Oxygen-sensitive Dehalococcoides bacteria play crucial roles in detoxification of chlorinated contaminants (e.g., chlorinated ethenes), and bioremediation monitoring relies on quantification of Dehalococcoides DNA and RNA biomarkers. To explore the effects of oxygen on Dehalococcoides activity, viability, and biomarker quantification, batch experiments with a tetrachloroethene-to-ethene dechlorinating consortium (Bio-Dechlor INOCULUM [BDI]) harboring multiple Dehalococcoides strains were performed to quantify the effects of < or = 4 mg/L dissolved oxygen. Oxygen inhibited reductive dechlorination, and only incomplete dechlorination to vinyl chloride (VC) occurred following oxygen consumption and extended incubation periods (89 days). Following 30 days of oxygen exposure and subsequent oxygen removal (i.e., reversibility experiments), all trichloroethene- (TCE-) fed cultures dechlorinated TCE to VC, but VC dechlorination to ethene occurred in only one out of fourteen replicates. These results suggest that Dehalococcoides strains respond differently to oxygen exposure, and strains catalyzing the VC-to-ethene dechlorination step are more susceptible to oxygen inhibition. Quantitative real-time PCR (qPCR) analysis detected a 1-1.5 order-of-magnitude decrease in the number of Dehalococcoides biomarker genes (i.e., 16S rRNA gene and the reductive dehalogenase [RDase] genes tceA, vcrA, bvcA) in the oxygen-amended cultures, but qPCR analysis failed to distinguish viable, dechlorinating from irreversibly inhibited (nonviable) Dehalococcoides cells. Reverse transcriptase qPCR (RT-qPCR) detected Dehalococcoides gene transcripts in the oxygen-amended, non-dechlorinating cultures, and biomarker transcription did not always correlate with dechlorination (in)activity. Enhanced molecular tools that complement existing protocols and provide quantitative information on the viability and activity of the Dehalococcoides population are desirable.

BackgroundGeobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae.ResultsSequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53–56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration.ConclusionsGene acquisitions have expanded strain SZ’s respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

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This study describes the design and implementation of remote Summer undergraduate research programs during the COVID-19 pandemic, including program strengths and recommendations for improvement from the perspectives of undergraduate researchers.