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