Redutive Dehalogenation of Chlorinated Alkanes by Novel Bacteria at the PetroProcessor of Louisiana Inc. Superfund Site

Abstract

A reductively dehalogenating enrichment culture was established using chloroalkane-contaminated groundwater from the PetroProcessors of Louisiana, Inc. (PPI) Superfund site. Two novel, strictly anaerobic bacterial strains, designated as BL-DC-8 and BL-DC-9, were isolated from the enrichment culture. These strains represent the first bacteria known to anaerobically dehalogenate 1,2,3-trichloropropane, the degradation pathway of which was determined. Both strains could be cultured in completely defined basal medium and were also able to dehalogenate a variety of other vicinally chlorinated alkanes including 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, and 1,2-dichloropropane via dichloroelimination reactions. Chlorinated alkanes containing only a single chlorine substituent (1-chloropropane, 2-chloropropane), chlorinated alkenes (tetrachloroethene, trichloroethene, 1,2-dichloroethenes, vinyl chloride), and chlorinated benzenes (1-chlorobenzene and 1,2-dichlorobenzene) were not dehalogenated. Phylogenetic analysis based on 16S rRNA gene sequences showed these isolates to represent a new lineage within the Chloroflexi. Their closest previously cultured relatives are “Dehalococcoides” strains with 16S rRNA gene sequence similarities of only 90%. A quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes indicated that both strains couple reductive dechlorination to cell growth. Growth was not observed in the absence of hydrogen (H2) as an electron donor and a polychlorinated alkane as an electron acceptor. Oligonucleotide primers targeting signature 16S rRNA gene sequences of the novel isolates were used in conjunction with primers targeting “Dehalococcoides” strains to investigate spatial distribution and relative abundance of dehalogenating bacteria within the dense non-aqueous phase liquid (DNAPL) source zone at the PPI site. Results revealed the presence of bacteria similar or identical to strains BL-DC-8 and BL-DC-9 as well as “Dehalococcoides” strains throughout the DNAPL source zone. 16S rRNA gene copy concentrations as high as 1.88 ± 0.07×106 copies/mL groundwater for the isolates and 5.84 ± 0.20×105 copies/mL for “Dehalococcoides” strains. The new genus represented up to 18.6% of total bacterial 16S rRNA gene copies at some locations, and it may play an important role in natural attenuation at this site. These results have the potential to improve decision making regarding remediation strategies and may aid in development of waste treatment and monitoring approaches.