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Electronic Supplementary Material

Benzoate-Driven Dehalogenation of Chlorinated Ethenes in Microbial Cultures from a Contaminated Aquifer

Michael Bunge†*, Jutta Kleikemper†, Ciro Miniaci†, Laurence Duc†, Margje G. Muusse†, Gerd Hause‡, and Josef Zeyer†

†Soil Biology, Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland

‡Biocenter, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany

*Corresponding author e-mail:

Table S1: 16S rDNA-directed primers for the second amplification step in the nested PCR.

Primer
/ Sequence (5'-3') / T (°C)a / Target siteb / Target organismsd and GenBank accession numbers
DCH 204 / AACCTTCGGGTCCTGCCGTC / 58 / 204-222 / Desulfuromonas chloroethenica (U49748)
DCH 1034 / GCCGAACTGACCCCTATGTTc / 1034-1015
DRE 446 / GGAAGAACGGCATCTGTG / 54 / 446-462 / Dehalobacter restrictus (U84497)
DRE 1265 / GGCTTCCGTTCCGTCTG / 1265-1249
DTI 179 / ATGAGACCACATGAGCTC / 52 / 179-193 / Desulfomonile tiedjei (M26635)
DTI 1017 / GTTTCCACGACTGTCCG / 1017-1001
SMU 176 / CCCATACTCCTTCTTGTC / 52 / 176-193 / Sulfurospirillum multivorans (X82931)
SMU 641 / TTCGAGAGCAGTTCAACG / 641-624
DET 731 / GCGGTTTTCTAGGTTGTC / 52 / 731-748 / all Dehalococcoides spp.
DET1368w / CACCWTGCTGATATGCGG / 1368-1351
DES 437 / TGTCTTCAGGGACGAACG / 55 / 437-454 / Desulfitobacterium hafniense strains DCB-2T (X94975), PCP1 (U40078), TCE1 (X95742), TCP-A (AJ404686); D. dehalogenans (L28946); D. chlororespirans (U68528); D. sp. strain PCE1 (X81032)
DES 1043 / CTCATAGCTCCCCGAAGG / 1043-1027
SYN82 / GCTTCGGCGGGTGAGTA / 54 / 82-98 / Syntrophus gentianae HQgoe1 (X85132), Syntrophus buswellii DSM 2612 (X85131)
SYN604 / CATCTGACTTGCCTAGCC / 604-587

aAnnealing temperature, bcorresponding to E. coli-positions (Brosius et al. 1981), caccording to Löffler et al. (2000), dThe primers selected for this study were designed using the ARB program package (Ludwig et al. 2004) and candidate primer sequences were subsequently examined by using the analysis tools provided by the Ribosomal Database Project (RDP-II, http://rdp.cme.msu.edu/). Target organisms represent bacteria with exact matches to the sequences of both primers, D. Desulfitobacterium.

Table S2: Dehalogenation end products in serial dilutions of dechlorinating cultures amended with hydrogen, acetate, and PCE or cis-DCE.

Dilution
Treatmenta / 100 / 10-1 / 10-2 / 10-3 / 10-4 / 10-5 / 10-6 / 10-7
PCE/–0.45µm filtrate / ETH / ETH / ETH / ETH / ETH / ETH / ETH / ETH / DCE / DCE / DCE / DCE / DCE / PCE / PCE / PCE
PCE/+0.45µm filtrate / ETH / ETH / ETH / ETH / ETH / PCE / PCE / PCE
DCE/–0.45m filtrate / ETH / ETH / ETH / ETH / ETH / ETH / ETH / ETH / DCE / DCE / DCE / DCE / DCE / DCE / DCE / DCE
DCE/+0.45 µm filtrate / ETH / ETH / ETH / ETH / DCE / ETH / ETH / ETH

aThe sample 100 was inoculated with 10% (v/v) of a culture from the first transfer grown with benzoate, acetate and PCE (see Figure 1b). Cultures that did not show complete dechlorination within 160 days (10-4 to 10-7 of both culture sets) were selected for a second inoculation with 0.45 µm filtrate (Schleicher & Schuell 0.45 µm filters: FP30/0.45 CA-S) of the original undiluted culture. Duplicate cultures for each dilution step were established. ETH, ethene; DCE, cis-dichloroethene; PCE, tetrachloroethene.