United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-88/054 Jan. 1989
f/EPA Project Summary
Determination and
Enhancement of Anaerobic
Dehalogenation: Degradation of
Chlorinated Organics in
Aqueous Systems
Donna T. Palmer, Timothy G. Linkfield, Jayne B. Robinson,
Barbara R. Sharak Genthner, and George E. Pierce
Anaerobic degradation is poten-
tially an efficient means to destroy or
detoxify many environmental pollu-
tants. Anaerobic degradation of
halogenated organic compounds is
especially interesting, because many
of these compounds are toxic and
apparently resistant to aerobic
degradation. The full report sum-
marizes our initial efforts to isolate
microorganisms capable of anaero-
bic dehalogenation; to examine the
nutritional requirements of dehalo-
genating enrichments and a
dehalogenating consortium and to
study the genetics of dehalo-
genation.
Anaerobic enrichments were
established in which 3-chloro-
benzoate (3CB but not 4-chloro-
benzoate was degraded. Studies
using a 3CB degrading consortium,
showed that specific manipulations
of the growth medium could
eliminate some members of the
consortium while maintaining the
organisms capable of dehalo-
genation. Such manipulations are
useful in efforts to isolate organisms
in pure culture. Genetic studies were
begun using the anaerobic dehalo-
genator, strain DCB-1 (obtained
from Dr. J. M. Tiedje). No plasmids
were found in this strain, therefore, it
was presumed that the dehalogenase
activity was chromosomally encoded.
Genomic DMA was extracted and
purified. A partial library was
generated by cloning DNA fragments
into the cosmid pHC79 and into the
plasmid pUC8. A rapid dehalogenase
assay was developed for the purpose
of screening recombinants for
dehalogenase activity.
This Project Summary was devel-
oped by EPA's Risk Reduction Engi-
neering Laboratory, Cincinnati, OH, to
announce key findings of the re-
search project that is fully docu-
mented in a separate report of the
same title (see Project Report
ordering information at back).
Introduction
Many halogenated organic compounds
pose a serious environmental problem
because of their persistence and toxicity.
It may be possible to employ microbial
degradation to detoxify or destroy these
compounds either in situ or during waste
treatment. Recent investigations have
shown that many halogenated organic
compounds, including halogenated ben-
zoates and halogenated phenols, can be
anaerobically degraded. Some of these
compounds are not known to be aero-
bically degraded; thus, evidence of
anaerobic degradation is extremely
important. In contrast to the mechanism
of aerobic degradation of many haloaro-
-------
matics, the first step in anaerobic
degradation involves the removal of the
halogen leading immediately to the
formation of a generally less toxic, more
biodegradable compound.
The overall objective of the proposed
program is the development of
engineered microorganisms capable of
destroying hazardous organic com-
pounds (e.g., chlorinated organics) under
anaerobic conditions. An understanding
of microbially mediated anaerobic
dehalogenation and the exploitation of
this process may result in the significant
reduction of toxic and hazardous wastes
in the United States. Therefore, this
program would assist in detoxifying
recalcitrant halogenated organic com-
pounds that have not been biode-
1 gradable or accessible to chemical and
physical destruction techniques in the
past.
In order to study the genetics of
anaerobic dehalogenation, it would be
useful to clone the gene or genes
responsible for this activity. If plasmids
are found in dehalogenating micro-
• organisms and if dehalogenase activity is
encoded by a gene or genes carried by
the plasmid, plasmid genes specifying
the anaerobic degradation or bio-
transformation of chlorinated organics
could be introduced into suitable hosts
, using genetic engineering techniques.
These modified strains could then be
examined to study and enhance the
degradation of organic chemical con-
taminants present in hazardous waste
sites. However, if dehalogenase activity
is chromosomally encoded, a genomic
library must be constructed in order to
isolate the dehalogenase gene.
In order to initiate the cloning effort
and study the genetics and biochemistry
of dehalogenation, a pure culture of an
anaerobic dehalogenating organism was
needed. In the first phase of this study,
an effort was made to isolate a
dehalogenating enrichment and a pure
culture of a dehalogenating micro-
organism from a secondary sludge in
Columbus, OH. In the second phase, we
obtained a 3-chlorobenzoate (3CB)
degrading consortium from Dr. J. M.
Tiedje. This consortium was the first
anaerobic dehalogenating consortium to
be reported. It served as a model
system for the isolation and identification
of the organisms responsible for
dehalogenation. In the final phase of this
study, genetic studies began on a pure
culture of strain DCB-1, the organism
responsible for dehalogenation in
Tiedje's consortium. Since plasmid DNA
was not detected in DCB-1, efforts were
undertaken to construct a library of
DCB-1 genomic DNA in Escherichia
co//. A partial genomic library has
potentially been cloned using a cosmid
cloning system. Further effort is needed
to characterize the recom-binants.
Materials and Methods
Growth Conditions and Strains
The anaerobic techniques employed
for the handling of the inocula,
preparation of media, and handling of
enrichments and cultures had been
previously established. Enrichments
were prepared by adding sterile
anaerobic solutions of 3-chlorobenzoate
(3CB) or 4-chlorobenzoate (4CB) to the
basal medium containing 10% secondary
anaerobic digester sewage sludge
(Jackson Pike Plant, Columbus, OH) as
inoculum. For the work with the
enrichments and the consortium, the
basal medium contained rumen fluid (or
yeast extract), B-vitamins, minerals,
NaHCOa, Na2S reducing solution,
resazurin redox indicator, and a 90%
N2:10% C02 gas phase (final pH 7.0).
The terminal electron acceptor was C02
for methanogenic media, while 20 mM
NaS04, 15 mM KNOa, or 20 mM sodium
fumarate was added for sulfate, nitrate or
fumarate enrichments, respectively.
Fermentative enrichments were prepared
by adding the carbohydrates used in the
Complete Carbohydrate medium (CCM)
of Leedle and Hespell (I980).
Stock cultures of DCB-1 were
maintained in basal medium containing
10-20% (v/v) clarified rumen fluid and
0.2% (w/v) pyruvate. For DNA extraction,
DCB-1 was grown in basal medium
consisting of 20% clarified rumen fluid,
and 0.2% pyruvate. The cultures were
grown in an atmosphere of 80% N2:20%
CO2.
The E. co// strains and vectors used in
this work are listed in Table 1. E. co//
strains were grown on LB plates, LB
broth or nutrient broth with the appro-
priate antibiotic, as necessary, for
selecting recombmants. Antibiotics were
used at a final concentration of 30-40
ng/ml for ampicillin and 15 ng/ml for
tetracycline. The bacteria were incubated
at 37°C.
Dehalogenase Screening Assay
Using a glass pipet, 1 drop each of the
following reagents was added to the well
of a white porcelain well plate: 0.2%
KNO2, 0.4% starch, 2% ZnCI2 solution,
and 1.9N HCI. A drop of DCB-1 liquid
culture medium (which included 3-
iodobenzoate as a substrate for dehalo-
genation) was then added to the
containing the reagents. A bluish-pi
color indicated a positive reaction for
presence of iodide and, theref<
evidence for dehalogenation.
Genomic DNA Isolation and
Purification
In order to obtain sufficient quant
of purified genomic DNA, a modifies
of a published procedure was u;
Additional modifications were made
follows: Proteinase K (Sigma) was i
at a final concentration of 1 mg/m
optimize cell breakage. RNase (Sigm.
a final concentration of 20 ng/ml
employed to reduce the high com
(ration of contaminating RNA; ethyl'
diaminetetraacetic acid (EDTA) con
trations in both the storage buffer anc
dialysis buffer were increased to 10
to minimize nuclease activity; and
TgES storage buffer (6 mM Tris pH
0.1 mM EDTA, 10 mM NaCI),
replaced by TE buffer (10 mM Tris
7.4, 10 mM EDTA) to decrease the
content. For cloning, DNA of a spe
size range was isolated on a suci
gradient, usually 10% to 40% (w/v).
Cloning Techniques
Restriction enzymes (DNA modif
enzymes) were purchased from se>
manufacturers. Ligations were d
overnight at either 12° or 4°C. Cells v
made competent and transformed u
standard procedures (as in the Ir
national Biotechnologies, Inc. catal
Competent DH5a cells were purchi
from BRL. In vitro packaging kits \
purchased from Strategene. Reag
were used according to the mi
facturer's instructions.
High Pressure Liquid
Chromatography
Benzoate, 3CB, and 4CB w
separated, identified, and quantifiee
high pressure liquid chromatogra
(HPLC). A reverse phase C18 Lichro
column (10 n, 4.6 mm [ID] x 25
Alltech Associates, Inc., Deerfield, IL)
used. The ratio of the solvent c
ponents used during most of the s
was 60.40:5 methanol/H2O/acetic
and the flow rate was normally
ml/min. Sample detection was achii
by U.V. adsorption at a wavelengt
284 nm. These compounds were q
tified by comparing the integrated
under the curve produced by
compound in the culture sample to
produced by a 500 pM sample of
authentic compound.
-------
Table 1. Microorganisms and Vectors
Strain Genotype
Source
DCB-1
E. coli
AC80
JM83
MM294
DH5o
Vectors
pBR322
pHC79
pUC8
thr leu met hsdR-hsd *
ara 4 (lac-pro) strA thi
(080 dlac rZA M15)
endAl thi-1 hsdflt7 sup£44
endAl hsdfll? sup£44 thi-1 recAl
gyrA96 relAl SOdlacZA M15
J. 7/ed/e
L Bopp
B. Bachmann
Ap* TcR cos
/acZa
BRL
BflL
BflL
§ Bethesda Research Laboratories.BRL
" ampicillin resistance, ApR,; tetracycline resistance, TcR
6 Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
Results and Discussion
In order to properly examine the
genetics of anaerobic dehalogenation,
defined pure cultures were highly
desirable. Several different approaches
were used to obtain a pure culture of an
anaerobic dehalogenator. Because Dr.
Tiedje's dehalogenating consortium and
he pure culture DCB-1 (from this
consortium) were not available at the
initiation of this project and because it
was of interest to determine whether
additional anaerobic dehalogenators
could be isolated from an area other than
the Michigan location where Dr. Tiedje
obtained his inoculum source, an effort
was begun to examine Columbus
sewage for dehalogenation activity. The
successful demonstration of anaerobic
dehalogenation by Columbus enrich-
ments led to an effort to isolate the
microorganism responsible for this
activity. When we received Dr. Tiedje's
consortium, work began in parallel to
isolate the dehalogenating organisms
from the Tiedje and Columbus consortia,
and to study some of the characteristics
of the dehalogenating microorganisms.
Finally, Tiedje's dehalogenating orga-
nism, strain DCB-1, was sent to us.
Since DCB-1 was a pure culture,
studies with DCB-1 assumed highest
priority. The goal of producing a superior
dehalogenating anaerobic organism
could be best approached by studying
the genetics and biochemistry of
anaerobic dehalogenation. This study
required the use of a pure culture. The
ivailability of DCB-1 increased the
speed at which we could move toward
our goal.
Enrichments from Columbus
Sewage
At the initiation of this program, there
had been only one report of anaerobic
dehalogenation; it was highly probable
that other anaerobic microorganisms/
consortia capable of similar activities
would also be found. The parameters of
enrichment were varied in an effort to
obtain these other anaerobic dehalo-
genating organisms in laboratory pure
cultures. The following enrichments were
undertaken.
Two compounds, 4CB and 3CB, were
used in the attempt to isolate different
anaerobic dehalogenating organisms.
Methanogenic enrichments were pre-
pared because the dehalogenating con-
sortium developed by Dr. Tiedje came
from a methanogenic environment.
Enrichments were also prepared in which
nitrate, sulfate, and fumarate served as
terminal electron acceptors instead of
carbon dioxide. A fermentative enrich-
ment was also examined.
The basal medium was used for
methanogenic enrichments. Nitrate re-
duction enrichments used a medium
similar to the basal medium. Cysteine
(2.5%) replaced the cysteine/sulfide
reducing solution. The N2/CC-2 gas
phase was retained as some strict
anaerobes known to reduce nitrate also
require CC-2. In the sulfate reduction
enrichments, the basal medium with 20
mM NaS04 and 20 mM NaCI was used.
Also, the sodium sulfide reducing
solution replaced the cysteine/sulfide
solution.
M, P. Bryant reported that fumarate
could serve as a terminal electron
acceptor for microorganisms that de-
grade benzoate. Since the use of
fumarate might eliminate the need for an
additional H2 utilizing organism as the
terminal electron sink, an enrichment was
made with fumarate added to the basal
medium. The ability of fermentative
organisms to dehalogenate 3CB was
examined. The basal medium was used,
with the addition of Neopeptone (0.1%,
Difco), Tryptone (0.1%, Difco), and the
carbohydrates used in CCM medium.
Benzoate, 3CB, or 4CB was added to all
the enrichments.
The effect of Hg on the development of
consortia capable of degrading chlori-
nated organic compounds was of special
interest because anaerobic dehalogen-
ation is a reductive process. It has been
observed that the chlorine must be
removed before further degradation of
the ring can occur; thus, this may be an
obligatory first step. The degradation of
the dehalogenated intermediate requires
that the H2 concentration be kept very
low (less than 1 x 10-5 atm) to have
degradation become thermodynamically
feasible (i.e., a negative Gibbs Free
Energy [G]). Therefore, while the pre-
sence of some hydrogen might stimulate
reductive dehalogenation, the presence
of too much hydrogen would inhibit the
degradation of the organic intermediate.
Including just enough \\% to provide
-------
reducing equivalents for dehalogenation
might result in a decrease in the lag
phase before dehalogenation is observed
without leaving excess hydrogen to
inhibit further degradation of the com-
pound.
The enrichments were periodically
sampled during this study in order to
determine some of the biological
processes which were occurring. HPLC
analyses of culture fluid from those
enrichments containing benzoate indi-
cated that the benzoate was completely
transformed within one month. The HPLC
results did not indicate total utilization,
but rather that all of the benzoate in the
system had been at least partially
degraded or transformed.
The degradation of 3C8 and 4CB in the
enrichments was periodically monitored
by HPLC during an 11-month period.
No degradation of 4CB was detected in
any of the enrichments (Table 2). A
nitrate enrichment incubated without
hydrogen was the first enrichment to
show significant 3CB degradation (Table
2). Once the initial amount of 3CB was
no longer detectable, an additional 800
nmoles 3CB was added to the culture to
confirm its ability to degrade this
compound. The 3CB added to the
enrichment was utilized within one week.
Initially, the rate of disappearance of 3CB
was 54 iimoles/liter/day, but after the
third day, the rate increased to 145
nmoles/liter/day. Degradation after the
third day was linear (R = 0.999).
Table 2. Develpment of 3-CI-Benzoate
Degrading Consortium Under
Various Enrichment Conditions
Enrichment Type
3-CI-benzoate
methanogenic
sulfate
nitrate
fumarate
CCM
4-CI-benzoate
methanogenic
sulfate
nitrate
fumarate
Time
no H2
I0-23f
NDO§
6-10
70-23
A/DO
A/DO
A/DO
A/DO
A/DO
(Weeks)"
+ H2
70-23
A/DO
70-23
70-23
A/DO
A/DO
A/DO
A/DO
A/DO
* Weeks of incubation before degration
observed.
t Degradation not observed at 10 weeks, but
apparent at 23 weeks.
§ A/DO - no degradation observed
A Gram stain of the enrichment was
prepared. Gram-negative short rods and
cocci were present as well as refractile
bodies, (i.e., spores). Gram-negative,
thin, extremely long rods, similar to
Methanospirillum hungatei, were also
present. The presence of M. hungatei
suggested that a methanogenic enrich-
ment had become established.
The fumarate and methanogenic
enrichments, after 28 weeks of incu-
bation, showed the complete absence of
3CB (Table 2). Further examination of the
enrichments showed that neither the
terminal electron acceptor present in the
fumarate and nitrate enrichments nor
hydrogen was required for degradation.
Microscopic examination of the enrich-
ments revealed a mixture of Gram-
negative rods of varying shape and
lengths (from coccobacillus to long rod
similar to M. hungatei). There was no
degradation of 3CB in either the sulfate
or CCM enrichments.
Examination of 3-CI-Benzoate
Consortium
The microbial consortium capable of
degrading 3-chlorobenzoates was sup-
plied by Dr. James Tiedje. Previous
workers indicated that the dehalo-
genating organism isolated from the
consortium grew slowly in a medium
containing pyruvate and that it reduced
nitrate to nitrite. This suggested that it
might be possible to improve the growth
rate of the dehalogenating organism by
providing nitrate as a terminal electron
acceptor. Selectively improving the
growth rate of this dehalogenating
organism would aid in an attempt to
isolate this organism in pure culture. The
consortium was inoculated (10% v/v) into
the following three variations of basal
medium in order to establish a pure
culture of the dechlorinating organism:
1. 800 uM 3CB and 15 mM sodium
nitrate,
2. 800 jiM 3CB, 15 mM sodium nitrate,
and 0.3% sodium pyruvate,
3. 800 nM 3CB, 15 mM sodium nitrate,
and 50% hydrogen.
The basal medium contained yeast
extract instead of rumen fluid. Pyruvate
could serve as an energy source and as
reducing potential for the reductive
dechlorination of 3CB. Nitrate could
serve as a terminal electron sink to pro-
duce energy for growth.
The 3CB concentration was deter-
mined at 0, 3, 16, and 44 days. At 44
days, the culture containing 3CB and 15
mM NC>3 showed no detectable 3CB, t
a large peak was observed with a low
retention time (8.36 minutes) corr
spending to 694.2 ^M benzoate. Tf
indicated that under these conditio
3CB was being dechlorinated, but r
degraded. A Gram stain of the culti
showed that about 95% of the ce
present were small Gram-negati
coccobacilli found mostly in pairs. The
were also some large Gram-negati
rods. Cells with the morphology of
hungatei were not seen. It appeared tt
under these conditions the benzos
degrader and methanogens we
selected against and were now abse
Thus, the dechlorinating organism w
thought to be one or both of the c
types present. The large Gram-negat
rod observed corresponded to t
dechlorinating organism described
Tiedje, but the small coccobacillus w
not described in his report. Further wt
indicated that it was most likely that t
rod and not the coccobacillus was I
dehalogenating organism.
At this point in the research, t
dehalogenating organism, strain DCB
was received from Dr. Tiedje and t
isolation effort was discontinued.
seemed likely that the organi;
responsible for dehalogenation in the
experiments was the same as or vi
similar to strain DCB-1.
Genetics of DCB-1
Dehalogenation
DCB-1 is thought to be related to
genus Desulfovibrio. This strain v\
originally isolated from anaerol
digester sludge from a sewage treatm
plant in Holt, Ml. It is a Gram-negal
non-sporeforming obligate anaerc
capable of dehalogenating haloarom;
compounds by removing haloge
(chloro, bromo, and iodo but not flue
from meta-substituted benzoate cc
pounds.
DCB-1 is the first anaerot
bacterium to be isolated in pure cult
which possessed dehalogenase activ
The dehalogenase activity of DCB-1
interesting because the mechanism «
conditions of anaerobic dehalogenat
are different from the mechams
observed in many aerobic deha
genating microorganisms. Because sti
DCB-1 grows very slowly and beca
strain DCB-1 is a fastidious sti
anaerobe, it was decided that the t
opportunity for studying anaero
dehalogenation would be achie\
through the cloning and expression of
dehalogenase encoding gene or ge
-------
from DCB-1 in an alternative host
nicroorganism.
Rapid Screening Method for
Dehalogenase Activity-
When a large number of recombinant
bacteria are made in an effort to find a
gene present only once in the genome, it
is necessary to efficiently and rapidly
screen the recombinant bacteria to find
those recombinants carrying the gene of
interest.
A rapid qualitative assay was devel-
oped that could detect the presence of
free iodide ions in liquid culture. The
procedure is a modification of the
starch-iodide spot test for nitrites. The
otiginal test depends on the formation of
nitrous acid and its subsequent reaction
with potassium iodide to liberate iodine,
which turns the starch blue. By providing
a source of nitrite as a 0.2% aqueous
solution of KNOa the presence of iodide
ions in the medium due to the
dehalogenation of the substrate 3-
iodobenzoate can be detected. Quanti-
tative analysis of the loss of 3-iodo-
benzoate and concomitant appearance of
benzoate as determined by HPLC was
used as evidence of dehalogenation. The
results of the HPLC analysis were
compared with the spot test reactions in
jrder to determine the sensitivity of the
spot test (Table 3).
After 23 days of incubation, all DCB-1
samples were positive for dehalogenation
as determined by the rapid starch spot
test. A very strong positive reaction was
evident in sample (a), the only sample
shown to completely dehalogenate the
3-iodobenzoate based upon HPLC data.
The remaining samples were all positive
using the spot test, with 20.4% - 51.3%
of 3-iodobenzoate remaining based on
HPLC data. The development of a rapid
technique to assess anaerobic dehalo-
genation, using 3-iodobenzoate, was a
significant achievement. With this
technique, large numbers of clones can
be screened under a variety of
environmental conditions.
Isolation of Plasmid DNA--
Initially, it was hoped that DCB-1
might carry the genes encoding
dehalogenase activity on a plasmid, the
cloning of a gene carried by a plasmid
would be much simpler than cloning a
chromosome encoded gene. However,
all attempts to isolate plasmid DNA from
DCB-1 were negative. Since there was
no evidence to suggest the existence of
any plasmids in DCB-1, it was
concluded that the dehalogenase activity
was chromosomally encoded. In order to
clone the gene or genes responsible for
the dehalogenase activity, it was
necessary to generate a complete
genomic library of DCB-1 DNA and
search for the gene or genes of interest.
Preparation of DCB-1 Genomic
Library--
The goal of the cloning effort was to
generate a DCB-1 genomic library and
to screen the library for the gene (or
gene complex) that encodes the
dehalogenase activity. The initial set of
experiments was performed in order to
show that a DCB-1 genomic DNA
library could be constructed using an £
coli vector and host. For these experi-
ments, purified genomic DNA was
digested with either Pst1 or EcoR1 and
hgated to Pst1 restricted pBR322 and
EcoR1 restricted pUC8, respectively.
Recombinant E. coli with DCB-1 inserts
were isolated. These clones were grown
anaerobically and tested for dehalo-
genase activity using the 3-iodo-
benzoate screen. However, dehalo-
genase activity was not detected in any
of the clones.
The successful cloning of DCB-1
DNA (small fragments) indicated that the
DNA was suitable for a more extensive
cloning effort Because the isolation of
DCB-1 was a slow process and the
yield was relatively poor, it seemed
desirable to clone large DNA pieces into
a vector so that DCB-1 DNA could then
be produced in the recombinant host, E.
coli, and subclones could be made then
from these large inserts. Cosmid vectors
were designed for the purpose of cloning
large DNA fragments. A genomic library
was partially generated in the cosmid,
pHC79, by the method outlined in Figure
1.
Banked cosrnids can be tested for the
presence of the gene responsible for
dehalogenation by screening the
recombinant bacteria for expression of
dehalogenase activity Because of the
large size of the cloned DNA fragments,
expression of these recombinants will
depend almost entirely on the ability of
DCB-1 promoters and translation initi-
ation sites to function in E. coli. (A
complementation study, as indicated in
Figure 1, is useful for determining if
foreign promoters and translation initia-
tion sites function in £. coli).
Since an £. coli promoter may be
needed to express DCB-1 DNA in E.
coli, we plan to purify DCB-1 DNA from
Table 3. Comparison of Deba/ogenase Acf/v;fy by Standard HPLC Methods and the Rapid Spot Screening Assay.
Dehalogenation as Measured by HPLC
Dehalogenation as Measured by
the Spot Test
3-iodobenzoate
(% remaining)
Sample
DCB-1 a
b
c
d
e
0
roo
too
roo
roo
roo
(days)
9
743
95.6
89.4
862
88.9
23
0
51.3
20.4
29.5
28.1
0
0
0
0
0
0
Benzoate
% formed
(days)
9
NT§
NT
NT
NT
NT
23
100
43.3
100
53.8
50.0
Starch Iodine
Reaction"
(days)
0 9 23
— — + + +
— — -4-
— — +
— — +
— — -t-
Each value represents the mean of duplicate samples.
§ Not tested; NT
* A negative reaction is indicated by a minus sign and a positive reaction is indicated by one or more plus signs. A three plus
reaction is one that occurred rapidly and results in a very dark blue color.
-------
EcoR1
Pst 1 »
Large DNA Fragments
Fill in
with
Klenow
AddC Tail
Restrict with Pst 1
and G Tail
Total Genomic
DNA
f
Sucrose
Gradient
Package in vitro into infectious
particles, infect
Screen for Recombmants (Tcf
Aps)
Isolate cosmids with inserts
Pool cosmids
Transform auxotrophic
hosts
Test for dehalogenase
activity
Examine recombmants
lor complementation of
auxotrophic mutations
Figure 1. Construction of genomic library in cosmid vector pHC79 and screening of the
library for gene expression in E coli
the cosmid recombinants and then
subclone smaller fragments of DCB-1
DNA (3-9 kb) into the vector pUC8.
Expression of fragments cloned into
multiple restrictions site in the lacZ gene
may occur from the lac promoter of
pUC8. Recombmant clones will be
pooled and screened for dehalogenase
activity.
In order to generate a genomic library
with a 95% probability of containing any
particular single-copy gene, approxi-
mately 380 recombinants (with DNA
inserts of 35 kb) would have to be
isolated (This is a library of about five
genomic units). At this point we have 57
potential cosmids containing clones
(about 15% of the library) These
potential recombinants must be analyzed
further to verify the presence of an insert
and to determine the size of the insert
We are somewhat concerned by the lack
of vigor shown by these potential
recombinants These bacteria grow very
slowly and some were found to be
sensitive to preservation by freezing
Conclusions and
Recommendations
The ultimate goal of our work with
anaerobic dehalogenatmg bacteria is to
develop engineered microorgamsr
capable of degrading hazardous orgar
compounds (e.g., chlorinated organics)
environmentally safe forms under s
aerobic conditions This work will al
provide information on the process
anaerobic dehalogenation To achie
this goal, we recommend completing t
generation of the DCB-1 library and t
cloning of the dehalogenase gene
When Columbus sewage was used
an inoculum for the various ennchmei
discussed in this report, dehalogenati
of 3-chlorobenzoate (3CB) was c
tected The Battelle laboratory was t
second laboratory to report this activi
-------
Anaerobic dehalogenation, therefore, is
an activity found in multiple sewage
samples; it is not an isolated activity.
Dehalogenation of 4-chlorobenzoate
(4CB) was not detected in the primary
enrichment; this result is in agreement
with the work of others.
A hypothesis that Ha at a concentration
of 10% would aid in the establishment of
a 3CB degrading consortium was shown
not to be true. Experiments with the
dehalogenating consortium of Tiedje
showed that varying the growth condi-
tions (change in the terminal electron
acceptor) could enhance the growth of
the dehalogenating bacteria relative to
other bacteria originally present in the
consortium.
Pure cultures of DCB-1, the micro-
organism responsible for the dehalo-
genation of 3CB in the Tiedje consorti-
um, were examined for the presence of
plasmids by a variety of methods; no
plasmids were observed. The absence of
plasmid DMA indicated that a total
genomic library of DCB-1 needed to be
generated in order to clone the gene or
genes responsible for anaerobic
dehalogenation. Our efforts have shown
that genomic-DNA isolated from DCB-
1 can be cloned in and banked in E. coli.
The full report was submitted in ful-
fillment of CR811120-02-4 by Battelle
Columbus Division under the spon-
sorship of the U.S. Environmental
Protection Agency.
-------
Robinson, Barbara R.
Battelle Columbus Division,
Donna T. Palmer, Timothy G. Linkfield, Jayne B.
Genthner, and George E. Pierce are with
Columbus, OH 43201.
Albert D. Venosa, is the EPA Project Officer (see below).
The complete report, entitled, "Determination and Enhancement of Anaerobic
Dehalogenation: Degradation of Chlorinated Organics in Aqueous Systems,"
(Order No. PB 89-110 2821 AS; Cost: $15.95, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, V'A 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-88/054
0000329 PS
U S EKVIR PROTECTION AGENCY
CHICAGO
------- |