Metabolic Interactions Supporting Effective TCE Bioremediation under - PowerPoint PPT Presentation

Metabolic Interactions Supporting Effective TCE Bioremediation under Biogeochemical Conditions Grant 1R01ES024255-01 Lisa Alvarez-Cohen Presenter: Shan Yi 04/22/2019

Anaerobic Microbial Reductive Dechlorination PCE TCE cis -DCE VC ETH Cl - Cl - Cl - Cl - H 2 H 2 H 2 H 2 Dehalococcoides mccartyi (Dhc) Clostridium, Dehalobacter, Complete dechlorination Dehalospirillum, Desulfitobacterium, Desulfomonile, Desulfuromonas, Sulfurospirillum, Geobacter, etc Partial dechlorination • Electron acceptors: chlorinated ethenes • Electron donor: H 2 • Carbon source: acetate, CO 2 • Coenzymes: corrinoids (vitaminB 12 ) • Toxic waste: CO

Interactions in Dechlorinating Communities Dhc does not live alone in nature. Organic Substrate (lactate/whey/molasses) CO 2 CO 2 Hydrogenotrophic Fermenters Methanogens Acetogens Corrinoids CO Acetate H 2 (e.g., Vitamin B 12 ) TCE Ethene Dehalococcoides mccartyi 3

Geochemical Perturbations on TCE Bioremediation Important to determine how environmental conditions affect material exchanges in TCE-dechlorinating communities. Injection wells Soil Groundwater Cl - Chlorinated solvents Na + 4

Technical Objectives and Approach 1) Construct defined 2) Investigate consortia consortia representing performance in the presence of major interactions crucial to sulfate reduction or high salinity TCE-bioremediation qPCR Defined consortia TCE Ethene Expression array Insight into Cell activity & engineering metabolite solutions exchange RNA-seq analysis 3) Apply either microarray or RNA-seq to elucidate the 4) Possible solutions to effects of perturbation on metabolism and functions of Dhc. 5 overcome the perturbation.

Effects of Sulfate Reduction on TCE-dechlorination 6

Sulfate Effects • Sulfate is prevalent in groundwater. • Sulfate-reducing bacteria often occur in the same niche with dechlorinating bacteria. 2- +4H 2 +H + à HS - + 4H 2 O SO 4 • Lack of consistent understanding of sulfate’s effects on TCE dechlorination. • Two testing hypotheses: Ø Inhibitory effects of sulfate or sulfide Ø Competition of electron donor (H 2 ) Electron donor competition Toxicity effects Two scenarios: 1) electron acceptor limiting, 2) electron donor limiting Complex Pure culture Consortia enrichment 7

Two Types of Syntrophic Consortia Bacterium Function Desulfovibrio vulgaris Hildenborough (DvH) Fermentation, sulfate reduction Syntrophomonas wolfei (S. wolfei) Fermentation Dehalococcoides mccartyi strain 195 (Dhc 195) TCE dechlorination Scenario 2: electron donor limiting Scenario 1: electron acceptor limiting Lactate Butyrate Butyrate Sulfate DvH S. wolfei S. wolfei Sulfide Sulfate Sulfate DvH DvH Acetate + H 2 Acetate + H 2 H 2 CO CO Sulfide Sulfide CO Dhc195 TCE TCE TCE Dhc195 Dhc195 VC & Ethene VC & Ethene VC & Ethene 8

Inhibitory Effects on Syntrophic Consortia Members 2- à HS - SO 4 Axenic cultures Function Sulfate Sulfide Decreased Dhc 195 TCE dechlorination 5 mM No effects 5 mM yield by 65% Decreased S. wolfei Fermentation 5 mM No effects 5 mM yield by 40% Fermentation, sulfate Cell growth DvH reduction N/A >10 mM inhibited 9 Mao et al. Appl. Environ. Microbiol. 2017

Sulfide Inhibition on Dhc195 • Decreased TCE dechlorination rates. • Decoupled growth from dechlorination when sulfide was introduced. • Transcriptomic analysis using microarray indicates the gene expression changes in ATP synthase, biosynthesis, and metal-containing enzymes. Mao et al. Appl. Environ. Microbiol. 2017 10

Effects of Sulfate Reduction on TCE-dechlorination Dhc195:DvH: ~5:1 Scenario 1: electron acceptor limiting Lactate DvH Sulfate Dhc195 DvH Sulfide CO in pure Dhc195 Acetate H 2 CO Dhc195 TCE CO in VC & Ethene DvH/Dhc195 coculture 11 Men et al., ISME J, 2012 , Polasko et al., AGU,2014, Zhuang et al., PNAS 2014

Co-Culture DvH/Dhc195 under Electron Acceptor Limitation Unlimited H 2 Dhc195:DvH: ~1:6 production Inhibited TCE dechlorination 12 Mao et al. Appl. Environ. Microbiol. 2017

Tri-Culture S. wolfei /DvH/Dhc195 No Inhibition on TCE dechlorination under Electron Donor Limitation Butyrate S. wolfei Sulfate DvH Acetate + H 2 CO Sulfide CO TCE Dhc195 VC & Ethene Dhc195:DvH:S.wolfei: ~16:1:1 13 Mao et al. Appl. Environ. Microbiol. 2017

Effects of Sulfate Reduction on TCE- Dechlorinating Lactate Enrichment Culture • Enrichment culture showed similar inhibitory patterns as the defined consortia under the two limitation conditions. • Methane production occurred in the control culture but not in sulfate amended groups due to low H 2 concentration. 14 Mao et al. Appl. Environ. Microbiol. 2017

Effects of Salinity on TCE-dechlorination 15

Salinity Effects on TCE bioremediation • TCE is present at 389 National Priorities List (NPL) sites, many of which are along the coast. • Effects of salinity on TCE bioremediation are unknown. • Two testing hypotheses: Ø Salt stress at the cellular level of Dhc Ø Salt stress on the metabolic interactions Bacterium Function DvH Fermentation Pelosinus fermentans R7 ( PfR7 ) Fermentation, corrinoid production Dhc 195 TCE dechlorination Salinity stress Inhibitory effects Two scenarios: 1) Existing salinity in groundwater 2) Salinity perturbation Pure culture Consortia 16

Tri-Culture of PfR7/DvH/Dhc195 under Salt Stress Lactate PfR7 DvH H 2 Propionate Corrinoids Acetate CO TCE Dhc195 VC & Ethene 17

Salt Stress on Consortium Members Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 PfR7 & DvH Dhc195 Limiting dechlorination Limiting Limiting Partially Significant 100% PfR7 DvH restored growth enhancement by GB by GB 80% Glycine betaine 13% (GB) 2 mM 18 Sun et al. In preparation 2019

Overall Salt Stress Response of Dhc195 Pure Culture 80 Mass of TCE (µmole) per bottle No effects 60 40 20 MIC 0 0 1 2 3 Time (day) Control 470 mM 554 mM MIC = Minimum inhibitory concentration 383 mM 513 mM 595 mM stressor concentration that decreases the overall yield by 50% Ectoine Proline Glycine betaine 19 Sun et al. In preparation 2019

Transcriptional Responses of Dhc195 Pure Culture under Salinity Perturbation • Biosynthesis: • Acetyl-CoA synthesis • Pyruvate synthesis • Glutamate/glutamine biosynthesis • DNA/RNA synthesis • Riboflavin metabolism 383 mM • tRNA synthetase • Energy metabolism: • NADH dehydrogenases Schematic diagram for Dhc195 pure culture • ATP synthases salt perturbation experiment • ABC transporters 20 Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (I) Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 PfR7 & DvH Dhc195 Limiting dechlorination Limiting Limiting 1 st dose of TCE + salt perturbation Amount of TCE/bottle Time Compare dechlorination kinetics & cell growth during this period 21 Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (I) Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 PfR7 & DvH Dhc195 Limiting dechlorination Limiting Limiting 100 GB slightly improved 80 TCE (µmole/bottle) TCE dechlorination 60 40 20 0 0 2 4 6 8 10 12 14 Time (day) Control A B C D D+GB 22 Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II) Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 PfR7 & DvH Dhc195 Limiting dechlorination Limiting Limiting 1 st dose of 2 st dose of TCE + salt Amount of TCE/bottle TCE perturbation + GB Time Compare dechlorination kinetics & cell growth during this period 23 Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II) Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 Limiting PfR7 & DvH Dhc195 Limiting dechlorination Limiting 100 Amendment with GB 80 TCE (µmole/bottle) 60 40 20 0 0 1 2 3 4 5 6 7 Time (day) Control+GB E+GB G+GB D+GB F+GB 24 Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II) Group Control A B C D E F G H I J Na + conc. after perturbation 50 183 227 271 315 359 404 448 492 536 580 (mM) Limiting factor for TCE N/A PfR7 PfR7 & DvH Dhc195 Limiting dechlorination Limiting Limiting Amendment with GB + vitamin B 12 Severe inhibition 25 Sun et al. In preparation 2019