microbiological tools for contaminated site monitoring and remediation

Incorpora(ng Molecular Biological Tools (MBTs) into Site Management

Why do we need MBTs?

Contaminant concentra,ons and geochemistry don’t always provide the complete picture. Plate counts do not accurately reflect in situ microbial community

< 1 % of bacteria can be cultured in the laboratory

Ques(ons that MBTs can answer

What is the concentra,on of contaminant degraders?

qPCR

QuantArray

Is biodegrada,on occurring?

Stable Isotope Probing (SIP)

Compound Specific Isotope

Analysis (CSIA)

What microorganisms are present?

Next Genera,on Sequencing

(metagenomics)

What treatment strategy should be selected?

In Situ Microcosms

(ISMs)

CENSUS® qPCR and QuantArray® What is the concentra(on of contaminant degraders?

•  qPCR Amplifica,on –  Primers & probe bind to target gene –  Fluorescence signal increase

propor,onal to concentra,on

•  Two main types of target genes –  Taxonomic (16S rRNA gene) –  Func,onal (Reductases, oxygenases)

qPCR Basics Rapidly detect and quan,fy a target gene or microbial popula,on

CENSUS qPCR Approach

DNA Extrac(on

Sample Collec(on

TOD

PHE

BSS

QuantArray®

DNA Extrac(on

Sample Collec(on

SubArray Amplifica(on

TOD

RMO

BSS

ABC

NAH

ANC

MNSS

QuantArray®-‐Petro Aerobic BTEX and MTBE (cells/mL) Toluene 3-‐ and 4-‐Monooxygenases (RMO) Toluene 2 Monooxygenase (RDEG) Phenol Hydroxylase (PHE) Toluene/Benzene Dioxygenase (TOD) Xylene/Toluene Monooxygenase (TOL) Ethylbenzene/Isopropylbenzene Dioxygenase (EDO) Biphenyl/Isopropylbenzene Dioxygenase (BPH4) Methylibium petroliphilum PM1 (PM1) TBA Monooxygenase (TBA)

Aerobic PAHs and Alkanes (cells/mL) Naphthalene Dioxygenase (NAH) Phenanthrene Dioxygenase (PHN) Alkane Monooxygenase (ALK)

QuantArray®-‐Petro Anaerobic BTEX (cells/mL) Benzoyl Coenzyme A Reductase (BCR) Benzylsuccinate synthase (BSS) Benzene Carboxylase (ABC)

Anaerobic PAHs and Alkanes (cells/mL) Benzoyl Coenzyme A Reductase (BCR) Naphthylmethylsuccinate Synthase (NMS) Naphthalene Carboxylase (ANC) Alklysuccinate Synthase (ASSA)

Other (cells/bead) Total Eubacteria (EBAC) Sulfate Reducing Bacteria (APS)

Benzene Carboxylase (ABC)

Naphthalene Carboxylase (ANC)

QuantArray®-‐Chlor

Reductive Dechlorination Dehalococcoides PCE, TCE, DCE, VC, CPs, CBs, PCBs TCE Reductase TCE BAV1 Vinyl Chloride Reductase DCE, VC Vinyl Chloride Reductase DCE, VC Dehalobacter spp. PCE, TCE, TCAs, DCAs chloroform reductase CF Dehalobacter DCM DCM Dehalogenimonas spp. TeCA, 1,1,2,2-‐TCA, 1,2-‐DCA, DCP Desulfitobacterium spp. PCE, TCE, DCA*, CPs Desulfuromonas spp. PCE, TCE 1,1-‐Dichloroethane reductase 1,1-‐DCA 1,2-‐Dichloroethane reductase 1,2-‐DCA Total Bacteria & Competitors Total Eubacteria Total Sulfate Reducing Bacteria Compe,tors Methanogens Compe,tors

14

QuantArray®-‐Chlor

Aerobic (Co)Metabolic Soluble Methane Monooxygenase TCE, DCE, VC, CF, 1,2-‐DCA Par,culate Methane Monooxygenase TCE, DCE, VC Toluene Dioxygenase TCE Phenol Hydroxylase TCE Toluene Monooxygenase 2 TCE Toluene Monooyxgenase TCE, 1,2-‐DCEs, 1,1-‐DCE, VC, CF Ethene Monooxygenase VC Epoxyalkane transferase VC

14

O2

Es(ma(ng Cometabolism Contribu(on

0.01

0.1

1

10

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05

TCE De

grad

a(on

Rate Co

nstant (p

er year)

PHE (gene copies/mL)

PHE is ND (X) or Low PHE is Average

PHE is High

Significant Degrada(on

No Degrada(on

Some Degrada(on

Faster Degrada(on

ESTCP ER-‐201584

•  Former chemical manufacturing facility

•  Superfund site

•  Groundwater impacted by

–  Chloroethanes

–  Chloroethenes

–  Chloropropanes

QuantArray®-‐Chlor Case Study

Site Management Ques(ons

qPCR

Is complete reduc,ve dechlorina,on likely?

Should an electron donor be

added?

Was electron donor injec,on effec,ve?

Is bioaugmenta,on needed?

What is the concentra,on of contaminant degraders?

qPCR

QuantArray

28%

79%

0%

20%

40%

60%

80%

100%

<1 1-‐2 2-‐3 3-‐4 4-‐5 >5

Percen

t with

VC or Ethen

e De

tected

Log Dehalococcoides cells/mL Vinyl chloride Ethene

Threshold Target Gene Concentra(on

1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08

DHC TCE BVC VCR DHBt DHG DSB DSM

Cells or g

ene copies/m

L

Baseline

QuantArray-‐Chlor® & Reduc(ve Dechlorina(on

25th 24th

21st

1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08


Cells or g

ene copies/m

L

Baseline Post-‐Injec,on

Post-‐Injec(on

92nd

>98th >97th

90th

1.00E+00

1.00E+02

1.00E+04

1.00E+06

1.00E+08


Cells or g

ene copies/m

L


Dehalococcoides and vinyl chloride reductases

19th

68th

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

EBAC APS MGN

Cells or g

ene copies/m

L


QuantArray® & Compe(ng Electron Acceptors

63rd 81st

83rd

Sulfate Reducers

Methanogens

•  Applicable to all enhanced biodegrada,on products •  Baseline (pre-‐treatment) samples

–  Evaluate MNA

–  Quan,fy baseline concentra,ons of contaminant degraders

•  Post-‐Treatment performance monitoring –  Document growth of contaminant degraders in response to treatment

–  Direct evidence of treatment effec,veness

Links -‐ qPCR and QuantArray®

•  Inoculum Injec,on –  qPCR or QuantArray to assess DHC


•  Ac,vated Carbon product w/ monitored natural amenua,on (MNA) –  QuantArray® or qPCR

•  Ac,vated Carbon product w/ enhanced biodegrada,on product –  qPCR or QuantArray®

•  Recover samples with visual evidence of Ac,vated Carbon


Ac(vated Carbon

Dehalococcoides

Decrease in electron donor

Low vinyl chloride concentra(ons (<5 µg/L) Re

scaled

Y axis

TOC decreases to Non-‐Detect

DHC con(nues to decrease with consump(on of

e-‐donor

Vinyl chloride detected

•  Effec,ve adsorp,on and biodegrada,on –  Dehalococcoides is an obligate halorespiring microbe –  Dehalococcoides decreased when e-‐ donor was consumed –  Daughter products only detected when Dehalococcoides had likely dropped to low concentra,ons

•  Microbial monitoring cri,cal aoer Ac,vated Carbon –  Daughter products not detected during biodegrada,on –  Daughters only detected aoer biodegrada,on slowed (e-‐ donor consumed and redox condi,ons less favorable)

Conclusions

Metagenomics & Next Genera(on Sequencing Who is there?

Next Genera(on Sequencing

What microorganisms are present?

Next Genera,on Sequencing

(metagenomics)

Microbial Insights EMD Webinar Series

hmp://www.microbe.com/webinars/

Metagenomics & Next Genera(on Sequencing: How to Make the Most of Your Data without Jumping to Conclusions

“Sequencing”

“Sequencing”

Amplicon Sequencing (16S rRNA gene)

Specific target region is amplified to improve coverage level

Who is there? (Taxonomy Classifica,on)

What do you get?

Sample ID Reads Passing Quality Filtering

% Reads Classified to Genus

Shannon Genus Diversity

MW6 607,795 92.2% 3.0 MW7 577,170 93.6% 2.9 MW8 719,650 93.7% 2.3 MW9 736,200 94.1% 2.3 MW10 734,080 93.6% 2.7

99.6% 97.8% 97.3% 95.4% 94.5% 92.2%

49.2%

0%

20%

40%

60%

80%

100%

Kingdom Phylum Class Order Family Genus Species

% Total Reads Classified

Top Genus Classifica(on Results Classifica(on Number of

Reads % Total Reads Descrip(on

Dechloromonas 146,290 24.1% Faculta,ve anaerobic bacteria (uses oxygen as electron acceptor when available). Some strains u,lize nitrate as an electron acceptor and some can reduce perchlorate and chlorate.

Geobacter 108,799 17.9% Anaerobic, gram-‐nega,ve, iron reducing bacteria. Some species can also reduce sulfur.

Unclassified at Genus Level 74,511 12.3% Pseudomonas 26,248 4.3% Pseudomonas is a metabolically diverse genus of aerobic organisms. Some

species can also denitrify. Some strains use common hydrocarbons as carbon sources.

Rhodoferax 25,011 4.1% anaerobic genus that oxidizes acetate with the reduc,on of Fe (III). Gallionella 23,727 3.9% Aerobic, iron oxidizing bacteria Sulfuritalea 18,234 3.0% Genus of faculta,ve anaerobes bacteria (uses oxygen as electron acceptor

when available) that also reduce nitrate. Grows chemolithoautotrophically by oxida,on of reduced sulfur compounds and hydrogen under anoxic condi,ons. Heterotrophic growth on organic acids.

Methylotenera 16,927 2.8% Facultative methylotrophs that utilize methylamine. Some may utilize methanol, ethanol and pyruvate.

Hierarchical Clustering

T2 T1 T5 T4 T3

Baseline Post-‐Treatment

PCA Biplot – Samples and Variables

•  Emerging Contaminants •  Chlorinated hydrocarbon degrada,on

–  Enhanced anaerobic biodegrada,on –  In situ chemical reduc,on

•  Petroleum hydrocarbons –  BTEX, MTBE and TBA

Links -‐ NGS

SIP & CSIA Is Biodegrada(on Occurring?

Ques(ons MBTs can answer



Compound Specific Isotope

Analysis (CSIA)

Microbial Insights EMD Webinar Series

hmp://www.microbe.com/webinars/

CSIA vs. SIP What is the difference and how do I use them?

Compound Specific Isotope Analysis (CSIA)

•  As organic compounds degrade, the ra,o of stable isotopes (13C/12C, 2H/H, 37Cl/35Cl) in the frac,on remaining aoer degrading can change in a predictable way.

•  CSIA can provide a conserva,ve boundary on the extent of degrada,on

EPA Guidance

Unit of measure Amount of 13C rela,ve to 12C is expressed by the δ13C nota,on

The standard is a specific carbon-‐containing mineral from a

specific loca,on: Pee Dee Belimnite (PDB)

Units of δ13C are o/oo or “per mill”

[ ] 10001)/()/(

‰ Standard

1213Sample

121313 ⋅⎟

⎟⎠

⎞⎜⎜⎝

⎛−=

CCCC

Cδ

•  Chemical bonds with the lighter isotope (12C) are slightly weaker than those formed with the heavier isotope (13C) and react more quickly.

•  The parent compound becomes enriched in the heavier isotope (increasing δ13C).

•  The daughter product is ini,ally very depleted in the heavy isotope (lower or “more nega,ve” δ13C).

CSIA – Why it works

13Chocolate Frac(ona(on

Decreasing total M & M’s

Decreasing ra,o M : M (Increasing ra,o M : M)

12C 13C

Time

•  Conclusive evidence of biodegrada,on •  Broad applicability

–  Chlorinated ethenes -‐ PCE, TCE and daughter products –  Chlorinated ethanes -‐ TCA, DCA –  Chlorinated methanes -‐ Carbon tetrachloride, chloroform –  Petroleum hydrocarbons -‐ BTEX, MTBE and TBA –  Emerging contaminants (1,4-‐Dioxane)

•  Es,mate extent of parent compound degrada,on

CSIA Strengths

•  Evalua,ng abio,c degrada,on –  Zero valent iron (ZVI) –  Iron bearing minerals (FeS, pyrite) –  Permanganate and Fenton’s-‐like reagents

•  Rela,vely inexpensive •  Environmental forensics (source iden,fica,on)

CSIA Strengths

•  Specially produced “heavy” compounds which are composed of 99+% 13C –  Natural compounds are 99% 12C –  Same characteris,cs as original compound –  Behave similar to the natural compound

•  Used as a “probe” or “tracer” to determine if biodegrada(on is occurring –  If biodegrada,on occurs, the 13C will be

incorporated biomass or mineralized to 13CO2.

Stable Isotope Probes

Overview of Bio-‐Trap SIP Approach

13C labeled Benzene

Beads loaded with 13C compound

Bio-Trap® with 13C-benzene loaded beads

In-Situ deployment in monitoring well

Beads analyzed following deployment

•  Passive microbial sampling tool

•  Colonized by active microbes

•  25% Nomex and 75% PAC

•  Used in conjunction with

–  Stable isotope probing

–  qPCR and QuantArray

–  Other MBTs

What Are Bio-‐Trap® Samplers?

Bio-‐Trap SIP Analysis

Residual 13C-‐Compound

13C/12C Dissolved Inorganic Carbon

13C/12C of Biomarkers

U(liza(on

Mineraliza(on (C for energy)

Metabolism (C for growth)

PLFA DNA RNA

ü Contaminant concentra,ons ü Geochemistry •  Molecular Biological Tools

MNA Assessment -‐ SIP Case Study

Concentra,ons of contaminant degrading

microorganisms?



QuantArray & qPCR

Study Wells – Weathered Limestone

Well Naphthalene 2-‐Methylnaphthalene

UMW-‐7C 13 1,000

Well Naphthalene 2-‐Methylnaphthalene

UMW-‐44 15 100

Well

MMW-‐17D

Is naphthalene biodegrada(on occurring?

-‐50

0

50

100

150

200

250

Background UMW-‐7C

DIC δ1

3 C (‰

) 13C naphthalene mineralized to CO2

UMW-‐7C

-‐50

150

350

550

750

950

Background UMW-‐7C

PLFA

δ13C (‰

) Is naphthalene biodegrada(on occurring?

13C incorpora(on into biomass

UMW-‐7C

QuantArray-‐Petro

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

NAH PHN ARH NID BCR MNSSA ANC

Cells/m

L

MMW-‐17D UMW-‐7C UMW-‐44

Aerobic PAHs

Anaerobic PAHs

MNA Assessment

Chemical Microbiological

Decreasing contaminant concentra,on?

Stable Isotope Probing Did biodegrada,on occur?

QuantArray Concentra,ons of

contaminant degraders?

Naphthalene

•  Conclusive evidence of in situ biodegrada,on •  Don’t need to know organisms or pathways involved •  Broad applicability (carbon and energy sources)

–  BTEX, MTBE, TBA –  Naphthalene –  Chlorobenzene –  Emerging contaminants (dioxane, sulfolane)

•  Inexpensive for many common contaminants

SIP Strengths

Links – SIP & In Situ Microcosms (ISMs)

-‐500

0

500

1000

1500

2000

2500

3000

Average Background MNA ORC Advanced

PLFA

Del (‰

)

13C Incorpora(on into Biomass

In Situ Microcosms Screening Remedia(on Op(ons

In Situ Microcosms (ISMs)

What treatment strategy should be selected?

In Situ Microcosms

(ISMs)

Control (MNA)

Treatment Op,on

1

Treatment Op,on

2

Unit Samplers Assembly

Control (MNA)

Treatment Op,on

1

Treatment Op,on

2

GEO

COC

Bio-‐Trap

Supplier

Supplier

•  Shallow aquifer impacted by TCE.

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

•  DCE appears to be accumula,ng (“DCE stall”)

•  Considering –  Bios,mula,on (BioS,m) with electron donor

–  Bioaugmenta,on (BioAug) w/culture and electron donor

Site Background

ISM Study – Microbial Lines of Evidence

qPCR

MNA (Control) Are halorespiring bacteria (e.g. Dehalococcoides) present?

BioS(m Will electron donor addi,on s,mulate growth of halorespiring bacteria?

Bioaugmenta,on needed?

BioAug Will a bioaugmenta,on culture survive?

ISM Study – Chemical Lines of Evidence

qPCR

MNA (Control)

BioS(m

BioAug

Contaminant concentra,ons under exis,ng condi,ons?

Will electron donor addi,on enhance daughter product forma,on?

Ethene? Full dechlorina,on?

Will bioaugmenta,on enhance biodegrada,on compared to electron donor alone?

qPCR -‐ MNA vs BioS(m vs BioAug

4.51E+01

7.18E+02

2.30E+07

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

Cells/bd

Dehalococcoides spp. tceA Reductase vcrA Reductase

MNA

BioS(m

BioAug

ISM Study – Microbial Lines of Evidence

qPCR

MNA (Control)

Are halorespiring bacteria (e.g. Dehalococcoides) present?

Yes, but at a low concentra,on

BioS(m Will electron donor addi,on

s,mulate growth of halorespiring bacteria?

Yes, a noteworthy increase was observed

Bioaugmenta,on needed?

Probably not

BioAug Will a bioaugmenta,on culture survive?

Yes, DHC remained high during the deployment period

VOCs – MNA vs BioS(m vs BioAug MNA BioS(m

0.0

0.2

0.4

0.6

0.8

1.0

Mole Frac(o

n

TCE 1,2 DCE Vinyl Chloride Ethene

BioAug

BioS(m Enhanced daughter product forma,on

BioAug Further enhanced daughter product forma,on but…

ISM Study – Chemical Lines of Evidence

qPCR

MNA (Control)

BioS(m

BioAug

Contaminant concentra,ons under exis,ng condi,ons? Mainly TCE (60%) with DCE (40%) – no vinyl chloride, ethene

Will electron donor addi,on enhance daughter product forma,on? Yes, enhanced DCE produc,on (90%)

Ethene? Full dechlorina,on?

Yes

Will bioaugmenta,on enhance biodegrada,on compared to electron donor alone?

Yes but not substan,ally

Site Management Decision

Overall Ques,on

MNA or Bios,mula,on or Bioaugmenta,on?

Bios,mula,on

Client’s Ac,on

•  Chlorinated hydrocarbon degrada,on –  Enhanced anaerobic biodegrada,on

•  Petroleum hydrocarbons –  BTEX, MTBE and TBA

Links -‐ ISM

A litle info about Microbial Insights

Founded in 1992 as a technology transfer company based on the research of Dr. D.C. White at the University of Tennessee

•  Experience •  Accuracy, precision and quality control •  Innova,on

–  Comprehensive suite of MBT analyses – Microbial Insights Database –  QuantArray & con,nuous assay development –  Next Genera,on Sequencing

•  Customer Service

A litle more about Microbial Insights

•  www.microbe.com

•  Contacts –  Kate Clark ([email protected]) –  Casey Brown ([email protected])

•  Telephone (865) 573-‐8188

For more informa(on

microbiological tools for contaminated site monitoring and remediation

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