microbiological tools for contaminated site monitoring and remediation
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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
DHC TCE BVC VCR DHBt DHG DSB DSM
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
DHC TCE BVC VCR DHBt DHG DSB DSM
Cells or g
ene copies/m
L
Baseline Post-‐Injec,on
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
Baseline Post-‐Injec,on
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
Links -‐ qPCR and QuantArray®
• 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
Links -‐ qPCR and QuantArray®
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
Is biodegrada,on occurring?
Stable Isotope Probing (SIP)
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
Stable Isotope Probing (SIP)
• 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?
Is biodegrada,on occurring?
Stable Isotope Probing (SIP)
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 (kclark@microbe.com) – Casey Brown (cbrown@microbe.com)
• Telephone (865) 573-‐8188
For more informa(on
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