A fascinating webinar with Dora Taggert of Microbial Insights discussing the range of sophisticated yet accessible microbiological tools for contaminated site monitoring.

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

2. 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

3. 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)

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

5. • 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

6. CENSUS
qPCR
Approach
DNA
Extrac(on
Sample
Collec(on
TOD
PHE
BSS

7. QuantArray®
DNA
Extrac(on
Sample
Collec(on
SubArray
Amplifica(on
TOD
RMO
BSS
ABC
NAH
ANC
MNSS

8. 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)

9. 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)

10. 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

11. 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

12. 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
Degrada(on
Rate
Constant
(per
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

13. • Former
chemical
manufacturing
facility
• Superfund
site
• Groundwater
impacted
by
– Chloroethanes
– Chloroethenes
– Chloropropanes
QuantArray®-­‐Chlor
Case
Study

14. 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

15. 28%
79%
0%
20%
40%
60%
80%
100%
<1
1-­‐2
2-­‐3
3-­‐4
4-­‐5
>5
Percent
with
VC
or
Ethene
Detected
Log
Dehalococcoides
cells/mL
Vinyl
chloride
Ethene
Threshold
Target
Gene
Concentra(on

16. 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
gene
copies/mL
Baseline
QuantArray-­‐Chlor®
&
Reduc(ve
Dechlorina(on
25th
24th
21st

17. 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
gene
copies/mL
Baseline
Post-­‐Injec,on
Post-­‐Injec(on
92nd
>98th
>97th
90th

18. 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
gene
copies/mL
Baseline
Post-­‐Injec,on
Dehalococcoides
and
vinyl
chloride
reductases
19th
68th

19. 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
gene
copies/mL
Baseline
Post-­‐Injec,on
QuantArray®
&
Compe(ng
Electron
Acceptors
63rd
81st
83rd
Sulfate
Reducers
Methanogens

20. • 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®

21. • Inoculum
Injec,on
– qPCR
or
QuantArray
to
assess
DHC
Links
-­‐
qPCR
and
QuantArray®

22. • 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®

23. Ac(vated
Carbon

24. Dehalococcoides

25. Decrease
in
electron
donor

26. Low
vinyl
chloride
concentra(ons
(<5
µg/L)
Rescaled
Y
axis
TOC
decreases
to
Non-­‐Detect
DHC
con(nues
to
decrease
with
consump(on
of
e-­‐donor
Vinyl
chloride
detected

27. • 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

28. Metagenomics
&
Next
Genera(on
Sequencing
Who
is
there?

29. 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

30. “Sequencing”
“Sequencing”
Amplicon
Sequencing
(16S
rRNA
gene)
Specific
target
region
is
amplified
to
improve
coverage
level
Who
is
there?
(Taxonomy
Classifica,on)

31. 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

32. 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.

33. Hierarchical
Clustering
T2
T1
T5
T4
T3
Baseline
Post-­‐Treatment

34. PCA
Biplot
–
Samples
and
Variables

35. • Emerging
Contaminants
• Chlorinated
hydrocarbon
degrada,on
– Enhanced
anaerobic
biodegrada,on
– In
situ
chemical
reduc,on
• Petroleum
hydrocarbons
– BTEX,
MTBE
and
TBA
Links
-­‐
NGS

36. SIP
&
CSIA
Is
Biodegrada(on
Occurring?

37. 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?

38. Compound
Specific
Isotope
Analysis
(CSIA)

39. • 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

40. 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
1213
Sample
1213
13
⋅
⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛
−=
CC
CC
Cδ

41. • 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

42. 13Chocolate
Frac(ona(on
Decreasing
total
M
&
M’s
Decreasing
ra,o
M
:
M
(Increasing
ra,o
M
:
M)
12C
13C
Time

43. • 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

44. • 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

45. Stable
Isotope
Probing
(SIP)

46. • 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

47. 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

48. • 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?

49. 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

50. 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

51. Study
Wells
–
Weathered
Limestone
Well
Naphthalene
2-­‐Methylnaphthalene
UMW-­‐7C
13
1,000
Well
Naphthalene
2-­‐Methylnaphthalene
UMW-­‐44
15
100
Well
MMW-­‐17D

52. Is
naphthalene
biodegrada(on
occurring?
-­‐50
0
50
100
150
200
250
Background
UMW-­‐7C
DIC
δ13C
(‰)
13C
naphthalene
mineralized
to
CO2
UMW-­‐7C

53. -­‐50
150
350
550
750
950
Background
UMW-­‐7C
PLFA
δ13C
(‰)
Is
naphthalene
biodegrada(on
occurring?
13C
incorpora(on
into
biomass
UMW-­‐7C

54. 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/mL
MMW-­‐17D
UMW-­‐7C
UMW-­‐44
Aerobic
PAHs
Anaerobic
PAHs

55. MNA
Assessment
Chemical
Microbiological
Decreasing
contaminant
concentra,on?
Stable
Isotope
Probing
Did
biodegrada,on
occur?
QuantArray
Concentra,ons
of
contaminant
degraders?
Naphthalene

56. • 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

57. 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

58. In
Situ
Microcosms
Screening
Remedia(on
Op(ons

59. In
Situ
Microcosms
(ISMs)
What
treatment
strategy
should
be
selected?
In
Situ
Microcosms
(ISMs)
Control
(MNA)
Treatment
Op,on
1
Treatment
Op,on
2

60. Unit
Samplers
Assembly
Control
(MNA)
Treatment
Op,on
1
Treatment
Op,on
2
GEO
COC
Bio-­‐Trap
Supplier
Supplier

61. • 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

62. 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?

63. 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?

64. 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

65. 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

66. VOCs
–
MNA
vs
BioS(m
vs
BioAug
MNA
BioS(m
0.0
0.2
0.4
0.6
0.8
1.0
Mole
Frac(on
TCE
1,2
DCE
Vinyl
Chloride
Ethene
BioAug
BioS(m
Enhanced
daughter
product
forma,on
BioAug
Further
enhanced
daughter
product
forma,on
but…

67. 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

68. Site
Management
Decision
Overall
Ques,on
MNA
or
Bios,mula,on
or
Bioaugmenta,on?
Bios,mula,on
Client’s
Ac,on

69. • Chlorinated
hydrocarbon
degrada,on
– Enhanced
anaerobic
biodegrada,on
• Petroleum
hydrocarbons
– BTEX,
MTBE
and
TBA
Links
-­‐
ISM

70. 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

71. • 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

72. • www.microbe.com
• Contacts
– Kate
Clark
(kclark@microbe.com)
– Casey
Brown
(cbrown@microbe.com)
• Telephone
(865)
573-­‐8188
For
more
informa(on