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 μ 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 Ⅰ - Dechlorination pattern of the mixed culture  Part Ⅱ - Organism involved in TCE reduction to 1,1-DCE  Part Ⅲ - 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 Ⅰ : Dechlorination pattern Methods and Analysis

Part Ⅰ : Dechlorination Pattern Results Ⅰ : 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 Ⅰ : Dechlorination Pattern Results Ⅱ : TCE reduction by the TCE-fed culture in the presence of ampicillin 1,1-DCE was the dominant DCE isomer.

Part Ⅰ : Dechlorination Pattern Results Ⅲ : 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 Ⅰ : Dechlorination pattern Results Ⅳ

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 Ⅱ : 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 Ⅱ 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 Ⅱ 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 Ⅲ : 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 Ⅲ : In vitro dehalogenation Inhibitory Effects of CN - :

A transition-metal cofactor may be involved in 1,1-DCE production. Part Ⅲ : In vitro dehalogenation Results Ⅰ : 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 Ⅲ : 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 Ⅲ : In vitro dehalogenation Results Ⅱ : 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 Ⅰ : Physiological study Methods and Analysis Ⅱ