Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

Abstract

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 mol liter ؊1 day ؊1 , respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ؎ 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.Chlorinated ethenes are pervasive groundwater contaminants resulting from extensive usage, improper disposal, and accidental spills, and the incomplete microbial dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) leads to dichloroethene and vinyl chloride (VG) accumulation. A breakthrough in the anaerobic treatment of chloroethene-contaminated sites was the discovery of bacteria that use chloroorganic compounds as electron acceptors to drive their energy metabolism. This metabolic reductive dechlorination process, also known as (de)chlororespiration, is a focus of current bioremediation approaches to contain or remediate chloroethene plumes.Numerous bacterial isolates that reductively dechlorinate chloroethenes have been described previously (26); however, no single organism has the ability to couple energy generation with each reductive dechlorination step leading from PCE to ethene. The majority of isolates dechlorinate PCE to cis-1,2-dichloroethene (cis-DCE), and the ability to respire PCE to cis-DCE is distributed among phylogenetic groups and includes members of the classes Deltaproteobacteria, Gammaproteobacteria, and Firmicutes (14). Bacteria with the ability for incomplete dechlorination of PCE to cis-DCE are often present at co...

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

“…CN-Cbl was depleted at higher rates in the 25 mg l 21 CN-Cbl-amended Dehalococcoides cultures. At day 17, when BAV1 cultures had completely dechlorinated cis-DCE to ethene, CN-Cbl concentrations dropped to 0.7 + 0.1 mg l 21 , averaging an uptake rate of 1500 ng l 21 qPCR analysis demonstrated that limiting amounts of 1 mg l 21 CN-Cbl affected Dehalococcoides growth yields, which were calculated as described [22]. For example, 1.4 + 0.8 Â 10 7 BAV1 cells per mmole of Cl 2 released were obtained in cultures amended with 1 mg l 21 CN-Cbl, whereas 1.3 + 0.1 Â 10 8 BAV1 cells per mmole of Cl 2 released (a 9.1-fold increase) were measured in cultures amended with 25 mg l 21 CN-Cbl (table 1).…”

“…Methane, ethene and chlorinated ethenes were quantified using an Agilent 7890 gas chromatograph equipped with a flame ionization detector and a DB-624 capillary column (60 m  0.32 mm  1.8 mm) as described [22]. Acetate was analysed using an Agilent 1200 series HPLC system equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA).…”

Identification of chlorinated solvents degradation zones in clay till by high resolution chemical, microbial and compound specific isotope analysis

“…(Dhc) (MaymoGatell et al, 1997). Dhc containing the gene tceA have been found to respire TCE and cis-DCE (Seshadri et al, 2005) whereas Dhc with the genes vcrA or bvcA respire cis-DCE and vinyl chloride (VC) (Muller et al, 2004;Sung et al, 2006). Several recent laboratory studies have also suggested that abiotic degradation pathways for chlorinated ethenes are of importance in the presence of iron minerals (Darlington et al, 2008;Ferrey et al, 2004;He et al, 2008He et al, , 2010Jeong et al, 2011;Lollar et al, 2010).…”

Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases

“…Genome sequences exist for several OHRB, including five Dehalococcoides mccartyi strains [13][14][15][16] and Dehalogenimonas lykanthroporepellens strain BL-DC-9 [17] within the Chloroflexi; Geobacter lovleyi strain SZ [18] and Anaeromyxobacter dehalogenans strains 2CP-C and 2CP-1 [19] from the Proteobacteria; and Desulfitobacterium hafniense strains Y51 and DCB-2 [6,20], Desulfitobacterium dehalogenans (NC_018017), Desulfitobacterium dichloroeliminans (NZ_AGJE00000000) and most recently two Dehalobacter strains [21] from the Firmicutes. Given the low cost and broad availability of sequencing from pure or mixed cultures, the number of OHRB genomes will increase rapidly.…”