A Vinyl Chloride-Dechlorinating Culture That Reduces Trichloroethene through Predominantly 1,1-Dichloroethene Jingjing Zhang, Andrew Joslyn, and Pei C. Chiu Department of Civil & Environmental Engineering University of Delaware July, 2005 Trichloroethene (TCE) and Perchloroethene (PCE) Extensively used in metal degreasing and dry cleaning Prevalent groundwater contaminants Potential human carcinogens (MLCs = 5 g/L for both TCE and PCE ) In situ bioremediation GW Table GW Flow (Clay/Bedrock) DNAPL (e.g., TCE, PCE) Pathway for biological reductive dechlorination of TCE C=C H HH H HCl TCE (trichloroethene) C=C H Cl H cis-DCE (dichloroethene) C=C H Cl H H VC (vinyl chloride) ethene 1,1-DCEtrans-DCE C=C H ClH C=C Cl H H cis-DCE is usually observed as the dominant DCE isomer. TCE Dehalogenators * TCEcis-DCE Dehalobacter, Desulfitobacterium, Desulfomonile, Desulfuromonas *Holliger, et al, 1999; Adrian, 2001; He, et al, 2003; Sung, et al, 2003; Griffin, et al, 2004; Miller, et al, Dehalococcoides ethenogenes strain CBDB1 TCEtrans-DCE TCEtrans-DCE/cis-DCE ( ) PCB-dechlorinating bacterium DF-1 Dehalococcoides ethenogenes strain 195 TCEcis-DCEVCethene Dehalococcoides ethenogenes strain BAV1 TCEcis-DCEVCethene 1,1-DCE has never been shown to be the dominant daughter product of TCE. Observation: A mixed culture derived from a landfill site in Dover, DE, which normally dechlorinated TCE to cis-DCE, was able to reduce TCE to predominantly 1,1-DCE (1,1-to-cis ratio = 2.5 0.4) under certain conditions. Experiments Conducted to Investigate This Further: Part - Dechlorination pattern of the mixed culture Part - Organism involved in TCE reduction to 1,1-DCE Part - Enzyme involved in 1,1-DCE production Methods Stock Culture: A groundwater culture that can completely dechlorinate TCE to ethene was obtained from Dover Air Force Base. Medium Lactate, acetate, propionate, sulfate, yeast extract, phosphate buffer, vitamin solution, mineral solution. Lactate, acetate, propionate, sulfate, yeast extract, phosphate buffer, vitamin solution, mineral solution. Experiments were conducted in an anaerobic glove bag under N 2. Typical inoculum = 8%. Analysis At different times, 0.1 mL headspace samples At different times, 0.1 mL headspace samples were drawn and analyzed using GC/FID. Part : Dechlorination pattern Methods and Analysis Part : Dechlorination Pattern Results : TCE reduction by the original TCE-fed groundwater culture cis-DCE was the dominant DCE isomer. Time (days) ,1-to-cis ratio Mean ratio 2.6 0.1 Part : Dechlorination Pattern Results : TCE reduction by the TCE-fed culture in the presence of ampicillin 1,1-DCE was the dominant DCE isomer. Part : Dechlorination Pattern Results : TCE reduction by the ampicillin-treated, TCE-fed culture in the absence of ampicillin There appeared to be at least two TCE-dechlorinating populations. In the absence of ampicillin, the one producing cis- DCE outcompeted the other that produced more 1,1-DCE. TCE dechlorination through predominantly 1,1-DCE was consistently observed in both 1,1-DCE-fed (15 transfers) and VC-fed cultures (5 transfers). This suggests that the cis-DCE-producing population in this culture is unable to utilize 1,1-DCE and VC. H 2 was a better electron donor, compared to the organic substrates, to support the TCE-to-1,1-DCE dehalogenation activity. Part : Dechlorination pattern Results 1,1-DCE-fed culture VC-fed culture VC-fed culture DNA PCR products Agarose Electrophoresis SequencingDGGE PCR: Polymerase chain reaction. DGGE: Denaturing Gradient Gel Electrophoresis. Part : Molecular characterization using primers specific to known dehalogenators No PCR products were obtained with primers targeting the following 5 species: Dehalobacter, Desulfitobacterium frappieri PCP- 1, Desulfomonile tiedjei, Desulfitobacterium dehalogenans, Desulfuromonas. PCR products of the same size were obtained with primers targeting Dehalococcoides. Primers for lanes 2,3 and 4 were designed by Hendrickson (AEM,2002); lanes 5,6 and 7: Loffler (AEM, 2000) bp Part PCR results DNA template lanes 2 and 6: 1,1-DCE-fed culture lanes 3 and 7: VC-fed culture lanes 4 and 5: water % 90% Primers lanes 1 and 2 were from Hendrickson (AEM, 2002) lanes 3 and 4 from Loffler (AEM, 2000); DNA templates lanes 1 and 3 from 1,1-DCE-fed culture; lanes 2 and 4 from VC-fed culture. Part DGGE results Gradient Results suggested only 1 Dehalococcoides species existed in these cultures. The 1,1-DCE-producing dehalogenator was likely a Dehalococcoides. C 2 HCl 3 C 2 H 2 Cl 2 + Cl - Crude cell extract Whole cellBroken cell French press 2e - + H + Part : In vitro dehalogenation Methods Transition metal cofactors, especially vitamin B 12 derivatives, are often involved in reductive dehalogenation. M n+ Co 3+ B 12 Fe 3+ Heme Ni 2+ F 430 dehalogenase enzyme :NN: :N M n+ cofactor Cyanide (CN-) is a strong ligand that binds transition metals. Part : In vitro dehalogenation Inhibitory Effects of CN - : A transition-metal cofactor may be involved in 1,1-DCE production. Part : In vitro dehalogenation Results : Effect of CN - TCE reduction in the presence of 0, 5, 10, 20 mM KCN Product distribution of TCE reduction in the absence of KCN Co( I )B 12 activate enzyme CH 4 +. CH 3 H + e e Co( II )B 12 CH 3 -Co ( III ) B 12 + I - inhibited enzyme CH3-I + Part : In vitro dehalogenation Inhibitory Effect of Methyl Iodide (CH 3 I): CH 3 I can inhibit super-reduced cobalamin (B 12s ) through methylation, and methylated B 12 can be reactivated by light. A cobalt corrinoid (B 12 ) cofactor may be involved. Part : In vitro dehalogenation Results : Effect of CH 3 I Product distribution of TCE reduction with photo-reversed cell extracts TCE reduction with untreated, photo-reversed, CH 3 I-treated cell extracts Results and Implications - I A distinct TCE dehalogenation pathway was observed, in which TCE was dechlorinated through predominantly 1,1-DCE, instead of the usual cis-DCE. Dechlorination of TCE and its daughter products was probably carried out by two different populations, one reducing TCE to cis-DCE and the other reducing TCE completely to ethene through primarily 1,1-DCE. The 1,1-DCE-producing population could grow on 1,1- DCE and VC but was outcompeted by the other (cis- DCE-producing) dehalogenator(s) when TCE was abundant (in the absence of ampicillin). Results and Implications - II Dehalococcoides 16S rRNA gene sequences were recovered from 1,1-DCE-fed and VC-fed cultures that reduced TCE to 1,1-DCE. A Dehalococcoides species was probably responsible for the novel regio-selectivity. A B 12 enzyme was probably involved in the preferential production of 1,1-DCE from TCE. This research provides a possible biological mechanism for the presence of 1,1-DCE at chlorinated ethene- contaminated sites. Acknowledgments National Science Foundation UD Davis/Bloc Graduate Fellowship Tim McHale (AFB) Tom Hanson (DBI) Thank You! Agilent 6890 gas chromatograph with a flame ionization detector and a 30 m GS-GasPro capillary column. Temperature program for the GC was 40 for 2 min, 25 /min to 115 , 10 /min to 200 , and 200 for 1 min. Quantification of peak area was based on external calibration standards. Part : Physiological study Methods and Analysis