Populations Implicated in Anaerobic Reductive Dechlorination of 1,2-Dichloropropane in Highly Enriched Bacterial Communities

Abstract

1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that Dehalococcoides populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of Dehalococcoides populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by Dehalococcoides species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of Dehalococcoides populations.The short-chain chlorinated aliphatic compound 1,2-dichloropropane (1,2-D) has been used as a fumigant to fight rootparasitic nematodes in the production of high-value crops, as an insecticide, and in numerous industrial applications (17). Although 1,2-D is no longer employed in agricultural applications and its industrial use has been reduced, these practices lead to substantial groundwater contamination. Thus, many sites with 1,2-D concentrations exceeding the U.S. Environmental Protection Agency enforced maximum concentration level of 5 ppb (www.epa.gov/safewater/dwh/c-voc.html) exist. Due to the toxicity (and potential for carcinogenicity) of 1,2-D to the liver, kidneys, adrenal glands, bladder, and gastrointestinal and respiratory tracts, its presence in the environment is of genuine public concern, and many of the existing contaminated sites require remedial action.No naturally occurring aerobic microbial pathways that efficiently transform 1,2-D to nontoxic products have been described (11), although enzymes that transform polychlorinated propanes were generated by in vitro molecular evolution (3, 32). Most of the contamination with 1,2-D has occurred in oxygen-limited or anaerobic subsurface environments, where reductive processes are more probable (17, 34). In a previous study, the reductive dechlorination (i.e., hydrogenolysis) of 1,2-D to monochlorinated propanes and subsequent dehydrochlorination to propene was demonstrated in microcosms derived from river sediment (17). Interestingly, in sediment-free, nonmethanogenic enrichment cultures derived from these microcosms, only vicinal reduction (dichloroelimination) of 1,2-D to propene without the intermediate formation of monochlorinated propanes occurred (17). Hydrogen consumption threshold measurements provided circumstantial evidence for the presence of populations in the enrichment cultures that gain energy ...

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

“…Dehalococcoides-containing consortia included cultures SZ(PCE) and SZ(VC), which were derived from SuZi Creek sediment (Seoul area, South Korea), with PCE and VC as electron acceptors, respectively. Cultures KS(1,2-D) and RC(1,2-D), which do not utilize chloroethenes as electron acceptors, were derived from Alaska and Michigan river sediments, respectively, and were grown with 1,2-dichloropropane (1,2-D) (39). Bio-Dechlor INOCULUM (BDI), a commercially available PCE-to-ethene-dechlorinating consortium that has been used successfully for bioaugmentation at chloroethene-contaminated sites (40), was maintained in MM amended with lactate and TCE.…”

“…Qualitative information regarding the presence of Dehalococcoides organisms has been obtained using PCR primers that targeted signature regions of the 16S rRNA gene (18,29). Sensitivity was further improved using an initial amplification of community 16S rRNA genes with Bacteria-targeted primers, followed by PCR with Dehalococcoides-specific, internal primers (i.e., nested PCR), effectively increasing the detection limit by 2 orders of magnitude (29,39). After the significance of Dehalococcoides to chloroethene detoxification was realized, quantitative PCR methods to enumerate Dehalococcoides 16S rRNA gene copies were developed and applied (8,15,16,26,47).…”

“…To overcome the limitations resulting from the high degree of Dehalococcoides 16S rRNA gene similarity (Ͼ98% identity) and an absence of correlation between 16S rRNA gene sequence and dechlorination activity (2,9,16,17,20,34,39,49), a quantitative real-time PCR (qPCR) approach targeting three Dehalococcoides strain-specific reductive dehalogenase (RDase) genes was developed to more comprehensively characterize the chloroethene-dechlorinating Dehalococcoides community. The approach selectively targeted the TCE-to-VC RDase gene (tceA) of Dehalococcoides ethenogenes strain 195 (3,32) and strain FL2 (17,21); the bvcA gene, implicated in VC-to-ethene reductive dechlorination in strain BAV1 (23); and the vcrA gene, first discovered in strain VS (36) and later identified in strain GT, a pure Dehalococcoides culture capable of metabolic TCE-to-ethene dechlorination (49).…”

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“…Culture broth was centrifuged for 15 min at 8650g and supernatant was collected and examined for CHE activity based on Gallo's method 1 . Bacterial isolates producing CHE were identified by using PCR amplification of 16 S rDNA gene employing bacterial universal primers Bac8F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and Bac1492R (5 -ACGGTTACCTTGTTACGACTT-3 ) 26 . Reaction mixtures were manipulated in 100 µl volumes containing 100 ng of DNA samples, 1 × PCR buffer, 20 µl of each primer, 10 mM dNTP, 1.5 mM MgCl 2 , and 1.5 units of Taq DNA polymerase.…”

Factors Affecting Cis-Dichloroethene and Vinyl Chloride Biological Transformation Under Anaerobic Conditions

“…Analyzing the strain diversity of Dehalococcoides species is a challenging task because cell abundance is low and species are very closely related based on 16S rRNA gene sequence comparison. Recent studies have demonstrated that common phylogenetic markers, like the 16S rRNA gene, are not sufficient to distinguish between closely related Dehalococcoides strains exhibiting different dechlorinating activities (9,15,42,43). However, molecular probing of genes that encode enzymes catalyzing different dechlorination reactions provides the resolution to distinguish between Dehalococcoides strains with different metabolic abilities to grow with chlorinated ethenes as electron acceptor.…”