Post on 16-Nov-2018
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Nucleic Acid-Based Tools for Monitoring
Bioremediation at Chlorinated Solvent Sites
Erik A. Petrovskis, Ph.D., P.E.
Geosyntec Consultants
Environment, Energy and SustainabilityDenver, May 2009
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4. TITLE AND SUBTITLE Nucleic Acid-Based Tools for Monitoring Bioremediation at ChlorinatedSolvent Sites
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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18
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What’s In It For Me?
• Learn the science behind MBTs
• Learn when to use MBTs
• Learn how to sample groundwater for MBTs
• Learn the “Rules of Thumb” for MBT data
• Learn how to save time and money with a smarter
bioremediation design and operation– do I
bioaugment?
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Types of MBTs
• Nucleic acid probes (DNA or RNA)
Various targets including
• 16S rRNA gene
• Functional genes (e.g., RDase, Hydrogenase, Oxygenase, etc.)
• Protein biomarkers
• Lipid biomarkers
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Purpose of MBTs
• Reduce remediation costs and increase effectiveness by
Supporting sites where MNA is being evaluated
Predicting sites where biostimulation will succeed
Identifying sites where bioaugmentation is required
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Chlorinated Ethene
Reductive Dechlorination
Desulfuromonas michiganensis
Sulfurospirillum, Desulfitobacterium,
Dehalobacter, Geobacter
Desulfitobacteriumsp. strain Viet1
Desulfitobacterium sp.strain PCE1
PCE TCE cis-DCE VC Ethene
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Dehalococcoides (Dhc) Involved
in Reductive Dechlorination
Dehalococcoides sp. strain BAV1
Dehalococcoides sp. strain VS
Dehalococcoides sp. strain GT
Müller et al., 2004, AEM, 70:4880
Sung et al., 2006, AEM, 72:1980
Dehalococcoides ethenogenes strain 195
Dehalococcoides sp. strain FL2
Maymó-Gatell et al. 1997. Science 276:1568
He et al. 2005. Environ. Microbiol. 7:1442
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16S rRNA Gene Targets for Dhc
• 16S rRNA found in all bacteria
• rRNA part of the ribosome; critical for protein
biosynthesis
• Contains variable regions which allows for the
differentiation of bacterial species
• Dhc has one 16S rRNA gene per cell
• Dhc 16S rRNA gene count = number of Dhc cells
The 16S rRNA molecule has insufficient
information to infer physiological traits
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Dhc Reductive Dehalogenases
tceA
vcrA
bvcADehalococcoides ethenogenes strain 195
Dehalococcoides sp. strain FL2
Dehalococcoides sp. strain BAV1
Dehalococcoides sp. strain VS
Dehalococcoides sp. strain GT
Dehalococcoides sp. strain KB-1/VS
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qPCR Sensitivity:
Detection vs. Quantification
Extract Community DNA
Amplification with universal
16S rRNA gene-targeted
primers (for nested PCR)
Dechlorinator
targeted primers
Dhc
Dsf
DhbSensitive detection
of dechlorinating bacteria
(~101 copies/L)
qPCR
Genomic
DNA
Sensitive quantification
of dechlorinating bacteria
(~103 copies/L)
PCR
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Dhc Rules of Thumb in the Field
Dhc 16S rRNA gene
copies per LInterpretation
103 or lower Suboptimal Dhc to sustain
dechlorination rates
104 – 106 May sustain appreciable
dechlorination rates
107 or greater
Often associated with high rates of
dechlorination and ethene
production
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MBT Sampling
• Groundwater sampling preferred
Difficulties with repetitive soil sampling
Spatial variability in soil
• Sampling method influences results
• Use SOP that complements VOC sampling methods
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Groundwater Sampling
It is not practical to
perform vacuum
filtration in the field
Shipping groundwater samples is problematic
•Heavy, costly
•Leakage/breakage
• Biomarker integrity
• Groundwater disposal
Improved procedure: Filtration in the field
•Economical
•No leakage/breakage
• Biomarker stability?
• GW remains on-site
DNA
RNA
vs.
DNA
RNA
ESTCP Project ER-0518:
Sterivex™ filters
are a viable alternative!
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13
MW-7
• Sampling biases
• Ratio of planktonic
vs. attached cells
RNA
DNA
Results
• Biomarker stability?
• Efficiency of each step?
• Method biases?
• No standards!
• No guidelines for
results interpretation
Sampling Considerations
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Groundwater Sampling
SOP (ER-0518)• Low-flow purge
Wait for stabilization of geochemical parameters to obtain a sample representative of formation groundwater
• Surging Increases particulate matter in sample for recovery of associated (i.e.,
attached) biomass
• Field filtration Sterivex™ filters for biomass collection in the field
• Economical, no leakage/breakage, groundwater remains onsite
• Shipping Secure samples for overnight shipment to laboratory
Maintain samples at 4°C
Sampling protocol should be defined and maintained for the duration of the monitoring effort for a particular site
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Sampling Locations
• Key sampling locations
should include
Source area(s)
Downgradient plume
locations where biodeg
products observed
Vertical stratification
• Where possible, use discrete
sampling zones and avoid
sampling wells with extended
screen intervals
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Sampling Frequency
• Seasonal variability Geochemical conditions and biomarker abundance can be
affected by seasonal changes (e.g., rain events, temperature changes, etc.)…be aware!
• Bioremediation field implementation Baseline and 1-2 months after injections
Quarterly in first 12-18 months
Collect with VOC, geochemical and TOC data
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Cost
• Field labor
Biomass collection can be performed concurrently with
sampling events planned for assessment of contaminants
Minimal additional time is needed for collection of samples
for biomarker analysis
• Laboratory
Microbiology labs specializing in biomarker analysis are
typically independent from chemical laboratories used for
other analyses
Typical cost for quantification of Dehalococcoides in a
sample of groundwater is approximately $250
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Cost for Not Using MBTs
• Unnecessary bench tests
• Poorly designed pilot tests
• Inefficient full-scale treatment
Application of bioaugmentation and/or biostimulation when
MNA would be appropriate
Bioaugmentation when sufficient Dhc are present to meet
remediation goals
Failure to bioaugment when Dhc populations are insufficient
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Case Study – NASA MLP/VAB Site
• TCE Source Area (~4,000 gal release 1960’s)
• Biostimulation Ethyl lactate
• Performance monitoring Every other month
TCE, cDCE, VC, ethene
Dhc and vcrA
~6 data points from each well
Injection Well
Monitoring Well30,000 µg/L TCE
3,000 µg/L TCE
1,000 µg/L TCE
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Groundwater VOCs
and Dhc: SAMW-02
CV
OC
Con
centr
ation
(µg/L
)
Co
nce
ntr
atio
n (
µg
/L)
Dh
c (g
ene
cop
ies/
L)
TCE cDCE VC EthenetDCE DHC
Dhc data
indicated no
need to
bioaugment!
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Groundwater DHGs
and Sulfate: SAMW-02D
isso
lved
Gas
es (
µg
/L)
Su
lfat
e (m
g/L
)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
7/2/20
06
8/21/2
006
10/10
/2006
11/29
/2006
1/18/2
007
3/9/20
07
4/28/2
007
6/17/2
007
8/6/20
07
9/25/2
007
11/14
/2007
0
100
200
300
400
500
600
Ethane MethaneEthene Lactate injectionSulfate
Sulfate did not
inhibit
reductive
dechlorination!
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Correlation of Dhc/vcrA
to VOCs and Ethene
• Correlation results:
– Dhc or vcrA to TCE, DCE or VC
• Weak correlation (rs < 0.33) for all comparisons
– Dhc or vcrA to ethene
• Dhc to ethene = strong correlation (rs = 0.66; n = 10; p = 0.05)
• vcrA to ethene = strong correlation (rs = 0.67; n = 10; p = 0.05)
Spearman Test
TCE
DCE
Ethene
VCDhc vcrA
Ethene
VC
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Correlation of Dhc/vcrA
to Dechlorination Rates
Spearman Test
TCE rate (yr -1)
DCE rate (yr -1)
VC rate (yr -1)
Dhc vcrADCE rate (yr -1)
VC rate (yr -1)
• Correlation results:
– Strong correlations (rs = 1.00) for all comparisons to Dhc
– Medium correlations (rs = 0.50) for all comparisons to vcrA
• Limited validity to results (only three data points)
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Case Study – Milledgeville
• TCE Source (18,000 ppb)
• Bioaugmentation Pilot
Testing
3 injection wells and 2
recovery wells oriented
perpendicular to the
prevailing direction of
groundwater flow
(southwest)
Soluble electron donor
(lactate) and dechlorinating
culture distributed by recirc
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1.0E-04
1.0E-03
1.0E-02
1.0E-01
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
1.0E+11
0
5000
10000
15000
20000
15-Mar-00 1-Oct-00 19-Apr-01 5-Nov-01 24-May-02 10-Dec-02 28-Jun-03 14-Jan-04
ge
ne c
op
ies p
er
L
µg
pe
r L
ch
loro
eth
en
e
date
Dehalococcoides and chloroethenes
PCE
TCE
cis-DCE
VC
Ethene
Methane
16S rRNA
Dhc
VOC and Dhc Data
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Correlation of Dhc
or RDases to VOCs
• Dhc or RDases to VOCs
No correlations to Dhc
Strong correlation of bvcA (rs = 0, n = 10, p = 0.02)
Strong correlation of vcrA to cDCE (rs = -0.80, n = 10, p =
0.01)
Strong correlation of tceA to VC (rs = 0.76, n = 10, p = 0.02)
No other correlations identified
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Survey: Correlation of Dhc/vcrA to Ethene
0%
20%
40%
60%
80%
100%
Dhc Tests % with Ethene
vcrA Tests % With Ethene
ND-2x105 2x105-107 > 107 >1.8 x108
Gene copies/ L For vcrA testing:
- below 2 x105/L ethene not normally detected
- above 1 x107/L ethene commonly detected
- above 1.8 x108/L ethene always detected
Courtesy: SiREM
N= 121 Samples vcrA
N=244 samples Dhc
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Biostimulation/Bioaugmentation
Flowchart
Add donor and microbes
Add donor only
Geosyntec t> consultants
Wiii MBT analysis support biostimulation or
bioaugmentation?
No
Are geochemica
conditions favorable?
No egradation Biostimulation 1+--------< desired in < 6
months?
Yes
MBTs
voc Yes concentrations
> 100 IJQ/L?
Dhc >
1 04/L - 1 05/L in
groundwater?
No
Bioaugmentation
No
iodegradation products?
~~ Sample for MBTs
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Reminder-Dhc Rules of Thumb
Dhc 16S rRNA gene
copies per LInterpretation
103 or lower Suboptimal Dhc to sustain
dechlorination rates
104 – 106 May sustain appreciable
dechlorination rates
107 or greater
Often associated with high rates of
dechlorination and ethene
production
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Summary
• MBTs are valuable tools to monitor biodegradation of
chlorinated ethenes
• SOPs are available for MBT sampling. Field filtration is
reliable.
• Biomarker genes (bvcA, vcrA) are indicators of field
dechlorination activity
• Rules of thumb and draft guidance are available
• Understand limitations of the data