Enhanced In Situ Biodegradation of
Trichloroethene/1,1,1 – Trichloroethane in Fractured
Rock Groundwater
Michael S. Kozar, PG
© 2011 O’Brien & Gere
Introduction
Michael Kozar, PG, LSRP | Vice President, O’Brien & Gere
22 years of professional experience
2
including 20 years with O’Brien & Gere
Provide client–focused solutions, technical consulting and
project management services throughout the U.S., focusing on
environmental site characterizations, soil and groundwater
investigations and subsurface remediation
Conducted environmental programs for industrial/federal sites
under CERCLA programs including several USEPA Regions and
various state regulatory programs
Managed complex environmental investigation and subsurface
remediation projects including in situ technologies, such as
enhanced in situ bioremediation for hydrocarbons and
chlorinated solvents in groundwater
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BS in Geological
Sciences from
Pennsylvania State
University
Registered
Professional
Geologist in
Pennsylvania
Licensed Site
Remediation
Professional in
New Jersey
Bioremediation Consulting Inc
bioremediation@bciLabs.com, www.bcilabs.com
Lab-Based ConsultingTM
Microcosm Experiments
Remediation Monitoring Analysis
Designer BacteriaTM Bioaugmentation Cultures
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Enhanced In Situ
Biodegradation (EISB*) of Chlorinated Organics
in Groundwater
*also commonly called Accelerated In Situ Bioremediation (AISB)
Optimizing groundwater redox conditions to sustain
the microorganisms that facilitate biodegradation
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Biodegradation Mechanisms
Reductive dechlorination
The sequential replacement of chlorine atoms on the alkene
molecule with hydrogen atoms
Trichloroethene (TCE)
1,1,1-Trichloroethane (TCA)
Aerobic cometabolism
The process by which certain VOCs (e.g., TCE or its daughter products)
are degraded by an enzyme or cofactor produced during microbial
metabolism of another compound
Cometabolism is limited to chlorinated solvents that have at least one
hydrogen atom attached to the carbon (i.e., PCE cannot be co-metabolized)
Direct oxidation
cis-1,2-dichloroethene (DCE) and vinyl chloride serve as
electron donors by bacteria
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PCE / TCE Degradation Pathways
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1,1,1 – TCA Degradation Pathways
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Groundwater Contaminants: EPA MCLs/RSLs
Contaminant
Drinking Water Level - µg/L (ppb)
1,1,1-Trichloroethane (TCA)
200
1,1-Dichloroethane (DCA)
2.4 1
21,000 1
Chloroethane (CA)
Tetrachloroethene (PCE)
5
Trichloroethene (TCE)
5
cis-1,2-Dichloroethene (DCE)
70
Vinyl Chloride (VC)
2
Ethene
NS
MCL – drinking water Maximum Contaminant Level
1 No specified MCL, value is EPA’s 2010 Regional Screening Level (RSL) for Tap Water
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Hydrogen Generation from Different Electron Donors
OH
CH3CHCOOH + H2O
(lactate)
HO
CH3COOH + CO2 + 2H2
(acetate)
OH
CH3CHCH2 + 2H2O
(propylene glycol)
CH3COOH + CO2 + 4H2
(acetate)
PCE / TCE / TCA: Electron Acceptors
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Dechlorination of Chlorinated Ethenes
Various anaerobes
PCE
TCE
DCE
Dehalococcoides ethenogenes (Dhc)
TCA to DCA to CA: Dehalobacter (Dhb)
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VC
Ethene
Microbial Community in Groundwater
Physiological Group
ORP mv
Aerobes
+ 800
Denitrifiers
+ 650
Mn Reducers
+ 500
Iron Reducers
+0
Fermentors
- 100
Sulfate Reducers
- 180
Acetogens
- 200
Dehalococcoides
- 200
Methanogens
- 300
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Anaerobic Microbial Community Supports Dhc Dechlorination
Nitrate Reducers NO3 + Organics
N2
Sulfate Reducers SO4 + Organics
SH
Lower ORP
Organic Acids
H2
Fermentors + Organics
Methanogens (Low ORP)
B12
D. Ethenogenes + DCE +B12 + H2 + Low ORP
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Ethene
Dhc
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Example Biological Reductive Dechlorination: Chloroethenes
Will accumulate if requisite bacteria like certain
strains of Dehalococcoides are absent
Anaerobic process that can be stimulated by injection of:
Nutrients
Alcohols, sugars, edible oils
biostimulation
Naturally occurring bacteria (e.g., Dehalococcoides culture)
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Or bioaugmentation
EISB Case Study
Superfund Site, Southeast Pennsylvania
USEPA Region 3
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Solvent Recycling Facility (Carbonate Valley, PA)
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Site Geology
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The Challenge
Aquifer Characteristics
Fractured dolomite setting
Depth to groundwater: ~ 90 ft
Source area aquifer thickness ~ 35 ft
Travel time ~ 2.7 ft/day (bromide tracer)
Groundwater pH < 6 (source area) to > 8 (downgradient groundwater)
~ 80,000 ppb VOCs
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The Challenge (2004 – 2005)
Jump start stalled
AISB field pilot
test to
dechlorinate TCE
and TCA, after two
prior
bioaugmentation
attempts and the
following
conditions:
Dechlorination stalled at DCE
DCE
67 ppm
VC
0.01 ppm
ethene
0.003 ppm
Dechlorination stalled at TCA
TCA
6 ppm
DCA
0.3 ppm
chloroethane
< 0.01 ppm
Toxic Donor Levels
methanol
15000 ppm
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ethanol
27000 ppm
organic acids
300 ppm
Bioaugmentation Inhibition Defined
Duhamel, M., S.D. Wehr, L.Yu, H. Rizvi, D. Seepersad, S. Dworatzek, E.E. Cox,
and E.A. Edwards. 2002. “Comparison of anaerobic dechlorinating
enrichment cultures maintained on tetrachloroethene, trichloroethene, cisdichloroethene, and vinyl chloride.” Wat. Res. 36:4193-4202
Co-Contaminant Inhibition
Dehalococcoides cultures reported to be inhibited by:
TCA > 0.7 ppm
Chloroform > 50 ppb slows dechlorination
Complete inhibition with chloroform > 300 ppb
No commercially available culture that degrades TCE and TCA
concurrently (2004 – 2005)
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Laboratory Approach
Objective: Culture a TCA tolerant TCE degrading consortium using Site
groundwater – capable of degrading methanol and ethanol and
TCE/TCA
Culturing Process - Bioremediation Consulting, Inc. (BCI)
Site ground water amended with ammonia, phosphate, and minor and
trace elements and vitamin B12
pH maintained at 6.9
Added Bacterial consortium was initially inhibited by methanol but
eventually degraded the methanol and ethanol
Sodium lactate was added as donor after the culture utilized organic acid
already present in groundwater
TCE, cDCE, VC, 1,1,1-TCA & 1,1-DCA were spiked in after these VOCs
were dechlorinated and methanol was degraded
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Laboratory Microcosms
Killed control
Live – native bacteria
donor: lactate
Live –
bioaugmented
donor: lactate
Headspace
Contaminated site
groundwater +
native bacteria
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Soil / Groundwater Microcosm Set-Up in Anaerobic Glove Box
Atmosphere:
96% N2
4% H2
Sieve and weigh soil
Add to microcosms
H2 + O2
+ catalyst
--> H2O
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Microcosm Results Summary
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Conducted with Site groundwater and TCE, cDCE, and TCA
concentrations much higher than in field
(residual source concentrations)
Bacteria culture added - contained 1,1,1-TCA-tolerant D. ethenogenes,
Dehalobacter, fermenters, acetogens, sulfate-reducing bacteria, and
methanogens
Complete degradation of TCE/cDCE and TCA (concurrently)
demonstrated using lactate and BCI culture
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Microcosm Study (2005)
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Culturing for AISB Pilot and Full-Scale
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Culture Delivery (“Kegs”)
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Bioaugmentation Culture
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AISB Pilot Testing (2006 – 2010)
Biostimulation/REDOX & Donor Management
AISB pilot test operated in a manual, daily batch delivery mode
Daily anoxic sodium lactate (Wilclear) injections in GW-7
ranged from ~ 3,000 to 5,000 mg/L
50 mg Vitamin B12 was added on daily basis
Amendments adjusted based on monthly redox, organic acid, hydrogen,
pH and nutrient groundwater results
Ammonia and phosphate additions to maintain 1 mg/L each
Later expanded injections to 2 additional wells
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AISB Pilot Test (“Phase 2”)
GW-7
Anoxic Batch Injection System
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AISB Pilot System (“Phase 3”)
GW-7
Direct Injection System
metering pump
sodium lactate
flow meter/valves
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AISB Full-Scale Design (2009 – 2010)
Pumping to control and facilitate distribution of donor / nutrients for
source treatment through AISB
USEPA issues remedy change document (August 2009) based on
laboratory/pilot results!
Accelerated in situ biological treatment (AISB) to degrade chlorinated
VOCs throughout the source zone (and enhance dissolution)
Groundwater extracted and amended with nutrients and re-injected in
the source area creating some groundwater recirculation / treatment
“cells”
Groundwater to be bioaugmented with TCE/TCA degrading culture
initially and as necessary
Natural Attenuation to address downgradient VOC plume
beyond AISB treatment zone
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AISB Injection and Extraction Wells
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Remediation Well Cross Section
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Installation of Injection / Extraction Wells
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Extraction Vault
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AISB Full-Scale: Year 1 Amendments (3/2010 – 2/2011)
3 to 5 gallons per minute (gpm) extraction/injection system
Donor: Sodium Lactate (60%)
Target concentration 825 mg/L
Injection rate 22 mL/min
Daily usage: ~6.5 gal/day
Usage in 1 year ~ 1500 gallons
Nutrients: Miracle Gro™ and Potassium Tri-poly Phosphate
(source of ammonia and phosphate)
Target concentrations: 1 mg/L
Injection rate 22 mL/min
Usage in 1 year: Miracle Grow ~ 525 gallons; KTPP ~ 330 gallons
Vitamin B12 ~ 260 grams
76L of bacteria culture
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AISB Target Concentrations
Target concentrations in extraction wells
100 ppm (mg/L) of total organic acids
1 ppm each of nitrogen and phosphate
Geochemical conditions for anaerobic dechlorination
pH ~ 6.9 S.U. (acceptable range: 6.3* – 8.5 S.U.)
ORP ~ -200 mV
DO ~ 0 ppm (<0.2 ppm)
* 2011: new low-pH-tolerant culture available (pH 5.5)
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AISB Full Scale Remediation
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Full-Scale Treatment Building
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Total Organic Acids (1 Year Full-Scale)
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Organic Acid Concentration
Table 1: Organic Acid Concentration in ppm
Tank 1
IW-1
IW-2
IW-3
IW-4
IW-5
Lactate
704
361
129
18
395
87
Acetate
76
322
267
202
132
193
Propionate
146
659
512
394
257
391
0
0
0
0
0
0
Butyrate
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Microbial Community in Groundwater
Physiological Group
ORP mv
Aerobes
+ 800
Denitrifiers
+ 650
Mn Reducers
+ 500
Iron Reducers
+0
Fermentors
- 100
Sulfate Reducers
- 180
Acetogens
- 200
Dehalococcoides
- 200
Methanogens
- 300
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Oxidation-Reduction Potential (ORP)
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PCE/TCE Reductive Dechlorination and Daughter Products molar and ppm / ppb concentrations
1 mM PCE → 1 mM TCE→
→ 1 mM DCE → 1 mM VC → 1 mM ethene
166 ppm PCE→
→131.5 ppm TCE→
→97 ppm DCE→
→62.5 ppm VC→
→28 ppm ethene
1 µM PCE → 1 µM TCE→
→ 1 µM DCE → 1 µM VC → 1 µM ethene
166 ppb PCE→
→131.5 ppb TCE→
→97 ppb DCE→
→62.5 ppb VC→
→28 ppb ethene
EXAMPLES:
100 µM TCE = 13,150 ppb
5,000 pbb total CVOCs = 38 µM TCE = 51 µM DCE = 79 µM VC
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PCE + TCE + DCE / VC + Ethene in Extraction & Monitoring Wells
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Ethene Production from PCE/TCE/DCE/VC 2006-2011
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AISB Groundwater Data –
GW-1 (Downgradient/Source Area Well) Chlorinated Ethenes
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AISB Groundwater Data –
GW-1 (Downgradient/Source Area Well) Chlorinated Ethenes
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AISB Groundwater Data –
GW-1 (Downgradient/Source Area Well) Chlorinated Ethanes
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Source Area Extraction Well – GW-10 Chlorinated Ethenes
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Source Area Extraction Well – GW-10 Chlorinated Ethanes
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Downgradient Monitoring Well GW-17 - Chlorinated Ethenes
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Upcoming Webinar
REMEDIATION ROUNDTABLE
Wednesday June 8th | 2pm
webinars@bnpmedia.com
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Microcosm Test
MW-11 plus Donor. No N&P
90
TCE
Effect of N and P
60
µM
30
PCE
IESI Site
0
0
30
60
Donor, no N&P no dechlorination
days
90
120
150
MW-11 with Donor and N&P
Donor and N&P PCE -> ETHENE
DCE
90
VC
60
µM
TCE
DCE
30
VC
PCE
ETHENE
0
0
30
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60
days
90
120
150
Requirement for B-12
Microcosm: GW7 Well Sample During
Remediation. pH 6.6, donor present,
add N & P
Microcosm: GW7 Well Sample During
Remediation. pH 6.6, donor present,
add N & P & B12
150
150
VC
DCE
100
100
VC
µM
µM
DCE
50
50
0
0
0
20
40
60
0
20
40
days
days
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AISB Groundwater Data – GW-7(Source Area Well) Chlorinated Ethenes
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Source Area / Downgradient Extraction Well – GW-18 Chlorinated Ethenes
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Downgradient Extraction Well – GW-12A Chlorinated Ethenes
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AISB Groundwater Data – GW-12A pH vs GWE
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Bioaugmentation Inhibition Defined
Low pH Microbial Inhibition – acid production processes
lactic acid degradation results in acid production
H+ + lactate ---> acetate - 2H+ + 2 H2 + HCO3Methanol and ethanol degradation result in acid production
2 methanol ---> acetate- + H+ + 2 H2
ethanol + H2O ---> acetate - + H+ + 2 H2
Methanogens can remove CO2 and acetate
CO2 + 4 H2 ---> CH4 + 2 H2O
acetate- + H+ ---> CH4 + CO2
methanogens are inhibited by high DCE
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Bioremediation Produces Acid
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Effect of pH…
…on dechlorination and methanogenesis in
groundwater microcosms Site M
Three microcosms, Three different pH
(1) pH 6.9 (2) pH 6.6 (3) pH 6.3
Ground water contains TCE, TCA, methanol
BCI culture contains
Dehalococcoides
Dehalobacter
Methanol utilizers and methanogens
Analyzed by removing 100 uL headspace samples through septa and
injecting into gas chromatograph
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Three Diagnostic Microcosms for site A
#1
#2
#3
Conclusions from microcosm #3:
Adjusting pH and amendments restored bioremediation in 60 days
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BCI Designer Bacteria™ Low-pH-Tolerant Cultures
BCI Culture # 1
TCE to Ethene
BCI Culture # 2 PCE to
Ethene and TCA to CA
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BCI’s Solution to Low pH Problems
2009
• Initiated work on low-pH-tolerant Dhc
with a grant from the National
Science Foundation
2010
• Developed 2 low-pH-tolerant
Dhc cultures
2011
• Started commercial production
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CC-7 Aerobic
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