metabolic interactions supporting effective tce bioremediation … · 2019. 4. 22. · metabolic...

Metabolic Interactions Supporting Effective TCE Bioremediation under Biogeochemical Conditions

Grant 1R01ES024255-01Lisa Alvarez-Cohen

Presenter: Shan Yi04/22/2019

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

Anaerobic Microbial Reductive Dechlorination

cis-DCE VCTCEPCE 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

TCE EtheneDehalococcoides mccartyi

Methanogens

Corrinoids(e.g., Vitamin B12)

H2

CO2

Hydrogenotrophic Acetogens

Fermenters

Organic Substrate (lactate/whey/molasses)

Acetate

Interactions in Dechlorinating CommunitiesDhc does not live alone in nature.

CO

3

CO2

Geochemical Perturbations on TCE BioremediationImportant to determine how environmental conditions affect material

exchanges in TCE-dechlorinating communities.

Soil

Chlorinated solvents

Groundwater

Na+

Cl-

Injection wells

4

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 intoengineering

solutions

qPCR

4) Possible solutions to overcome the perturbation. 5

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.

SO42- +4H2 +H+à HS-+ 4H2O• Lack of consistent understanding of sulfate’s effects on TCE dechlorination.

• Two testing hypotheses:Ø Inhibitory effects of sulfate or sulfideØCompetition of electron donor (H2)

Consortia Complex enrichment

Electron donor competitionTwo scenarios: 1) electron acceptor limiting, 2)

electron donor limiting

Pure culture

Toxicity effects

7

Bacterium FunctionDesulfovibrio vulgaris Hildenborough (DvH) Fermentation, sulfate reductionSyntrophomonas wolfei (S. wolfei) FermentationDehalococcoides mccartyi strain 195 (Dhc 195) TCE dechlorination

Two Types of Syntrophic Consortia

Lactate

Dhc195

DvH

Acetate + H2

VC & EtheneTCE

Sulfate

Sulfide

Scenario 1: electron acceptor limiting

Butyrate

S. wolfei

Acetate + H2

VC & EtheneTCE

Sulfate

Sulfide

DvH

Dhc195

Scenario 2: electron donor limiting

CO COCO

Butyrate

S. wolfei

H2

VC & EtheneTCE

Sulfate

Sulfide

DvH

Dhc195 8

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 Decreasedyield by 65%

S. wolfei Fermentation 5 mM No effects 5 mM Decreasedyield by 40%

DvHFermentation, sulfate

reduction N/A >10 mM Cell growthinhibited

SO42-à HS-

9

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

Lactate

Dhc195

DvH

Acetate H2

VC & EtheneTCE

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 11

Co-Culture DvH/Dhc195 under Electron Acceptor Limitation


Unlimited H2production

Inhibited TCE dechlorination

Dhc195:DvH: ~1:6

12

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

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

No Inhibition on TCE dechlorination


COCO

Butyrate

S. wolfei

Acetate + H2

VC & EtheneTCE

Sulfate

Sulfide

DvH

Dhc195

13

Effects of Sulfate Reduction on TCE-Dechlorinating Enrichment Culture

• Enrichment culture showed similarinhibitory patterns as the definedconsortia under the two limitation conditions.

• Methane production occurred in thecontrol culture but not in sulfateamended groups due to low H2concentration.

Mao et al. Appl. Environ. Microbiol. 2017 14

Lactate

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 DhcØ Salt stress on the metabolic interactions

Pure culture Consortia

Bacterium FunctionDvH FermentationPelosinus fermentans R7 (PfR7) Fermentation, corrinoid productionDhc 195 TCE dechlorination

Inhibitory effects Salinity stressTwo scenarios:

16

1) Existing salinity in groundwater2) Salinity perturbation

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

Lactate

PfR7

Dhc195

DvH

Propionate Acetate H2Corrinoids

VC & EtheneTCE

CO

17

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 & DvHLimiting

Dhc195 Limiting

PfR7Partially

restored growth by GB

Significant enhancement

by GB

Glycine betaine

(GB)2 mM

13%

80%

100%

18

Overall Salt Stress Response of Dhc195 Pure Culture

Ectoine Glycine betaineProline

No effects

Time (day)0 1 2 3

Mas

s of

TC

E (µ

mol

e) p

er b

ottle

0

20

40

60

80

Control383 mM

470 mM513 mM

554 mM595 mM


MIC

MIC = Minimum inhibitory concentrationstressor concentration that decreases the overall yield by 50%

19

Transcriptional Responses of Dhc195 PureCulture 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


383 mM

20

1st dose of TCE + salt perturbation

TimeAmou

nt o

f TC

E/bo

ttle

Compare dechlorination kinetics & cell growth during this period

Effects on Metabolic Interactions under Salt Stress (I)

Sun et al. In preparation 2019 21



50 183 227 271 315 359 404 448 492 536 580


N/A PfR7 Limiting

PfR7 & DvHLimiting

Dhc195 Limiting

Time (day)0 2 4 6 8 10 12 14

TCE

(µm

ole/

bottl

e)

0

20

40

60

80

100

Control A B C D D+GB

GB slightly improved TCE dechlorination


Effects on Metabolic Interactions under Salt Stress (I)

22



50 183 227 271 315 359 404 448 492 536 580


N/A PfR7 Limiting

PfR7 & DvHLimiting

Dhc195 Limiting

1st dose of TCE

2st dose of TCE + salt perturbation + GB

TimeAmou

nt o

f TC

E/bo

ttle

Compare dechlorination kinetics & cell growth during this period


Effects on Metabolic Interactions under Salt Stress (II)

23



50 183 227 271 315 359 404 448 492 536 580


N/A PfR7 Limiting

PfR7 & DvHLimiting

Dhc195 Limiting

Time (day)0 1 2 3 4 5 6 7

TCE

(µm

ole/

bottl

e)

0

20

40

60

80

100

Control+GB D+GB

E+GB F+GB

G+GB



50 183 227 271 315 359 404 448 492 536 580


N/A PfR7 Limiting PfR7 & DvHLimiting

Dhc195 Limiting



24

Amendment with GB

Severe inhibition

Amendment withGB + vitamin B12



25



50 183 227 271 315 359 404 448 492 536 580


N/A PfR7 Limiting

PfR7 & DvHLimiting

Dhc195 Limiting

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.

26

AcknowledgementsAlvarez-Cohen’s lab at UC Berkeley

Thank you!Questions?

[email protected]

Prof. Lisa Alvarez-Cohen Mohan Sun Dr. Xinwei Mao Alexandra Polasko

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metabolic interactions supporting effective tce bioremediation … · 2019. 4. 22. · metabolic...

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