in bioremediation of · bioremediation of benzene sandra dworatzek, jeff roberts and jennifer webb...
TRANSCRIPT
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
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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
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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])
siremlab.com1-866-251-1747