Single-Well Tests for Evaluating the Bioremediation of Chlorinated
Solvents in the SubsurfaceLewis Semprini
School of Chemical, Biological and Environmental Engineering
Oregon State University
Subsurface Chlorinated Solvent Contamination
Probability of Detecting VOCs in Ambient Untreated Groundwater Squillace et al., Environ. Sci. Technol. 1999
Butane plus Peroxide Butane plus Peroxide0
Meters
Monitoring Wells
Vadose ZoneTreatment Well
BioactiveZone
Pump
Aquitard
Lower Aquifer
10 Meters
4
8
12
16
20
24
Bioaugmentation to Recirculation Well SystemMcCarty et al. ES&T 1998)
Rationale
Information on contaminant transformations inthe subsurface is typically obtained through acombination of long-term groundwater sampling,laboratory microcosm studies, and numericalmodeling.
This approach is expensive and generallyprovides only qualitative information ontransformation rates.
New technologies are needed for detecting andquantifying contaminant transformations in situ.
Push-Pull Test ProtocolsTo meet this need we have developed asuite of single-well, push-pull test fieldassays for evaluating anaerobic andaerobic transformations of chlorinatedsolvents:
•Substrate Utilization Potential
•Anaerobic Transformation Potential
•Aerobic Transformation Potential
Single-Well, “Push-Pull” Test
During the injection phase, a prepared test solution
containing tracer, substrates, and/or contaminant surrogates
is injected into the saturated zone using an existing
monitoring well
During the extraction phase, samples are collected
to develop breakthrough curves for tracer, substrate, and/or surrogates and their
transformation products
Carboy 1
Carboy 2
Peristalticpump
Injection/extraction
well
Field Equipment is Simple and Inexpensive
TCE cis-DCE Vinyl Chloride (VC)
Ethene
C=CHCl
Cl ClC=C
HH
Cl Cl
H
ClCH2 =C CH2 = CH2
H2 HCl H2 HCl H2 HCl
§ TCE/ cis-DCE/ VC serve as electron acceptors§ Carbon and Energy Source (electron donor) required to
support the reductive dechlorination reaction.§ Fermentation of organic compounds produce H2 which serves
as the electron donor (Gossett et al., 1997)
Anaerobic Reductive Dechlorination of TCEAnaerobic Reductive Dechlorination of TCE
Substrate Transformation Potential
• Can indigenous organisms utilize exogenous substrates ?
• What is the initial rate of substrate utilization ?
• Can substrate utilization be stimulated by repeated substrate additions ?
• Are diagnostic substrate transformation products formed ?
Tests have been successfully conducted to answer the following questions:
Example 1
• Inject water containing tracer and lactate
• Collect groundwater samples and analyze forinjected tracer and lactate and fermentationproducts formed in situ
• Adjust measured concentrations for dilutionusing tracer concentrations and computetransformation rates
Question: Can indigenous microorganisms ferment lactate ?
Test Design:
Field Results: Lactate Fermentation
Time (days)0 10 20 30 40 50 60 70
Con
cent
ratio
n (m
g/L
)
0
20
40
60
80
100
120
LactateAcetatePropionate
10 mg/L•day
Fumarate Reduction
• Fumarate is a non-toxic organic acid that isexpected to undergo reduction to succinate underredox conditions similar to those under which TCEis reduced.
• Laboratory and field studies suggest fumaratereduction is correlated with TCE dechlorination
C C CC-O O-
O
H H
O
C C CC-O O-
O
H H
OH H
Fumarate Succinate
+ 2 e- + 2 H+
Example 2
1. Inject water containing tracer and fumarate
2. Collect groundwater samples and analyze forinjected tracer and fumarate, and succinateformed in situ
3. Adjust concentrations for dilution usingtracer concentrations and computetransformation rates
Question: Are redox conditions favorable forreductive dechlorination of TCE ?
Test Design:
Time (days)0 5 10 15 20 25 30
(mM
)
0.0
0.2
0.4
0.6
0.8
1.0
Field Results: Fumarate Reduction
TCE 1.5 2500cis-
DCE3800 ND
trans-DCE
23 ND
CE 327 NDethen
e35 ND
Fumarate
Succinate
Time (days)0 5 10 15 20 25 30
(mM
)
0.0
0.2
0.4
0.6
0.8
1.0
Fumarate
Reductive dechlorinationlikely rapid in this well
Reductive dechlorinationlikely slow in this well
(µM) (µM)
Anaerobic Transformation Potential
is Investigated Using Contaminant Surrogates• Trichlorofluoroethene
(TCFE) is reductivelydechlorinated by a pathwayanalogous to that of TCE
• In laboratory microcosmexperiments and field tests,TCFE and TCE aredechlorinated at similar rates
• In field tracer tests TCFE andTCE are transportedsimilarly
TCE
C CHH
Cl Cl
C CCl
HClCl
cis -DCE
C CH
H HCl
C CHH
H H
CE
Ethene
C CFH
Cl Cl
cis-DCFE
C CF
H HCl
C CFH
H H
(Z)-CFE
FE
TCFEC C
Cl
FClCl
PCE and TCFE Transformation in a Batch Reactor by the Evanite Enrichment.
A
0
4
8
12
0 50 100 150 200
Time (days)
Mas
s ( µ
mol
es )
PCE TCE c-DCE VC Ethene
B
0
4
8
12
0 50 100 150 200
Time (days)
Mas
s ( µ
mol
es )
TCFE c-DCFE 1,1-CFE FE
Pon
& S
empr
ini,
ES&
T 20
04
Example 3
• Measure initial rates of TCFE reductivedechlorination by injecting tap water, tracer, andTCFE
• Repeated injections of fumarate
• Measure post-fumarate addition rates of TCFEreductive dechlorination
Question: Is it possible to detect and stimulate reductive dechlorination in a TCE-contaminated aquifer ?Approach:
Time (days)0 10 20 30 40 50 60 70 80
TCFE
etc
. (µΜ
)
05
101520253035
Well 21C
Field Results:Initial Absence of Dechlorination in C-
Zone
TCFE
cis-DCFE
Time (days)0 1 2 3 4 5 6 7 8 9 10
Fum
arat
e, S
ucci
nate
(mΜ
)
0.0
0.2
0.4
0.6
0.8
1.0Well 21C
Field Results: Favorable Conditions Created by Repeated Fumarate Addition
Fumarate –1st fumarate
addition
5th
5th
Succinate
trans-DCFE TCFE cis-DCFETime (days)0 10 20 30 40 50 60 70 80
TCFE
etc
. (µΜ
)
05
1015202530
Well 21C
1.4 µM/day (16-30 days)
Field Results:Detection of Reductive Dechlorination in C-Zone Following Fumarate Additions
cis-DCFETCFE
TCE TCE epoxide
Cl
Cl
C
H
Cl
C
O2
Cl-CO2+
Cl
Cl
C
H
Cl
C
O
CO2Organic Growth Substrate(Ex: propane, butane)
Intermediate Products
Mono-oxygenase enzyme
Metabolism
Cometabolism
Aerobic Cometabolism of TCEAerobic Cometabolism of TCEDan Arp, Circa 2000Dan Arp, Circa 2000
Example 4
• Inject water containing tracer, propane, oxygen, and nitrate
• Collect groundwater samples and analyze for tracer, propane, oxygen, and nitrate
• Adjust measured concentrations for dilution using tracer concentrations and compute utilization rates
• Repeated substrate addition if necessary todemonstrate stimulate of propane oxidizers
Question: Is it possible to detect the presence ofpropane oxidizing bacteria in a TCE-contaminated aquifer ?
Test Design:
Field Results: Detection of Propane Oxidizers
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50 60 70 80Time (day)
DO
and
Bro
mid
e (m
g/L)
0
2
4
6
8
10
12
14
Prop
ane
and
Nitr
ate
(mg/
L)
BromideDOPropaneNitrate (as N)
1st Biostimulation 2nd 3rd 4th 5th
TCE, Ethylene and Isobutene Transformation by Toluene-Utilizing Microorganisms Expressing a Monooxygenase Enzyme
Monooxygenase O2 H2O NADH NAD+
Monooxygenase O2 H2O NADH NAD+
Monooxygenase O2 H2O NADH NAD+
Cl Cl C = C Cl H
Cl O Cl C C Cl H TCE Epoxide
H H C = C H H
H O H C C H H
H CH3 C = C H CH3
H O CH3 C C H CH3
TCE
Ethylene
Isobutene Oxide
Ethylene Oxide
Isobutene
Example 5
• Inject water containing tracer, toluene, oxygen, and isobutene, trans-DCE
• Collect groundwater samples and analyze for tracer, toluene, o-cresol, isobutene, isobutene oxide, cis-DCE, and trans-DCE
• Adjust measured concentrations for dilution using tracer concentrations and compute transformation rates
Question: Is it possible to measure rates of cometabolismby toluene oxidizing bacteria in a TCE-contaminated aquifer?
Test Design:
Push-pull Field Test Set-up at Fort Lewis, WA
Carboy 1
Carboy 2Pump 1
Pump 3
CollapsibleTeflon Bag
Pump 2
Isobutene
Push-Pull Toluene Activity Test
0.0
2.0
4.0
6.0
8.0
10.0
0.0 0.5 1.0 1.5 2.0 2.5Time (hours)
Tolu
ene
(mg/
L)
0
5
10
15
20
25
30
TolueneO-Cresol
o-C
reso
l (ug
/L)
LC192-P1: Rest Phase 25 hrs
Isobutene Transformation to Isobutene Oxide in a Push-Pull Activity Test
0
10
20
30
40
50
0.0 0.5 1.0 1.5 2.0 2.5 3.0Time (hours)
0.0
1.0
2.0
3.0
4.0
5.0
IsobuteneIsobutene Oxide
LC192-P1: Isobutene Activity Test Rest Phase: 22.5 hrs
Isob
uten
e O
xide
Pro
duce
d (µ
M)
Isob
uten
e Ex
trac
ted(
µM)
Natural-Gradient Activity TestEstimated Zero-Order Rates of Transformation
0
1
2
3
4
0 10 20 30 40 50 60Time (hours)
cis-DCEtrans-DCE
LC192-P1: Natural-Gradient Activity Test
Dilu
tion-
Adju
sted
(uM
)
Summary• Push-pull tests can detect and quantify rates of
microbial activity in situ:– Lactate fermentation– Fumarate reduction– Propane oxidation – Reductive dechlorination (trichlorofluoroethene surrogate)– Aerobic cometabolism of cis-DCE, trans-DCE, TCE by
toluene utilizing microorganisms, using isobutene as a surrogate, and 1-butyne as an inhibitor.
• Push-pull tests can monitor enhanced bioremediation and bioaugmentation
Other Push-Pull Test Technologies
http://web.engr.oregonstate.edu/~istokj/grl-manuscripts.htm• Anaerobic Transformations of BTEX• Aerobic Transformations of PAHs• Rates of Denitrification • Anaerobic Transformations of Radionuclides• Colloid Transport• Dispersivity and Groundwater Velocity• Mass Transfer • Sorption/Cation Exchange• Partitioning Tracers
Acknowledgements
Dr. Kim Hageman, Dr. Young Kim,
Dr. Mohammad Azizian, Dr. Jack Istok, and
Dr. Jennifer Field
Environmental Security Technology Certification Program (ESTCP)
National Institute of Environmental Health Sciences (NIEHS)
Western Region Hazardous Substance Research Center
Chevron Environmental Management Company
Textron