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 ------- |