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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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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18


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?


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


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


Chlorinated Ethene

Reductive Dechlorination

Desulfuromonas michiganensis

Sulfurospirillum, Desulfitobacterium,

Dehalobacter, Geobacter

Desulfitobacteriumsp. strain Viet1

Desulfitobacterium sp.strain PCE1

PCE TCE cis-DCE VC Ethene


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


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


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


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


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


107 or greater

Often associated with high rates of

dechlorination and ethene

production


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


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!


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


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


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


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


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


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


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


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!


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!


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


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)


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


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


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


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


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


Reminder-Dhc Rules of Thumb

Dhc 16S rRNA gene

copies per LInterpretation

103 or lower Suboptimal Dhc to sustain


104 – 106 May sustain appreciable


107 or greater

Often associated with high rates of

dechlorination and ethene

production


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


Acknowledgments

Geosyntec Dr. Rebecca Daprato

Dr. Wayne Amber

Georgia Tech Dr. Frank Löffler

Dr. Kirsti Ritalahti

Tetra Tech Keith Henn

NAVFAC Carmen Lebrón

Support provided by ESTCP project ER-0518 and NAVFAC SE