Bio-traps and Site Assessment Strategies for Groundwater Impacted by Chlorinated

Hydrocarbons

Kerry SubletteUniversity of Tulsa

Dora Taggart, Brett Baldwin, Anita Biernacki, Kate Clark

Microbial Insights, Inc

Passive sampling tool for microbes

Collects active microbes

Integrated sample vs. “snapshot”

Analyzed using molecular biological tools, analytical chemistry, and stable isotope analysis

What Are Bio-Trap® Samplers?

Bio-Sep

How Do Bio-Traps Work?

– 3-4 mm in diameter

– 25% Nomex and 75% PAC

– 74% porosity

– 600 m2 of surface area/g

– Heat sterilized 270 oC

– Colonized by activemicrobes

Properties of Bio-Sep Beads

Standard Bio-Trap– Basic design

– Sampling groundwater, surface

waters, sediments, soils

– Compatible with all MBTs,

analytical chemistry, and stable

isotope techniques

Types of Bio-Trap Samplers 250 Bio-Sep beads

Unit Samplers

GEO

COC

MICRO(Bio-Trap)

Supplier

Supplier

In Situ Microcosm

Unit SamplersAssembly

Control(MNA)

TreatmentOption

1

TreatmentOption

2

GEO

COC

MICRO(Bio-Trap)

Supplier

Supplier

Unit SamplersAssembly

Control(MNA)

TreatmentOption

1

TreatmentOption

2

GEO

COC

MICRO(Bio-Trap)

Supplier

Supplier

Amendments Include:

Vegetable oil Molasses HRC EOS Lactate And more

Benzene Toluene p-Xylene MTBE TBA Naphthalene Chlorobenzene 1,4-Dioxane Sulfolane And more

Stable Isotope Compounds (13C)Electron Donors

Electron Acceptors Oxygen (PermeOx, ORC) Nitrate Iron (III) Sulfate And more

Purge monitoring well

Suspend from top of casing

Deploy within the screened interval at depths of interest.

If large fluctuations in the water level are anticipated suspended from a float.

How Are Bio-Trap Samplers Deployed?

Monitoring Well

Screened Interval

Molecular Biological Tools– PLFA– CENSUS (qPCR)– QuantArrays– DGGE– Stable Isotope Probing (SIP)

Chemical Analysis– Compound specific isotope analysis

(CSIA)– Dissolved Inorganic Carbon (DIC)– Contaminant Concentrations

How Are Bio-Trap Samplers Analyzed:

• Determine if known degraders of a COC are present

• Evaluate monitored natural attenuation versus enhanced bioremediation

• Compare effectiveness of amendments designed to stimulate bioremediation

• Prove that bioremediation of a specific compound is occurring

What Can I Do With a Bio-Trap Sampler?

• Determine if known degraders of a COC are present

• Evaluate monitored natural attenuation versus enhanced bioremediation

• Compare effectiveness of amendments designed to stimulate bioremediation

• Prove that bioremediation of a specific compound is occurring

What Can I Do With a Bio-Trap Sampler?

Microbial Insights Database - Dehalococcoides

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Log cells/bead

0%

1%

2%

3%

4%

5%

6%

7%

8%

9% Dehalococcoidesspp. have no known

mode of motility

Brownian motion

Determine If Known Degraders Are Present

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

MW-14 MW-16

Cells

/bea

d

Dehalococcoides spp. Vinyl Chloride Reductase (bvcA)

CENSUS® - Dehalococcoides populations indicate the potential for complete reductive dechlorination of PCE to ethene but

stimulation needed

• Determine if known degraders of a COC are present

• Evaluate monitored natural attenuation versus enhanced bioremediation

• Compare effectiveness of amendments designed to stimulate bioremediation

• Prove that bioremediation of a specific compound is occurring

What can I do with a Bio-Trap Sampler?

Unit Samplers

GEO

COC

MICRO(Bio-Trap)

Supplier

Supplier

In Situ Microcosm

Samplers

Supplier

Analysis Lines of Evidence

COC VOCs • Contaminant concentrations• Daughter product formation

MICRO(Bio-Trap)

CENSUS® • Quantify specific microbialpopulations and processes

SIP

• Prove biodegradation• Compare relative rates• Compare degree of

contaminant incorporation

GEO AnionsDissolved Gases

• Redox conditions

• Ethene & Ethane production

Screening Remediation Options: Chlorinated hydrocarbon impacted site

Control(MNA)

Control Unit• Evaluate MNA as treatment alternative• Baseline for enhanced remediation options

BioStim(Electron Donor)

BioAug(Electron donor + culture)

BioStim Unit – Electron Donor Addition• Enhanced anaerobic bioremediation• Lactate, HRC, EOS

BioAug Unit – Culture and Electron Donor• Bioaugmentation (culture impregnated in beads in the

bio-trap)

Case Study: Amended ISMs

Chlorinated Solvent Site

• Shallow aquifer impacted by chlorinated solvents, primarily trichloroethene (TCE).

• Daughter product cis-1,2 dichloroethene (DCE) has been detected.

• DCE appears to be accumulating with no observed production of vinyl chloride or ethene (“DCE stall”).

• Biostimulation (electron donor addition) and bioaugmentation (donor and culture) were being considered as remediation strategies.

Site Background

Site Specific Questions

Are organisms capable of complete reductive dechlorinationof TCE to ethene (Dehalococcoides) present under MNA

conditions?

Will addition of an electron donor stimulate growth of these key dechlorinating bacteria?

Is bioaugmentation necessary?Will a bioaugmentation culture survive?

Microbiology

Site Specific Questions

Chemistry

Will electron donor addition promote daughter product formation and stimulate complete reductive dechlorination?

Will bioaugmentation + biostimulation more effectively stimulate reductive dechlorination than biostimulation

alone?

Study DesignUnit

Control(MNA)

BioStim

Assembly Samplers Potential Lines of Evidence

BioAug

Supplier

VOCs: Differences in TCE and daughter product concentrationsCOC

CENSUS: Dehalococcoides and vinyl chloride reductase genes

MICRO(Bio-Trap)

Supplier

GEO Dissolved Gases: Ethene production

Control (MNA) Unit – CENSUS® qPCR Results

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

Cells

/bea

d

Dehalococcoides spp. tceA Reductase vcrA Reductase

MNA

MNA vs BioStim

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

Cells

/bd

Dehalococcoides spp. tceA Reductase vcrA Reductase

Order of magnitude increase in DHC concentration

MNA BioStim

MNA vs BioStim vs BioAug

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

Cells

/bd

Dehalococcoides spp. tceA Reductase vcrA Reductase

MNA BioStim BioAug

Control vs. BioStim – Impact on COCsMNA BioStim

0.0

0.2

0.4

0.6

0.8

1.0

Mol

e Fr

actio

n

TCE 1,2 DCE Vinyl Chloride Ethene

Enhanced reductive dechlorination of TCE to

cis-DCE

Reductive dechlorination of cis-DCE to vinyl chloride and

ethene

BioStim vs. BioAug – Impact on COCsMNA BioStim

0.0

0.2

0.4

0.6

0.8

1.0

Mol

e Fr

actio

n

TCE 1,2 DCE Vinyl Chloride Ethene

BioAug

• Standard bio-traps can measure degradation potential by quantifying Dehalococcoides and functional genes characteristic of reductive dechlorination (pre- or post-injection)

• Bio-traps coupled with in situ microcosms can compare effectiveness of amendments designed to stimulate bioremediation

Bio-traps and Chlorinated Hydrocarbon Impacted Sites

For a copy of this presentation email kerry-sublette@utulsa.edu

For more information go to microbe.com