Download - MI Webinar 13Mar2013
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Incorporating Quantitative Polymerase Chain Reaction (qPCR)
Analyses into Site Management
Kirsti M. [email protected]
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Introduction
Value of quantifying bacteria, their function and activity
Molecular Biological Tools (MBTs) and Environmental Molecular Diagnostics (EMDs)
Quantitative Real-Time PCR
Available Assays
Application to MNA
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Value of Quantitative Microbial Information via Molecular Tools
Dilute and culture on agar
Count colonies
Stain and Count by Epifluorescence
Microscopy
Count cells
Enumerating bacteria by traditional means is tedious
Molecular tools expedite analysis, provide quantitative information, and can target bacteria that cannot be grown in isolation
We cannot study bacteria in isolation andexpect to see their behavior in nature
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What are MBTs and EMDs
Molecular Biological Tools (MBTs)
Tools targeting biological function in an environment, that can be used to enumerate, quantify or inform on the potential to carry out some desired function
Environmental Molecular Diagnostics (EMD)
Utilizing the information from the MBTs to effectively predict, and manage contaminated sites or other microbial processes
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Benefits of MBTsSpecific, sensitive, quantitative information regarding organisms
and genes involved with bioremediation
Provide information about the relevant processes affecting contaminant longevity
Applicable to a wide variety of environmental samples
Predictive tool for site assessment
Is MNA (monitored natural attenuation) an option?
Is biostimulation sufficient?
Is bioaugmentation necessary?
What endpoints can be expected?
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Targets in a Cell
mRNA DNA
Lipid
Activity
rRNA
Ribosome and rRNA
Protein
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MBTs
Protein
RNA
DNA Potential, gene numbers
General metabolic activity
Specific metabolic activity
Direct measure of activity
Lipids General metabolic activity
rRNAmRNA
qPCR, TRFLP, DGGE, FISH
RT-qPCR, CARD-FISH
CSIA, proteins,enzyme probes
PLFA
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Quantitative Real-Time PCR (qPCR)
qPCR is a robust assay for quantifying nucleic acid targets in environmental samples
Both DNA and RNA can be analyzed in most samples
Used for site assessment and long term monitoring programs
Dynamics in gene abundances and gene activity (i.e., transcripts) serve as activity indicators
Correct QC protocols must be established from sample collection through qPCR analysis
Ideal QC adjusts for losses in target number during sample collection, shipping, preparation and analysis
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F MGBQ
F MGBQ
qPCR
1. Hybridization
F Primer
R Primer
TaqMan Probe
Template
2. Strand Displacement, Taq Polymerase Binds
F Primer
R Primer
Template
Taq
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F MGBQ
qPCR3. Primer Extension
4. Probe Displacement and Flurophore Release
F Primer
R Primer
MGBQ
Template
Taq
F
F Primer
R Primer
Template
Taq
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2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle
Nor
mal
ized
fluo
resc
ence
0.1
1.0
10.
0
qPCR
5. Fluorescence Curves for Standards
Ct
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qPCR
CT 17.5Log Dhc= 7.116S rRNA copies= 1.3 x 107
CT 34.5Log Dhc= 1.816S rRNA copies= 6 x 101
6. Analysis based on standard curve
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Many Available Assays
Target microbial activities relevant to bioremediation and other environmental processes:
Total Bacteria, Archaea, Methanogens
Reductive Dechlorination/Aerobic Oxidation
Petrolium Detoxification
Nitrogen Cycling
Microbially Induced Corrosion
Metal/Radionuclide Reduction
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Commercial Assays
qEBAC “General Bacteria”
qARC “General Archaea”
qAPS Sulfate Reducing Bacteria
qMGN Methanogens
qDHC DehalococcoidesqTCE TCE to cDCEqVC cDCE and VC to ethene
qDHB Dehalobacter
qDHG Dehalogenimonas*
qDSB Desulfitobacterium
Target microbial activities relevant to bioremediation and other environmental processes:
qRMO ring hydroxylating toluene monnox.
qPHE phenol hydroxylase: BTEX oxidation
qTOL tol monooxygenase: toluene, xylene
qBSS benzylsuccinate synthase: toluenexylene
qTOD tol dioxy: benzene toluene ethylbenzene
qNAH prnapthalene
qPM1 MTBE utilizing Methylibium PM1
qDSM Desulfuromonas
qGEO Geobacter
qAGN acid producing bacteria (MIC)
qARG archaeglogus (MIC)
qDNF nitrate reducing bacteria
qpcrA perchlorate reductase
qAOB ammonia oxidizers
qsMMO methane monooxygenase
qPPO propane monooxygenase
qBOM butane monooxygease
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PCE TCE cis-DCE
Example: Chloroethene Dechlorination
Dehalobacter, Desulfuromonas, Sulfurospirillum,
Geobacter (et al.)
pceA
For the rest of the story go to the MI website and watch the seminar from
Frank Loeffler on Feb. 11, 2013
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Dehalococcoides: Keystone Bacteria for Detoxification
PCE TCE cis-DCE VC Ethene
tceA bvc
A
pceA
vcrA
Five Dhc Gene Targets (Biomarkers)1. Dhc 16S rRNA gene2. pceA encoding a PCE to TCE RDase3. tceA encoding a TCE-to-VC RDase4. bvcA encoding a VC-to-ethene RDase5. vcrA encoding a VC-to-ethene RDase
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Dhb 2
Dhb 1
Dhb 3
dcrA
cfrA
cfrA
pceA
pceAtceA
tceAbvcAvcrA
bvcAvcrA
Dhc
Dhc
Dhb 1
GeoDsmDhbDhc
Anaerobic Dechlorination Biomarkers
???
Chlorinated Ethanes Chlorinated Ethenes Chlorinated Methanes
Functional gene biomarker
16S rRNA gene biomarker
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DhcDhgmdcpA
Anaerobic 1,2-D and 1,2,3-TCP Pathways
1,2-DCP 1,2,3-TCP(Yan et al. 2008; Dhgm strains BLDC-8 and BLDC-9)
(Löffler et al. 1997; Ritalaht et al. 2004; Padilla-Crespo et al. 2013; Dhc
strains RC and KS)
dichloropropane dehalogenase
allyl chloride(unstable)
diallyl sulfide
propene
diallyl disulfideS-
allyl alcohol
Fe2+
S-
S-
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Dehalogenimonas lycantrhoporepellens strain BLDC-9
Dichloroelimination to Propene
2H+ + 2e-
2H+ + 2Cl-
Dehalococcoides mccartyi strains KS, RC
SYBR Gold, 1000x
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Dichloroelimination of Chloro-Ethanes
Other dichloroelimination reactions involve transformation of TCA to VC and DCA to ethene by Dehalobacter spp.
PCE TCE cis-DCE VC Ethene
1,1,2-TCA
1,2-DCA
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Aerobic 1,2,3-TCP Biomarkers and Pathways1,2,3-
trichloropropane
dhaA
hheC
haloalkane dehalogenase
haloalcohol dehalogenase
echAepoxide hydrolase
(Bosma et al. 2002; TCP for Agrobacterium radiobacter strain
AD1)
2,3-dichloro-1-propanol
1,3-dichloro-2-propanol
1-chloro-2,3-epoxypropane 3-chloro-1,2-propanediol
TCP
1,3-DCP
2,3-DCP
3-CPD
1,2-propanediolpropylene glycol
Pseudomonas sp. strain OS-K-29
Pseudomonas sp. strain AD1,Arthrobacter sp. strain AD2, and a coryneform strain AD3
Pseudomonas spp.,
propanediol dehydratase
Salmonella, spp. (needs cobalamin),
biomass, mineralization
pdu
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Understanding the CommunityNeed to know accessory organisms that contribute to a specific activity
Dhc requirements:
H2
B12
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The Corrin-RingVery costly to make these compounds so why do so?
To find out more about the roles of corrinoids and the populations
that stimulate reductive dechlorination, tune into the
seminar by Dr. Jun Yan, University of Tennessee, this
coming fall.
Yan et al. 2012, Appl. Environ. Microbiol. 78:6630-6636
Yan et al. 2013. Phil. Trans. R. Soc. B. In press
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Corrinoid pathway
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Value of MBT analysis to MNA sites
Difficult to predict a priori what will happen within an MNA site from only looking at the geochemistry
nutrient limited
may have low O2
often very dilute in contaminant
Studying the bacterial communities allows for a more predictive understanding of the site
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This Study: qPCR Data from 869 Wells at 56 Sites
qPCR analysis was putkerformed using established methods at Microbial Insights (at MI/GT/UTK) and geochemical data were compiled by MI/GT/.
Only groundwater samples included
All analyzed for Dhc 16S rRNA gene
Most samples evaluated for RDase genes
Total Bacteria quantified in 337 samples
Ethene data collected at 625 wells
BioTraps deployed in 78 wells at 6 sites
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Distribution of Dhc in Groundwater Samples
ND = not detected
Dehalococcoides/L
Treatment Samples (wells) Sites ND or <103 103 to 104 104 to 106 > 106
TOTAL WELLS 869 56 198 191 275 205Bioaugmentation 92 6 16 15 8 53
Biostimulation 154 20 28 19 43 64MNA 578 54 144 144 202 88
Chem Ox 8 2 0 2 6 0ZVI 37 5 10 11 16 0
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Dhc abundance (log gene copies/L)
Ethe
ne c
once
ntra
tion
(ppb
)Lo
g et
hene
con
cent
ratio
n (p
pb)
Dhc abundance (log gene copies/L)
Ethene Formation Correlates with Dhc Abundance
Corresponds with bivariate comparison of ethene to Dhc in GW
n = 176F = 74.8501P = <0.0001R2 = 0.3008significant
Corresponds with bivariate comparison of ethene to Dhc on BioSep Beads (BioTraps)
n = 61F = 22.9258P = <0.0001R2 = 0.279836significant
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bvcA+vcrA Gene to Dhc Ratio
Highest ethene production when bvcA+vcrA to Dhc ratio is between 0.05 and 10 in GW
0.05 -10
ZVI
Beads
0.05 -10
Suggests additional VC RDase genes
Despite high ratio of VC-RDases, ethene not observed.
Ethe
ne c
once
ntra
tion
(ppb
)
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VC and the bvcA+vcrA Gene to Dhc Ratio
BeadsBeads
0.05 -10 High VC presence correlates with bvcA+ vcrA gene ratios near unity
Wells without bvcA or vcrA but with high numbers of tceA and Dhc
VC c
once
ntra
tion
(ppb
)
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RDase Genes can Outnumber Dhc Cells
The most ethene is produced when bvcA+vcrA to Dhc ratio is between 0.05 and 10
When RDase genes >10x Dhc, Dhc abundances tend to be lowUnknown reservoir for additional RDase genes
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Dhc to Bacterial 16S rRNA Gene Ratio
Dhc 16S rRNA and VC RDase ratios of 0.0005 - 0.001 (0.05 - 0.1%) of the total Bacterial 16S rRNA gene numbers correlate with ethene detection
ZVI
Dhc 16S rRNA / Bacteria
Ethe
ne (
ppb)
bvcA+vcrA / Bacteria
Ethe
ne (
ppb)
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Statistical Correlations with tDCE, cDCE, VC and Ethene
Pairwise Comparison Sample n F value P value R2 significancetDCE to bvcA GW 29 3.5535 0.070 0.116306 weak
cDCE to Dhc GW 338 37.6523 <.0001 0.100768 yescDCE to tceA GW 198 10.9785 <0.0011 0.053042 yescDCE to bvcA GW 109 0.0989 0.754 0.000924 nocDCE tp vcrA GW 136 3.394 0.068 0.024703 weak
VC to Dhc GW 238 75.3012 <0.0001 0.241892 yesBead 35 17.2963 <0.00001 0.343888 yes
VC to tceA GW 173 64.5744 <0.0001 0.274115 yesBead 28 13.6407 0.001 0.344109 yes
VC to bvcA GW 84 0.1949 0.660 0.002372 noBead 27 6.9106 0.014 0.216561 yes
VC to vcrA GW 112 11.6951 <0.0009 0.96101 yesBead 24 6.7682 0.016 0.235268 yes
ethene to Dhc GW 176 74.8501 <0.0001 0.300784 yesBead 61 22.9258 <0.00001 0.279836 yes
ethene to tceA GW 108 73.2656 <0.0001 0.408698 yesBead 41 5.8581 0.020 0.130592 yes
ethene to bvcA GW 77 9.8926 0.002 0.116531 yesBead 42 9.7342 0.003 0.195725 yes
ethene to vcrA GW 110 71.9905 <0.0001 0.399968 yes Bead 37 2.0281 0.163 0.054772 no
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Bioaugmentation Consortia
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ConclusionsEthene formation correlated with Dhc abundance
Highest ethene formation at sampling locations with near- equal VC RDase to Dhc ratios
High VC presence correlates with bvcA+vcrA gene ratios near unity
RDase genes outnumber Dhc in ≈10% of wells, suggesting an unrecognized reservoir for VC RDase genes
Ethene formation generally occurred when Dhc and VC RDase genes exceeded 0.01% of the total Bacterial 16S rRNA gene abundance
Bioaugmentation inocula infulenced the dominant VC RDase
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High Throughput qPCR for Site Analysis
Current approaches are toward high throughput tools with increased sample numbers and replication while monitoring more targets at a reduced cost.
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Objective: Design QuantStudio Array
Simultaneous monitoring of 56+ genes for 12-48 samples
Assays target specific microbial processes:Reductive dechlorination (RD-qCHIP)Petroleum remediationMetal reduction Nitrogen cycling… and many more
16S rRNA genes of relevant spp.
Functional genes related to desired activity
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QuantStudio 12K Flex System
Microfluidics Robot for loading the chip Thermocycler
Inside the robot
Tips
Tip Waste
Chip holder
384-well sample plate
Analysis
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39
33 nL volume
HydrophilicHydrophobic
OpenArray® (Empty) Plate Description
Hydrophilic and hydrophobic coatings enable reagents to stay in the bottomless through- holes via capillary action.
48 subarrays
x 64 through-holes
3072 through-holes per arrayFour plates can be cycled simultaneously, producing >12,000 digital data points per run.
A B C D E F G H I J K L
1 2
3 4
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Luc
Bac
16S rRNAgenes
Arch
RDase of known function
Aerobic VC oxidation
Hydrogenase genes
Corrinoid salvageLifeTech array controls
Design Probes and Primers, Verify with qPCR, Decide on Assay Format, and Order Chip.
Example layout for RD-qCHIP
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Open Array Workflow
TaqMan assaysordered on-line Primers and probe spotted on
Open Array plateOpen Array plate
Load sampleswith robot
Place lid onarray case Cycle and image up to
4 Open Array plates
Run Plates (UT/ORNL)
Synthesize and load primers and probes to through-wells of the chip: (Life Technologies, Woburn, MA)
Synthesize Array Chip: Overview
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Tandem Standard: synthetic DNA of target regions for each assay in KanR vector cloned in E. coli
DhcDhgm
Vector
LucBac
tceAvcrAbvcA
dcpA
Apply in dilution series to verify reproducibility and quantification results of the array
A B C D E F G H I J K L
1 2
3 4
106 copies 104 copies 102 copies
S1 S2
Each spot on the array yields all qPCR data at each cycle and allows replication and dilution of samples with multiple spots
NTCS1(1:10) S2(1:10) Plasmid
Example: Validate RD-qChip with Tandem Standard
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Valuable Information from MBTs
Presence/Absence of genes: potential to deal with the compound of interest
Quantitative information of relevance to bioremediation/MNA applications can be obtained with MBTs
Abundance of organisms, genes and transcripts; before, during and after treatment, can be correlated with geochemical data to inform on decision making as to appropriate site remediation action
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Final Remarks
If you are interested in great summary of bioremediation applications see Dr. Frank Loeffler’s seminar (Feb, 11, 2013) posted on the MI website.
In Fall 2013 stay tuned to Dr. Jun Yan’s seminar that will go into detail on the contribution of corrinoids and bacterial populations that contribute to organohalide respiration
For case studies on chlorinated ethenes and petroleum bioremediation projects, see the Microbial Insights Website, or contact them for further information at www.microbe.com
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Shandra Justicia LeonDarlene WagnerJanet Hatt
Elizabeth PadillaBurcu Simsir
Dora OglesBrett BaldwinAnita Biernacki
Kirsti RitalahtiJun YanCindy Swift
Elizabeth EdwardsAriel GrosternMelanie DuhamelWinnie Chan
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To all the people involved in the sampling in the field and in the laboratory analysis of these many samples
To the many collaborators who provided available site characterization information,
To SERDP and ESTCP for providing funding to do this research.
Thank You! Questions