anaerobes were enumerated in
medium amended with 15 mm KNO3 and 20 mm
Na2SO4. Hydrogen-utilizing anaerobes were enumer-
ated in medium containing 2X bicarbonate buffer
under two atmospheres of 20/80 CO2/H2. Growth in
MPN sets was measured as an increase in optical
density (600 nm) and monitored until a maximum op-
tical density was obtained after which nitrate, nitrite
and sulfate concentrations were determined using ion
chromatography (Sharak Genthner et al. 1994).
Information gathered on tolerance to 2CB and ma-
jor physiological types of bacteria present in consortia
derived from the highest actively dechlorinating dilu-
tion of the original 2CB consortia will be used to aid
in the isolation of the bacterial species responsible for
2CB dechlorination in our consortia.
Materials and methods
Cultivation techniques
The anaerobic techniques and medium used were
previously described (Sharak Genthner et al. 1989a,
1989b), except that the anaerobic medium was sup-
plemented with 0.02% yeast extract. Consortia were
derived from methanogenic enrichments inoculated
with freshwater sediment from either Site B of the
Escambia River in northwest Florida (consortia M34
and M50), or the Eleven Mile Creek (consortia P20
and P21) in southeast Alabama (Sharak Genthner et al.
1989a). M34 and P20 were originally methanogenic.
Stable bacterial consortia were obtained as previously
described (Sharak Genthner et al. 1989b). The origi-
nal M50 and P21 enrichment cultures contained 1 mm
Ortho-dechlorination
The ability of the four original 2CB consortia to de-
halogenate 2CB was monitored monthly in fresh and
refed laboratory transfers for several years. The effect
of yeast extract on 2CB dechlorination was determined
by transferring the consortia into medium lacking

29
yeast extract, or to medium in which yeast extract was
replaced by pyruvate.
The ability of consortia M50−7, M34−9, P20−9
,
and P21−9 to dehalogenate 2CB and other halo-
genated aromatic compounds was examined in anaero-
bic medium containing yeast extract. Test compounds
were added from concentrated (100×), anaerobic,
sterile stock solutions (pH 7.0). Compounds were
tested at 500 µM, except where indicated otherwise
in parentheses, in triplicate cultures with duplicate
uninoculated controls. Uninoculated controls were
prepared to monitor abiotic changes under◦reducing
conditions. Cultures were incubated at 30 C for up
to 9 months.
Figure 1. Accumulation of ortho dehalogenation product, benzoate,
−9
from reductive dechlorination of 2CB by consortium P21
.
Loss of the halogenated compound and formation
of the non-halogenated product were monitored by
High performance Liquid chromatography (HPLC).
HPLC samples were prepared and analyzed on a
Hewlett-Packard 1050 HPLC as previously described
(Kuo & Sharak Genthner 1996) using the acetoni-
trile/phosphate buffer at a ratio of 10/90 to 50/50 under
isocratic conditions. Absorbance wavelengths from
220 to 270 nm were chosen to optimize separation
and detection of parent compound and dechlorination
product. UV spectral and gas chromatographic-mass
spectral (GC-MS) analyses were used, as previously
described (Sharak Genthner, et al. 1989c), to identify
dehalogenation products.
ortho-dechlorinating bacteria/ml in the original 2CB
consortia. Consortium M50 contained 1 × 107 dechlo-
rinating bacteria/ml (Table 1). The other three consor-
tia, designated M50−7, M34−9, P20−9, and P21−9
,
contained 1 × 109 dechlorinating bacteria/ml each.
Microscopic examination indicated that these dilu-
tion consortia were mixed cultures containing several
morphologically distinguishable gram-negative rods.
After the 3rd laboratory transfer (10%), the bacterial
populations in M50−7, M34−9, P20−9, and P21−9
were characterized.
2CB dechlorination rates were determined from
the linear portion of the 2CB dechlorination curve.
Benzoate, the ortho dehalogenation product, accu-
mulated in the highest actively dechlorinating MPN
dilution, as shown for consortium P21−9 (Figure 1).
Results/discussion
The rates of 2CB dechlorination by M50−7, M34−9
,
2CB-dechlorination/degradation
P20−9 and P21−9 at increasing concentrations of 2CB
(500 µM − 4 mm) are shown in Figure 2. The max-
imum 2CB dechlorination rate of 29 µm/day was
observed with consortium P20−9 at 2 mm, followed by
M50−7 with 19 µm/day at 500 µm 2CB. Consortium
P20−9 dechlorinated 2CB at rates near maximum from
500 µm up to and including 2 mm, but dechlorination
rates abruptly declined to approximately 4 µm/day
from 2.5 to 4 mm 2CB. Consortia M34−9 and P21−9
dechlorinated 2CB at 2–5 µm/day over the range of
2CB concentrations tested with increasing 2CB con-
centrations having little effect on dechlorination rates.
In contrast dechlorination rates increasingly slowed in
consortium M50−7 above 1 mm. Thus, consortium
P20−9 was the most efficient in dechlorinating 2CB
at ≤ 2 mm.
We have been able to propagate all four 2CB consortia
while retaining their ability to dechlorinate and de-
grade 2CB for several years through numerous labora-
tory transfers in anaerobic medium containing 0.02%
yeast extract. Consortium M50 is currently in its 17th
laboratory transfer, while M34 and P20 are in their
11th transfer and P21 is in its 10th. We were unable
to propagate 2CB degradation by consortium P20 or
P21 in defined medium, lacking yeast extract, but
have maintained their activity through a 5th transfer
in defined pyruvate (0.2%) medium lacking yeast ex-
tract. Although degradation was much slower than
in medium containing yeast extract, M34 and M50
have remained active through their 3rd and 5th trans-
fers, respectively, in both defined medium and defined
pyruvate medium.
Gerriste et al. (1992) reported that their anaer-
obic freshwater sediment enrichments removed the
ortho chlorine from various tri- and dichloroben-
Most Probable Number (MPN) analyses (Rodina
1972) were performed to determine the number of

30
a
Table 1. Enumeration of physiological cell types in 2CB-dechlorinating anaerobic bacterial
consortia
Cell types (bt/ml)
b
Consortium
Benzoate
degraders
Acetate
utilizers
H /CO
Nitrate
Sulfate-reducing
bacteria
2
2
utilizers
reducers
−9
7
5
7
7
6
6
2 c
5
7
P20
1.6 × 10
<1 × 10
<1 × 10
<1 × 10
1 × 10
1.1 × 10
1.1 × 10
1.5 × 10
4.5 × 10
<1 × 10
4.5 × 10
<1 × 10
4.5 × 10
−9
2 c
2 c
2 c
2
6
P21
<1 × 10
2.5 × 10
−9
−7
5
2 c
7
M34
1 × 10
2.5 × 10
3
5 d
6
M50
1 × 10
1 × 10
9.5 × 10
a
−9
All consortia dechlorinated 2CB; P20 also degraded the benzoate produced.
b
Consortia were the highest dilution of the original consortium to dechlorinate 2CB; dilution is
indicated by superscript.
c
−2
.
Lowest dilution tested was 1 × 10
d
Nitrate was reduced to nitrite.
Hausinger 1996), also an aerobic bacterial species,
reductively dechlorinated 2,4-dichlorobenzoate and
2-chloro-4-fluorobenzoate to form 4-chlorobenzoate
and 4-fluorobenzoate, respectively. However, A. den-
itrificans NTB-1 did not reductively dechlorinate
2,6-dichlorobenzoate, and neither species reductively
dechlorinated 2CB. These observations indicate that
the ortho-dechlorinating activity in our consortia
was different from the ortho-dechlorination of multi-
chlorinated benzoates.
Some Pseuodomonas sp. have been shown to de-
grade 2CB (Engesser & Schulte 1989; Fetzner et al.
1989; Romanov & Hausinger 1994), but metabolism
is oxidative and chlorine release is fortuitous, i.e. a
dehalogenase enzyme is not involved.
−7
Figure 2. Tolerance of 2CB-dechlorinating consortia, M50
,
−9
−9
−9
M34 , P20 , and P21 toward 2CB as shown by dechlorination
rates at increasing concentrations of 2CB.
Other ortho-dechlorinating activities in consortia
M50−7, M34−9, P20−9, and P21−9
zoic acids. In contrast to our findings, 2,3- and
2,5-dichlorobenzoate were dechlorinated at the or-
tho position, but 2,6-dichlorobenzoate was not. 2CB
dechlorination was not observed in this study. In a
follow-up study 2CB was lost from one of six sedi-
ment slurry replicates (Van der Woude et al. 1996).
Haggblom et al. (1993) reported a similar loss of
2CB in a methanogenic sediment enrichment. Neither
research group detected benzoate as a product from
2CB, therefore, neither confirmed reductive dechlori-
nation of 2CB. Apparently neither group studied these
2CB-degrading enrichments further.
Alcaligenes denitrificans NTB-1, an aerobic bacte-
rial species, was reported to remove the ortho chlorine
from 2,4-dichlorobenzoate by reductive dechlorina-
tion forming 4-chlorobenzoatewhich was metabolized
aerobically (van den Tweel et al. 1987). Similarly,
Corynebacterium sepedonicum KZ-4 (Romanov &
Of the 32 halogenated aromatic compounds tested,
four halogenated benzoic acids, in addition to 2CB,
were dehalogenated at the ortho position by all
four consortia. These included 2-bromobenzoate, 2-
chloro-5-hydroxybenzoate, 2,4-dichlorobenzoate, and
2,6-dichlorobenzoate. Both ortho chlorines were
removed from 2,6-dichlorobenzoate. Benzoate, 4-
chlorobenzoate, or 3-hydroxybenzoate were detected
where appropriate indicating reductive dechlorination.
Of these dechlorination products, only benzoate was
further degraded. The consortia did not dehalogenate
the following compounds: 3 - and 4-bromobenzoate;
3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate;
2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-
chlorophenol; 2-chloroaniline (100 µM); 2-chloro-5-
methylbenzoate (50 µM); 2,3-dichlorobenzoate; 2,5-

31
dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid
(50 µM); and 2,4-dichlorophenoxyaceticacid (50 µM).
These dechlorination data indicate that dehalo-
genation by our consortia occurred only at the ortho
position; that benzoate, and some compounds closely
related to benzoate, are the preferred substrate; and
that the presence and position of additional side groups
on the benzoate molecule can affect the ability of these
dilution consortia to ortho-dehalogenate.
Several halogenated compounds were tested to
examine the effect of altering the aromatic car-
boxyl group on ortho-dechlorination in our consortia.
These included: 2-chlorobenzaldehyde (100 µm), 2-
chlorobenzyl alcohol (100 µM), 2-toluene (50 µM),
2-chloroanisole (50µM), and 2-chloroaniline (50µM).
2-Chlorobenzaldehyde was reduced to 2-chlorobenzyl
alcohol (2CBA) by all four consortia. 2CBA was
not observed to decline. The reduction of aro-
matic aldehydes to alcohols has been previously re-
ported (Neilson, et al. 1988). Both 2-chlorotoluene
(50 µM) and toluene concentrations declined, but
neither was completely degraded in any of the four
consortia and toluene was not detected as a product
from 2-chlorotoluene. Three consortia demethoxy-
lated 2-chloroanisole to 2-chlorophenol which was
not dechlorinated. Only consortium P21 did not de-
grade or transform 2-chloroanisole. It is apparent that
the carboxyl function is important to the particular
ortho-dechlorination process under study.
Two compounds, including 2-chlorophenylacetic
acid (200 µm; 2CPA) and 2-chlorocinnamate
(200 µM; 2CC), were chosen to investigate the ef-
fect of additional alkyl residues between the aro-
matic ring and the carboxyl side chain. A small
amount (39 µM) of a compound tentatively iden-
tified by co-chromatography as phenylacetate was
detected in consortium M34 incubated with 2CPA.
However, the concentration of the product was too
low to obtain an accurate UV spectrum to confirm
its identity. Phenylacetate was not detected in the
other three consortia. 2CC concentrations declined in
all four consortia, but cinnamate was not detected.
GC-MS analysis revealed the presence of an aro-
matic product, 2-chlorohydrocinnamate(2CHC), indi-
cating reduction of the double bond in the propenoic
side chain. This result was confirmed by testing 2-
chlorohydrocinnamate, cinnamate, and hydrocinna-
mate. 2CC and cinnamate were biotransformed to
2CHC and hydrocinnamate, respectively, confirming
reduction of the propenoic side group and indicat-
ing that 2CC was not dechlorinated. Hydrocinnamate
was degraded by consortium P21 to non-aromatic
end products. Anaerobic biodegradation of hydrocin-
namate has been previously reported (Barik, et al.
1985). However, 2CHC was neither dechlorinated nor
degraded. Results were similar with all four of our
consortia. Again, the data indicate that the carboxyl
group of the benzoate molecule is important to the
ortho dechlorination process under study.
Most Probable Number (MPN) analyses (Ro-
dina 1972) were performed to characterize other
physiological types of bacteria present in the origi-
nal 2CB-degrading consortia and the derived 2CB-
dechlorinating consortia, M50−7, M34−9, P20−9, and
P21−9. Consortium M50 and M34 contained 1 × 105
benzoate-degrading bacteria/ml, while P20 and P21
contained 1 × 104, and 1 × 109 per ml, respec-
tively. Consortia M50−7, M34−9, P20−9, and P21−9
were similar in containing H2-utilizing anaerobes and
sulfate-reducing bacteria as major physiological types
of bacteria, i.e. 1 × 106 to 1 × 107 (Table 1).
Benzoate-degrading bacteria were below detectable
levels (<1 × 102bt/ml) in consortia M50−7, M34−9
,
and P21−9. As expected, benzoate accumulated in
these three dilution consortia as shown for P21−9 in
Figure 1. Benzoate-degrading anaerobes were also
among the major physiological types of bacteria in
consortium P20−9 and, as expected, was also the only
dilution consortium to degrade the benzoate resulting
from dechlorination of 2CB. After several laboratory
transfers, P20−9 also lost its ability to degrade ben-
zoate which was likely the result of loss of the bacterial
strain(s) responsible for benzoate degradation.
Using the Gibbs free energies of formation (Dolf-
ing & Harrison 1992; Thauer & Morris 1984; van den
Tweel et al. 1987; Weast 1987), the thermodynamics
for 2CB reductive dechlorination (pH 7.0, 25 ◦C) were
calculated as:
0
1G0
2CB− + H2 → Benzoate− + H+ + Cl− −129 kJ/rx
The change in free energy indicates that 2CB could
serve as a terminal electron acceptor during reductive
dechlorination and provide enough energy for ATP
synthesis and, hence, for cell growth.
These thermodynamic facts and our current data
will allow us to design laboratory enrichment and iso-
lation schemes to aid in obtaining pure cultures of
the bacterial strains responsible for 2CB reductive de-
halogenation in our consotia. Having determined the
tolerance of our consortia toward 2CB, we will pass
the consortia on relatively high concentrations of 2CB

32
(>2 mm) at intervals determined from our rate studies
to take advantage of an initial increase in numbers of
2CB-dechlorinating bacteria during growth on 2CB as
the terminal electron acceptor, as well as a likely inhi-
bition of non-dechlorinating bacterial strains that may
not tolerate these higher concentrations of 2CB well.
This would provide consortia enriched in numbers of
2CB bacteria that would then be plated onto anaerobic
2CB agar medium or into anaerobic 2CB roll tubes
containing the same high concentration of 2CB. The
colonies that develop would have been selected for
their ability to tolerate and grow on high concentra-
tions of 2CB. The ability of the bacterial isolates to
dechlorinate 2CB to benzoate would be determined in
liquid anaerobic 2CB medium via HPLC.
anaerobic bacterial strains, which can include both
methanogens and acetogens, consortia will be inoc-
ulated into anaerobic H2/CO2 roll tubes for isolation.
Colonies will be selected from roll tubes and inocu-
lated into dechlorinating liquid medium, as described
above, to determine if the isolate has the capacity to
dechlorinate 2CB, and into liquid medium under a
H2/CO2 gas phase in which the major metabolic end-
product will be identified as either methane or acetate.
Thus, we will be able to confirm the identity of the any
isolate that dechlorinates 2CB as either a methanogen
or acetogen, respectively.
Conclusions
In addition to the above approach, our cur-
rent MPN studies revealed that sulfate-reducing and
H2/CO2-utilizing bacterial species were the major
physiological types of bacteria in the highest actively
dechlorinating dilutions of the 2CB consortia, de-
spite no enrichment pressure to select for either type
of bacteria in the consortia. The first observation is
of interest because Desulfomonile tiedjei, the only
3-chlorobenzoate-dechlorinating anaerobic bacterial
species in pure culture (DeWeerd et al. 1986), was
identified as a sulfate-reducing bacterial species after
its isolation as a dechlorinating species and with no
selective pressure for the isolation of sulfate-reducing
bacterial strains.
Sulfate-reducing bacteria will be isolated from the
2CB dilution consortia using our technique that has
proven successful in isolating a wide variety of new
strains of sulfate-reducing bacterial species from en-
richment of a salt marsh rhizosphere (Rooney-Varga
et al. in press). In this technique an array of sulfate-
reducing media that differ by the electron donors
included are incorporated into liquid medium. Dilu-
tions of the consortia, enrichment or culture under
study are prepared in these media to obtain the most
numerous of a particular physiological type of sulfate-
reducing bacteria. Similar to the above approach, iso-
lated sulfate-reducing bacterial strains will be tested
for their ability to dechlorinate 2CB in a variety of
liquid anaerobic medium containing a array of elec-
tron donors with the potential to support for 2CB
dechlorination.
The current study is the first to show reductive dechlo-
rination of 2CB by anaerobic bacterial consortia as
shown by the accumulation of benzoate as the dehalo-
genation product. Dehalogenation by our consortia oc-
curred exclusively at the ortho position. Benzoate was
the preferred substrate, although compounds closely
related to benzoate were also ortho-dehalogenated.
The carboxyl group of benzoate was important to this
ortho-dehalogenation process under study. The pres-
ence and position of additional side groups on the
benzoate molecule as well as the presence of addi-
tional alkyl units between the carboxyl group and
the aromatic ring affected the ability of our consortia
to ortho-dehalogenate aromatic compounds related to
benzoate. These observations indicate that the ortho-
dechlorinating activity in our consortia was unique and
different from reductive ortho-dechlorinationof multi-
chlorinated benzoates (Gerritse, et al. 1992; Van der
Woude et al. 1996).
Information gathered on tolerance to 2CB and ma-
jor physiological types of bacteria present in consortia
derived from the highest actively dechlorinating dilu-
tion of the original 2CB consortia will be used to aid
in the isolation of the bacterial species responsible for
2CB dechlorination in our consortia.
Acknowledgements
H2/CO2-utilizing bacterial strains may be present
in the dilution consortia due to the formation of hy-
drogen and interspecies hydrogen transport, but could
also be involved in reductive dechlorination. In or-
der to obtain pure cultures of the H2/CO2-utilizing
Appreciation is extended to Beat Blattmann and Mau-
reen Downey for UV spectral and GC-MS analyses.
This work was supported in part by the U.S. Environ-
mental Protection Agency under contract 68-03-3479
with Technical Resources, Inc.

33
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