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