Metabolic Interactions Supporting Effective TCE Bioremediation under - - PowerPoint PPT Presentation

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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 -

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Metabolic Interactions Supporting Effective TCE Bioremediation under Biogeochemical Conditions

Grant 1R01ES024255-01 Lisa Alvarez-Cohen Presenter: Shan Yi 04/22/2019

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Clostridium, Dehalobacter, Dehalospirillum, Desulfitobacterium, Desulfomonile, Desulfuromonas, Sulfurospirillum, Geobacter, etc

Anaerobic Microbial Reductive Dechlorination

cis-DCE VC TCE PCE ETH

Dehalococcoides mccartyi (Dhc)

  • Electron acceptors: chlorinated ethenes
  • Electron donor: H2
  • Carbon source: acetate, CO2
  • Coenzymes: corrinoids (vitaminB12)
  • Toxic waste: CO

Cl- H2 Cl- H2 Cl- H2 Cl- H2 Partial dechlorination Complete dechlorination

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TCE Ethene

Dehalococcoides mccartyi Methanogens Corrinoids (e.g., Vitamin B12) H2 CO2 Hydrogenotrophic Acetogens Fermenters Organic Substrate (lactate/whey/molasses) Acetate

Interactions in Dechlorinating Communities

Dhc does not live alone in nature.

CO

3

CO2

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SLIDE 4

Geochemical Perturbations on TCE Bioremediation

Important to determine how environmental conditions affect material exchanges in TCE-dechlorinating communities.

Soil

Chlorinated solvents

Groundwater Na+ Cl- Injection wells

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Defined consortia Expression array 1) Construct defined consortia representing major interactions crucial to TCE-bioremediation 2) Investigate consortia performance in the presence of sulfate reduction or high salinity 3) Apply either microarray or RNA-seq to elucidate the effects of perturbation on metabolism and functions of Dhc. TCE Ethene Cell activity & metabolite exchange

Technical Objectives and Approach

RNA-seq analysis Insight into engineering solutions qPCR 4) Possible solutions to

  • vercome the perturbation.

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SLIDE 6

Effects of Sulfate Reduction on TCE-dechlorination

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Sulfate Effects

  • Sulfate is prevalent in groundwater.
  • Sulfate-reducing bacteria often occur in the same niche with dechlorinating bacteria.

SO4

2- +4H2 +H+à HS-+ 4H2O

  • Lack of consistent understanding of sulfate’s effects on TCE dechlorination.
  • Two testing hypotheses:

ØInhibitory effects of sulfate or sulfide ØCompetition of electron donor (H2)

Consortia Complex enrichment

Electron donor competition

Two scenarios: 1) electron acceptor limiting, 2) electron donor limiting Pure culture

Toxicity effects

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Bacterium Function Desulfovibrio vulgaris Hildenborough (DvH) Fermentation, sulfate reduction Syntrophomonas wolfei (S. wolfei) Fermentation Dehalococcoides mccartyi strain 195 (Dhc 195) TCE dechlorination

Two Types of Syntrophic Consortia

Lactate

Dhc195 DvH

Acetate + H2 VC & Ethene TCE

Sulfate Sulfide

Scenario 1: electron acceptor limiting Butyrate

  • S. wolfei

Acetate + H2 VC & Ethene TCE

Sulfate Sulfide DvH Dhc195

Scenario 2: electron donor limiting

CO CO CO

Butyrate

  • S. wolfei

H2 VC & Ethene TCE

Sulfate Sulfide DvH Dhc195

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Inhibitory Effects on Syntrophic Consortia Members

Mao et al. Appl. Environ. Microbiol. 2017

Axenic cultures Function Sulfate Sulfide Dhc 195 TCE dechlorination 5 mM No effects 5 mM Decreased yield by 65%

  • S. wolfei

Fermentation 5 mM No effects 5 mM Decreased yield by 40% DvH Fermentation, sulfate reduction N/A >10 mM Cell growth inhibited

SO4

2- à HS-

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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

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Effects of Sulfate Reduction on TCE-dechlorination

Lactate

Dhc195 DvH

Acetate H2 VC & Ethene TCE Scenario 1: electron acceptor limiting

CO Sulfate Sulfide Dhc195 Dhc195:DvH: ~5:1 DvH CO in pure Dhc195 CO in DvH/Dhc195 coculture

Men et al., ISME J, 2012 , Polasko et al., AGU,2014, Zhuang et al., PNAS 2014

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Co-Culture DvH/Dhc195 under Electron Acceptor Limitation

Mao et al. Appl. Environ. Microbiol. 2017

Unlimited H2 production Inhibited TCE dechlorination Dhc195:DvH: ~1:6

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Tri-Culture S. wolfei/DvH/Dhc195 under Electron Donor Limitation

Dhc195:DvH:S.wolfei: ~16:1:1

No Inhibition on TCE dechlorination

Mao et al. Appl. Environ. Microbiol. 2017

CO CO

Butyrate

  • S. wolfei

Acetate + H2 VC & Ethene TCE

Sulfate Sulfide DvH Dhc195

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Effects of Sulfate Reduction on TCE- Dechlorinating 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 H2 concentration.

Mao et al. Appl. Environ. Microbiol. 2017

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Lactate

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SLIDE 15

Effects of Salinity on TCE-dechlorination

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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 Pure culture Consortia Bacterium Function DvH Fermentation Pelosinus fermentans R7 (PfR7) Fermentation, corrinoid production Dhc 195 TCE dechlorination

Inhibitory effects Salinity stress

Two scenarios:

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1) Existing salinity in groundwater 2) Salinity perturbation

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Tri-Culture of PfR7/DvH/Dhc195 under Salt Stress

Lactate

PfR7 Dhc195 DvH Propionate Acetate H2 Corrinoids VC & Ethene TCE CO

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Salt Stress on Consortium Members

DvH

Sun et al. In preparation 2019

Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting PfR7 Partially restored growth by GB Significant enhancement by GB Glycine betaine (GB) 2 mM

13% 80% 100%

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Overall Salt Stress Response of Dhc195 Pure Culture

Ectoine Glycine betaine Proline

No effects

Time (day) 1 2 3 Mass of TCE (µmole) per bottle 20 40 60 80 Control 383 mM 470 mM 513 mM 554 mM 595 mM Sun et al. In preparation 2019

MIC MIC = Minimum inhibitory concentration

stressor concentration that decreases the overall yield by 50% 19

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Transcriptional Responses of Dhc195 Pure Culture under Salinity Perturbation

  • Biosynthesis:
  • Acetyl-CoA synthesis
  • Pyruvate synthesis
  • Glutamate/glutamine biosynthesis
  • DNA/RNA synthesis
  • Riboflavin metabolism
  • tRNA synthetase
  • Energy metabolism:
  • NADH dehydrogenases
  • ATP synthases
  • ABC transporters

Schematic diagram for Dhc195 pure culture salt perturbation experiment

Sun et al. In preparation 2019

383 mM

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1st dose of TCE + salt perturbation Time Amount of TCE/bottle Compare dechlorination kinetics & cell growth during this period

Effects on Metabolic Interactions under Salt Stress (I)

Sun et al. In preparation 2019

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Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting

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Time (day) 2 4 6 8 10 12 14 TCE (µmole/bottle) 20 40 60 80 100 Control A B C D D+GB

GB slightly improved TCE dechlorination

Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (I)

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Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting

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1st dose of TCE 2st dose of TCE + salt perturbation + GB Time Amount of TCE/bottle Compare dechlorination kinetics & cell growth during this period

Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II)

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Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting

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Time (day) 1 2 3 4 5 6 7 TCE (µmole/bottle) 20 40 60 80 100 Control+GB D+GB E+GB F+GB G+GB

Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting

Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II)

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Amendment with GB

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Severe inhibition

Amendment with GB + vitamin B12

Sun et al. In preparation 2019

Effects on Metabolic Interactions under Salt Stress (II)

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Group Control A B C D E F G H I J Na+ conc. after perturbation (mM) 50 183 227 271 315 359 404 448 492 536 580 Limiting factor for TCE dechlorination N/A PfR7 Limiting PfR7 & DvH Limiting Dhc195 Limiting

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Summary

  • Sulfate effects
  • Sulfide (5mM) inhibited TCE dechlorination and growth of Dhc195.
  • When hydrogen was abundant, sulfate-reducing bacterial activity generated sulfide that

inhibited TCE dechlorination.

  • The sulfate-reduction activity can be limited by using slow fermentable substrates to prioritize

TCE dechlorination.

  • Salt stress
  • Dhc195 has a relatively higher tolerance to salt stress compared to supporting bacteria that

formed syntrophic interactions with Dhc.

  • The salt stress mostly caused the transcriptional changes in genes encoding catabolism, tRNA,

amino acid, and nucleic acid biosynthesis in Dhc.

  • Osmoprotectant, i.e., GB can be used to ameliorate the inhibition on the supporting bacteria.
  • Biostimulation with medium containing cobalamin and GB is necessary to sustain the

bioremediation performance under salt perturbation at concentrations up to 400 mM.

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Acknowledgements

Alvarez-Cohen’s lab at UC Berkeley

Thank you!

Questions?

shan_yi@Berkeley.edu

  • Prof. Lisa Alvarez-Cohen

Mohan Sun

  • Dr. Xinwei Mao

Alexandra Polasko

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