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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
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