Anaerobic bacteria that dehalogenate chlorinated propanes: Insights into the roles played by Dehalogenimonas spp. William M. Moe, Ph.D., P.E. Dept. of Civil & Environmental Engineering Louisiana State University [email protected] Acknowledgements Coauthors Funding / Other support Jie Chen NPC Services Trent Key LA GBI Kalpataru Mukherjee DOE JGI Fred Rainey ChunLab Other contributors Kimberly Bowman Jacob Dillehay Andrew Maness Jyoti Rao Jun Yan Overview Characteristics of genus Dehalogenimonas D. lykanthroporepellens D. alkenigignens Groundwater monitoring 16S rRNA gene based detection and quantification at a superfund site Significant findings Dehalogenimonas lykanthroporepellens Isolation: DNAPL contaminated groundwater (Yan et al., 2009) Strictly anaerobic, mesophilic, Gram negative, non motile, non spore forming 0.2 µm Electron donor: H2 Electron acceptors: Polyhalogenated alkanes Formal taxonomic description (Moe et al., 2009) D. lykanthroporepellens Dihaloelimination reactions 1,2-Dichloroethane → Ethene 1,2-Dichloropropane → Propene 1,1,2-Trichloroethane → Vinyl chloride 1,1,2,2-Tetrachloroethane → Dichloroethenes 1,2,3-Trichloropropane → Allyl chloride → other compounds (Yan et al., 2009) Biotic Abiotic D. lykanthroporepellens Genome sequence of BL-DC-9T 1,686,510 bp chromosome No plasmids ~24 rdhA genes (17 “full length”) 7 rdhB genes (1 orphan) (Sidaramappa et al., 2012) Data from PPI Brooklawn studies (Yan et al., 2009) 108 7.5 Total bacteria 107 Dehalogenimonas 7.0 Dehalococcoides 106 pH 6.5 105 6.0 104 5.5 3 10 5.0 Groundwater pH * * * 102 * 4.5 101 4.0 16S rRNA gene copies/16S rRNA mL groundwater W-0627-2 W-0721-1 W-0726-4 W-0820-1 W-0822-3 W-0823-2 W-0825-1 W-0828-1 Well ID number PPI Scenic Site Plume Groundwater contaminants: mixture of chlorinated alkanes and alkenes Enrichment cultures exhibited reductive dechlorination (1,2-DCA, 1,2-DCP, 1,1,2-TCA, TCE) 16S rRNA gene libraries (pyrosequencing) >165,000 sequences 16S rRNA gene libraries -021309 Actinobacteria -041509 Bacteroidetes -052109 Chloroflexi -062309 Firmicutes -072909 Spirochaetes Proteobacteria -090209 Other -100609 0% 20% 40% 60% 80% 100% Isolation approach Dilution-to-extinction Chlorinated solvents as electron acceptors H2 as potential electron donor Titanium citrate as reducing agent Terminal positives identified via gas chromatography Dehalogenimonas alkenigignens 96% 16S rRNA gene sequence identity with D. lykanthroporepellens 0.91 µm Strictly anaerobic Small, irregular cocci 0.87 µm H2 as electron donor Resistant to antibiotics (ampicillin, vancomycin) (Bowman et al., 2013) Phylogenetic relationship based on 16S rRNA gene sequences IP3-3T grown on 1,2-DCA 30 25 1,2-DCA Ethene 20 15 10 (umoles/bottle) 1,2-DCA or Ethene or 1,2-DCA 5 0 0 10 20 30 40 50 60 Time (days) IP3-3T grown on 1,2-DCP 30 25 20 15 10 5 [umol/bottle] 0 1,2 DCP or Propene 0 20 40 60 Time (days) Electron acceptors Chloromethanes Vicinally halogenated alkanes . 1,2-Dichloroethane Dichloromethane . 1,2-Dichloropropane Trichloromethane . 1,1,2,2-Tetrachloroethane Tetrachloromethane . 1,1,2-Trichloroethane Chlorinated alkenes . 1,2,3-Trichloropropane Tetrachloroethene Trichloroethene Other chlorinated alkanes cis-1,2-Dichloroethene 1-Chloropropane trans-1,2-Dichloroethene 2-Chloropropane Vinyl chloride 1,1-Dichloroethane 1,1,1-Trichloroethane Chlorinated benzenes 1-Chlorobenzene 1,2-Dichlorobenzene Growth conditions (laboratory) Temperature range: 18-42°C (optimum 30-34°C) pH range 6-8 (optimum 6.5-7.5) Salinity (m/v): ≤1% Strain IP3-3T with high concentrations of 1,2-DCP (Maness et al., 2012) 30 25 20 D. alkenigignens IP3-3T mol/bottle) µ 15 10 Abiotic neg. 5 Propene ( cont. 0 0 2 4 6 8 10 12 14 Initial aqueous-phase 1,2-DCP (mM) Contaminant Mixtures (0.5 mM each 1,2-DCA and 1,2-DCP) 300 250 200 150 1,2-DCP Propene 100 (umol/bottle) 1,2-DCA Concentration Ethene 50 0 0 5 10 15 20 25 Time (Days) (Dillehay et al., in press) Biostimulation: Field study 5000 105 4000 104 3000 g/L) 1,1,2-TCA µ ( 2000 1,2-DCA 103 Dhg (copies/mL) 1000 16S rRNA16S gene 1,2-DCA 1,1,2-TCA or 0 102 Time (months) Significant findings There is considerable phylogenetic diversity among reductively dehalogenating members of phylum Chloroflexi Dehalogenimonas spp. can dehalogenate polychlorinated alkanes, even at high concentrations and in the presence of mixtures Significant findings Dehalogenimonas spp. appear to respond to biostimulation as a remediation strategy in a manner similar to Dehalococcoides and other dechlorinators.