in Bioremediation of · Bioremediation of Benzene Sandra Dworatzek, Jeff Roberts and Jennifer Webb (SiREM) Kris Bradshaw (Federated Co‐operatives Limited) Courtney Toth, Nancy Bawa,

Phil DennisSiREM

Advances in Anaerobic Bioremediation of Benzene

www.vertexenvironmental.ca

SMART RemediationToronto, ON │ January 24, 2019

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Advances in Anaerobic Bioremediation of Benzene

Sandra Dworatzek, Jeff Roberts and Jennifer Webb (SiREM) Kris Bradshaw (Federated Co‐operatives Limited) Courtney Toth, Nancy Bawa, Shen Guo, and Elizabeth A. Edwards (University of Toronto)

Presented by: Phil Dennis, SiREM

SiREM Core Service Areas

BioaugmentationCultures 

Characterization/Monitoring

Molecular Testing 

Passive Samplers for Vapour and Pore Water    

Remediation Testing    

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Acknowledgements

• Fei Luo, University of Toronto

• Funding Partners:

BTEX/Benzene Challenges

•/

• 12,000 gas stations in Canada among potential sources

• BTEX comprises ~18% of gasoline– Benzene is typically around 1% 

Benzene:

• Potent carcinogen

• Particularly mobile in groundwater due to low sorption & high water solubility

• Most difficult BTEX compound to degrade anaerobically (unsubstituted ring structure)

• Under anaerobic conditions, bottleneck to site remediation

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Aerobic Benzene Degradation

•/

Aerobic Benzene Degradation – Energy Yield High

O2

Dioxygenase 

O2CO2 +H2OKrebs Cycle  Dioxygenase 

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Benzene

Benzoyl‐CoA

Fatty acids,Alcohols

H2 /Acetate

CH4, CO2

ORM2SO4/CH4 reducing conditions 

Methanogens

H2S

Anaerobic BTEX Degradation-a Team Effort

Benzene fermentation energetically viable only when metabolites (e.g., H2 and acetate)  removed by: 

• Methanogens

• Sulfate reducers 

• Nitrate reducers

Energy yield lower than aerobic pathways  

?

Benzoate

PeptococcaceaeNO3 reducing  conditions

Nitrate  Reducers 

N2

Sulfate  Reducers 

Syntrophs

Fermenters  

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Why Go Anaerobic for BTEX?

• Hydrocarbon sites are often anaerobic-high organic loading consumes O2

• Electron acceptors (NO3/SO4/CO2) often already present in subsurface

• Anaerobic electron acceptors soluble, easier to apply/distribute compared to O2

(e.g., epsom salts (sulfate)/sodium nitrate)

• Anaerobic processes less likely to cause biofouling

• May be viable in situ remediation option for deep contamination

Overview of Project

BTEX Culture Scale Up

Treatability Testing

Genomics/ Development of Molecular Tools

Federal NSN Approval

Field Pilot Application

1 2 3 4 5

*underway *planning stages

Genomic Applications Partnership Program Project

All bioaugmentation cultures and associated molecular tools will be commercialized by SiREM

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ORM2 Anaerobic Benzene Degrader

0 100 200 300 400103

104

105

106

107

co

pie

s/m

l cu

lture

ORM2

fed

starved

• Benzene specialist derived from an oil refinery site in 2003

• ORM2 is a Deltaproteobacterium

• Produces enzymes that ferment benzene

• Slow growing ~ 30 day doubling time

Time (days)

DGG-B Culture – ORM2’s Home

• DGG-B successfully scaled up to commercial volumes Benzene degradation rate = 0.3 mg /L/ day 1010 ORM2/L  1 L of culture can feasibly treat at least 1000 L of contaminated groundwater

05

10152025

0 200 400 600

Ben

zen

e m

g/L

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BTEX‐contaminated materials from 10 sites were assessed for their anaerobic benzene bioremediation potential

Tested:    Intrinsic bioremediation Biostimulation (nitrate or sulfate) DGG‐B bioaugmentation

Treatability Testing Scope

123 4

5

6

7

89 10

Crushed core sample(60 g)

Incubate and monitor

Treatability Testing

Groundwater sample

*Aqueous BTEX concentrations 

ranged between 0.1 – 20 mg/L, 

depending on site materials

Mix

200 mL groundwater slurries50 mL headspace (10% CO2 / 90% N2)

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Treatability Test Results (Site #3, ON)

*

Intrinsic Bioremediation

NitrateBiostimulation

DGG‐B Bioaugmentation

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

12 109

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

12 109 179

0

5

10

0 100 200

0

5

10

0 100 200

0

5

10

0 100 200

1.0E+00

1.0E+03

1.0E+06

12 109 179

Gene copies/L

Days

Benzene (mg/L)

Total b

acteria

abcA

ORM2

* **

= below quantifiable limits

1.0E+03

1.0E+05

1.0E+07

1.0E+09

1.0E+11

1.0E+03

1.0E+05

1.0E+07

1.0E+09

1.0E+11

1.0E+03

1.0E+05

1.0E+07

1.0E+09

1.0E+11

Lessons Learned• Effective benzene degradation may require pre‐treatment of TEX

• Other (unknown) factors can decrease degradation efficiency of DGG‐B e.g., Other petroleum hydrocarbons, salinity, metals

0

1

2

3

4

0 100 200 300Days

Mixed BTEX + DGG‐B Site #6 + DGG‐B

Undiluted GW slurries

40% diluted with dH2O 

60%80%

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Indigenous Benzene Degraders Gas Station Site, SK

• Anaerobic benzene degraders (ORM2, GenPepto) and functional genes (abcA) detected in all wells

• Intrinsic benzene degraders comprise < 0.01% of total bacterial populations (GenBac).

Example Anaerobic Field Approach for Benzene

GW Flow Direction

Spill Site

BioaugmentationDGG‐B (ORM2)  

Use existing CO2 or added sulfate  

Source Zone benzene degradation under sulfate reducing/ methanogenicconditions    

SO4‐

CO2‐

Bioaugmentation PeptococcaeaeSodium Nitrate Biostimulation

NO3‐

CH4/CO2/H2SCO2/N2

Downgradient benzene degradation nitrate reducing conditions 

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Some Lessons Learned Anaerobic BTEX

• Treatability testing indicates NO3/SO4/CO2 are suitable electron acceptors

• Indigenous benzene degraders widely detected but at low proportions (<0.01%) and much lower than optimal abundance (107-108/L)

• Bioaugmentation possibly required even where indigenous benzene degraders

present (slow growth rates) -Application volumes may be higher than other cultures

• Benzene degradation in the presence of TEX compounds slower than benzene alone-may need to treat TEX first

Upcoming Work…

• Identification of enzymatic pathways for benzene fermentation in ORM2 =improved molecular tools for monitoring anaerobic benzene

• Environment Canada New Substances Notification Application

• Field applications of ORM2 benzene culture (2019) NJ, NC, SK

• Scale-up of existing TEX cultures to commercial volumes+ development of associated molecular tests

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Thank you for your Attention!Further Information

Phil Dennis ([email protected]) Sandra Dworatzek ([email protected])

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