16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species

Members of the genera Desulfuromonas and Dehalococcoides reductively dechlo rinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specif ic to hypervariable regions of the 16S rRNA genes of the Dehalococcoides gr oup (comprising Dehalococcoides ethenogenes and Dehalococcoides sp, strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (co mprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) w ere designed. The detection threshold of a nested PCR approach using univer sal bacterial primers followed by a second PCR with the Desulfuromonas dech lorinator-targeted primer pair was I x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sedime nts of three Michigan rivers and six different chloroethene-contaminated aq uifer samples was used as template in nested PCR. All river sediment sample s yielded positive signals with the BB1- and the Dehalococcoides-targeted p rimers. One chloroethene-contaminated aquifer tested positive with the Deha lococcoides-targeted primers, and another contaminated aquifer tested posit ive with the Desulfuromonas dechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from othe r known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the s pecific primers.