This document summarizes a microcosm study of a butane-utilizing mixed culture for cometabolic degradation of 1,1-dichloroethene (1,1-DCE). Indigenous and bioaugmented microcosms were constructed using aquifer materials and groundwater from a contaminated site. Microcosms were fed butane and/or 1,1-DCE and degradation was monitored over time. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize changes in microbial community structure. A biotransformation model was developed and fit experimental data to simulate cometabolic degradation kinetics and inhibition effects. Results showed that indigenous and bioaugmented cultures could degrade butane

1. Microcosm Study of a BioaugmentedMicrocosm Study of a Bioaugmented
Butane-Utilizing Mixed Culture:Butane-Utilizing Mixed Culture:
Community Structure and 1,1-DCECommunity Structure and 1,1-DCE
CometabolismCometabolism
Hee Kyung LimHee Kyung Lim
Oregon State UniversityOregon State University
Department of Civil, Construction, andDepartment of Civil, Construction, and
Environmental EngineeringEnvironmental Engineering

2. OverviewOverview
• The Moffett Field Site (Mountain View, CA)The Moffett Field Site (Mountain View, CA)
-contaminated with various chlorinated aliphatic-contaminated with various chlorinated aliphatic
hydrocarbons (CAHs) including 1,1,1-trichloroethanehydrocarbons (CAHs) including 1,1,1-trichloroethane
(1,1,1-TCA)(1,1,1-TCA)
-CAHs are potential health hazards-CAHs are potential health hazards
• 1,1-dichloroethene (1,1-DCE)1,1-dichloroethene (1,1-DCE)
-one of the major toxic products of the abiotic-one of the major toxic products of the abiotic
transformation of 1,1,1-TCA (Vogel and McCarty,transformation of 1,1,1-TCA (Vogel and McCarty,
1987)1987)
-The extreme toxicity is due to a toxic product of 1,1--The extreme toxicity is due to a toxic product of 1,1-
DCE transformation rather than 1,1-DCE itselfDCE transformation rather than 1,1-DCE itself
(Dolan and McCarty, 1995).(Dolan and McCarty, 1995).

3. OverviewOverview cont’dcont’d
• Cometabolic transformation of 1,1-DCECometabolic transformation of 1,1-DCE
-process by-process by nonspecific oxygenases of aerobic bacterianonspecific oxygenases of aerobic bacteria
-negative effect-negative effect may cause damage tomay cause damage to
microorganisms due to toxicity resulting from themicroorganisms due to toxicity resulting from the
transformation process or from transformationtransformation process or from transformation
productsproducts
-no known microorganism can use 1,1-DCE as a-no known microorganism can use 1,1-DCE as a
primary substrateprimary substrate cometabolic transformation is thecometabolic transformation is the
only known way for aerobic biodegradation of 1,1-only known way for aerobic biodegradation of 1,1-
DCEDCE
-past studies demonstrated butane to be an effective-past studies demonstrated butane to be an effective
growth substrate to support CAH transformationgrowth substrate to support CAH transformation

4. OverviewOverview cont’dcont’d
• Needs for Defining Microbial CommunityNeeds for Defining Microbial Community
Structure and DiversityStructure and Diversity
-Negative selective pressure of 1,1-DCE cometabolism-Negative selective pressure of 1,1-DCE cometabolism
may occur during bioremediation associated withmay occur during bioremediation associated with
formation of a toxic product.formation of a toxic product.
-The ability of microorganisms to survive and adapt to-The ability of microorganisms to survive and adapt to
the toxic environment is an important concern inthe toxic environment is an important concern in
bioremediationbioremediation
-Identifying bacterial strains that can sustain high rates-Identifying bacterial strains that can sustain high rates
of 1,1-DCE degradation.of 1,1-DCE degradation.
• Molecular biological methods using 16s rRNA geneMolecular biological methods using 16s rRNA gene
has been establishedhas been established

5. OverviewOverview cont’dcont’d
• Modeling butane utilization and 1,1-DCEModeling butane utilization and 1,1-DCE
transformationtransformation
-Biodegradation may be simulated by mathematically-Biodegradation may be simulated by mathematically
assigning a modelassigning a model
-Modeling can enhance the design and the application-Modeling can enhance the design and the application
of bioremediation system for contaminated site withof bioremediation system for contaminated site with
CAH.CAH.

6. ObjectivesObjectives
The general objectives of this study were:The general objectives of this study were:
• To test the cometabolic transformation abilities ofTo test the cometabolic transformation abilities of
indigenous and bioaugmented microorganisms in theindigenous and bioaugmented microorganisms in the
microcosms constructed with aquifer materials from themicrocosms constructed with aquifer materials from the
Moffett Field siteMoffett Field site microcosm studymicrocosm study
• To characterize the microbial community structure in theTo characterize the microbial community structure in the
microcosms and possible community shift due to 1,1-microcosms and possible community shift due to 1,1-
DCE transformation stressDCE transformation stress Terminal RestrictionTerminal Restriction
Fragment Length Polymorphism (T-RFLP)Fragment Length Polymorphism (T-RFLP)
• To assess the applicability of modeling to bioremediationTo assess the applicability of modeling to bioremediation
system: to mathematically simulate the experimental datasystem: to mathematically simulate the experimental data
obtained from microcosm studiesobtained from microcosm studies biotransformationbiotransformation
model developed by Kimmodel developed by Kim et al.et al. (2002)(2002)

7. Microcosm PreparationMicrocosm Preparation
Open- hole screw cap
Gray butyl rubber septa
Headspace (76mL)
Groundwater(55 mL)
Wet soil (25mL)
Open- hole screw cap
Gray butyl rubber septa
)
Groundwater(55 mL)
Wet soil (25mL)
•156 mL serum bottle with open hole screw cap and rubb
•All materials used were autoclaved to ensure aseptic co
•Incubated at 20 °C on shaker table at 200 rpm

8. Matrix for the indigenous microcosm studiesMatrix for the indigenous microcosm studies
MicrocosmMicrocosm ButaneButane
(µmol)(µmol)
1,1-DCE1,1-DCE
(µmol)(µmol)
HgClHgCl22
(mg/L)(mg/L)
NitrateNitrate
I1I1 7070 0.260.26 2525 --
I2I2 -- 0.240.24 -- --
I3I3 7070 -- -- --
I4I4 7070 -- -- --
I5I5 7070 -- -- --
I6I6 7171 0.260.26 -- --
I7I7 7171 0.300.30 -- --
I8I8 6464 0.250.25 -- --

9. Matrix for the bioaugmented microcosm studiesMatrix for the bioaugmented microcosm studies
MicrocosmMicrocosm ButaneButane
(µmol)(µmol)
1,1-DCE1,1-DCE
(µmol)(µmol)
HgClHgCl22
(mg/L)(mg/L)
NitrateNitrate
B1B1 67.867.8 0.230.23 2525 --
B2B2 -- 0.250.25 -- --
B3B3 -- 0.250.25 -- --
B4B4 -- 0.250.25 -- --
B5B5 70.270.2 -- -- --
B6B6 70.870.8 -- -- --
B7B7 67.967.9 -- -- --
B8B8 68.568.5 0.230.23 -- --
B9B9 72.272.2 0.210.21 -- --
B10B10 67.567.5 0.220.22 -- --

10. T-RFLP methodT-RFLP method

11. Flow chart depicting T-RFLP analysis ofFlow chart depicting T-RFLP analysis of
a microcosm samplea microcosm sample
DNA extraction from soil slurry sample
PCR reaction using 5µL DNA and 27F-FAM/338R primers
Electrophoresis of 5µL of PCR product on 1% agarose gel
to quantify amplified DNA
Restriction enzyme digests of 10µL amplified DNA using
restriction enzymes MnlI or Hin6I
Dilution to 0.5ng DNA/L of H2O
Approximately 0.5ng DNA from each digest sent for fragment analysis
Fragment analysis using ABI 377 slab gel automated sequencer
Fragment analysis data tables and electropherogram results

12. Features of the biotransformation modelFeatures of the biotransformation model
• Monod / Michaelis-Menten kineticsMonod / Michaelis-Menten kinetics
• Competitive inhibition of 1,1-DCE on butaneCompetitive inhibition of 1,1-DCE on butane
utilizationutilization
• Mixed inhibition of butane on 1,1-DCEMixed inhibition of butane on 1,1-DCE
transformationtransformation
• Transformation product toxicityTransformation product toxicity

13. Equations used in the modelEquations used in the model
l
gButccl
But
gButccl
But
DCEButicgDCEccl
DCE
ButS
ButBut
V
VHV
M
VHV
M
KVHV
M
K
Xk
dt
dM






+






+
+





+
+
=
,
)(
1
,,,
,
max,






+
+
+
+





+
+






+
=
ButDCEiugButccl
But
gBuccl
DCE
ButDCEicgButccl
DCE
DCES
l
gDCccl
DCE
DCE
DCE
KVHV
M
tVHV
M
KVHV
M
K
V
EVHV
M
Xk
dt
dM
,,,,,
,
,
max,
)(
1
)(
1
where,
MBut = Total mass of butane, µmol
MDCE = Total mass of 1,1-DCE, µmol
Kmax,But = Maximum specific rate of butane, µmol/mg cell-hr
Kmax,DCE = Maximum specific rate of 1,1-DCE, µmol/mg cell-hr
KS,But = Half-saturation constant for butane, µmol/L
KS,DCE = Half-saturation constant for 1,1-DCE, µmol/L
Hcc,BUT = Henry partition coefficient of butane, µmol/L/µmol/L
Hcc,DCE = Henry partition coefficient of DCE, µmol/L/µmol/L
Vg = Gas volume, L
Vl = Liquid volume, L
X = Active microbial concentration, mg/L
t = Time, hr
Kic,DCEBUT = Competitive inhibition coefficient of 1,1-DCE on butane, µmol/L
Kic,BUTDCE = Competitive inhibition coefficient of butane on 1,1-DCE, µmol/L
Kiu,ButDCE = Uncompetitive inhibition coefficient of butane on 1,1-DCE,µmol/L

14. Equations used in the modelEquations used in the model cont’dcont’d
l
DCE
DCEcl
But
Vdt
dM
T
bX
Vdt
dM
Y
dt
dX 111
,
−−=
where,
Y = Cellular yield of butane, mg cells/µmol butane
b = Cell decay rate, hr-1
Tc,DCE = Transformation capacity for 1,1-DCE, µmol 1,1-DCE/mg cells

15. Results of microcosm studies andResults of microcosm studies and
determination of microbial communitydetermination of microbial community
structurestructure
• Results of microcosm studies were comparedResults of microcosm studies were compared
to microbial community analysis usingto microbial community analysis using
T-RFLP method with restriction enzyme MnlIT-RFLP method with restriction enzyme MnlI
- Indigenous microcosms- Indigenous microcosms
- Bioaugmented microcosms- Bioaugmented microcosms

16. Non-augmented, no-substrate addedNon-augmented, no-substrate added
indigenous cultureindigenous culture
NTa
NTb
NTc
T-RFL (base pair)
RelativeFluorescenceUnit
169.73 bp
NTa
NTb
NTc
T-RFL (base pair)
RelativeFluorescenceUnit
169.73 bp
0
20
40
60
80
100
NTa NTb NTc
Microcosm
Portionofpeakareaof
T-RFL(%)
169.73 bp 279.86 bp 281.73 bp others
•The peak at 169.73 bp is dominant
•Reproducibility within triplicates

17. Comparison of the source,Comparison of the source,
bioaugmentation and indigenous culturebioaugmentation and indigenous culture
*Source Culture
*Bioaugmentation Culture
No-manipulated
Indigenous culture
183 bp
167 bp
207 bp
100 bp
51 bp
47 bp 99 bp 167 bp
179 bp
183 bp
207 bp
210 bp
276 bp
48 bp 167 bp
169 bp
T-RFL (base pair)
RelativeFluorescenceUnit
*Source Culture
*Bioaugmentation Culture
No-manipulated
Indigenous culture
183 bp
167 bp
207 bp
100 bp
51 bp
47 bp 99 bp 167 bp
179 bp
183 bp
207 bp
210 bp
276 bp
48 bp 167 bp
169 bp
T-RFL (base pair)
RelativeFluorescenceUnit
•The T-RFL of 167 bp is shown in three cultures
•The T-RFL of 179, 183, 207 and 210 bp is shown in the source culture and bioaugmented culture
•The T-RFL of 169 bp is shown only in the indigenous culture

18. Indigenous microcosms I3, I4 and I5Indigenous microcosms I3, I4 and I5
: fed butane only: fed butane only
0
10
20
30
40
50
60
70
80
0 30 60 90 120 150 180 210
Time (days)
Butanemass(µmol)
Butane
I4
Day 29.1
Day 48.3
Day 177.1
Day 179.6
Day 193.4
T-RFL (base pair)
169.7 bp
179.06 bp
RelativeFluorescenceUnit
Day 29.1
Day 48.3
Day 177.1
Day 179.6
Day 193.4
T-RFL (base pair)
169.7 bp
179.06 bp
RelativeFluorescenceUnit

19. Indigenous microcosms I6, I7 and I8Indigenous microcosms I6, I7 and I8
: simultaneously fed butane and 1,1-DCE: simultaneously fed butane and 1,1-DCE
I6 I7
I8
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160 180
Time (days)
Butanemass(µmol)
0
0.1
0.2
0.3
0.4
1,1-DCEmass(µmol)
Butane
1,1-DCE
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160 180
Time (days)
Butanemass(µmol)
0
0.1
0.2
0.3
0.4
1,1-DCEmass(µmol)
Butane
1,1-DCE
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160 180
Time (days)
Butanemass(µmol)
0
0.1
0.2
0.3
0.4
1,1-DCEmass(µmol)
Butane
1,1-DCE
•Although there was a long lag period of more
than 25 days, complete depletion of butane and
1,1-DCE was observed
•I7 showed the fastest rate of butane utilization
and 1,1-DCE transformation
•Different results in the kinetic tests
•Soil samples were taken at around 85 days

20. Indigenous microcosms I6, I7 and I8Indigenous microcosms I6, I7 and I8
: simultaneously fed butane and 1,1-DCE: simultaneously fed butane and 1,1-DCE cont’dcont’d
Day 86.0
Day 83.4
Day 84.9
T-RFL (base pair)RelativeFluorescenceUnit
I6
I7
I8
85.01 bp 207.5 bp
277.9 bp169.72 bp
277.9 bp
207.6 bp
207.6 bp
Day 86.0
Day 83.4
Day 84.9
T-RFL (base pair)RelativeFluorescenceUnit
I6
I7
I8
85.01 bp 207.5 bp
277.9 bp169.72 bp
277.9 bp
207.6 bp
207.6 bp

21. Bioaugmented microcosm B1Bioaugmented microcosm B1
: poisoned-control: poisoned-control
0
10
20
30
40
50
60
70
80
0 30 60 90 120 150 180 210 240
Time (day)
Butanemass(µmol)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1,1-DCEmass(µmol)
Butane
1,1-DCE

22. Bioaugmented microcosm B1Bioaugmented microcosm B1
: poisoned-control: poisoned-control cont’dcont’d
• Day 0.1: T-RFL of 169.76 bp was dominatedDay 0.1: T-RFL of 169.76 bp was dominated
• Day 149.9: T-RFL of ~85 bp became dominant (43%)/ 169 76 bp (2%)Day 149.9: T-RFL of ~85 bp became dominant (43%)/ 169 76 bp (2%)
T-RFL (base pair)
RelativeFluorescenceUnit
169.76 bp
Day 0.1
Day 13.9
Day 149.9
84.75 bp
T-RFL (base pair)
RelativeFluorescenceUnit
169.76 bp
Day 0.1
Day 13.9
Day 149.9
84.75 bp
0
20
40
60
80
100
0.1 13.9 149.9
Time, daysPortionofpeakareafor
T-RFL,% 84.75 bp 169.76 bp 237.68 bp 253.37 bp
276.78 bp 279.71 bp 281.71 bp others

23. Bioaugmented microcosm B5, B6 and B7Bioaugmented microcosm B5, B6 and B7
: fed butane with no 1,1-DCE present: fed butane with no 1,1-DCE present
• Butane utilization occurred successfully for 5 successive feedings and 1 kinetic testButane utilization occurred successfully for 5 successive feedings and 1 kinetic test
• Similar trends within triplicatesSimilar trends within triplicates
B5
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160 180 200 220
Time (days)
Butanemass(µmol)
Butane

24. Bioaugmented microcosm B5, B6 and B7Bioaugmented microcosm B5, B6 and B7
: fed butane with no 1,1-DCE present: fed butane with no 1,1-DCE present cont’dcont’d
T-RFL (base pair)
RelativeFluorescenceUnit
B5
Day 0.1
Day 5
Day 162
Day 164
Day 215
169.73 bp
183.31 bp
210.22 bp
T-RFL (base pair)
RelativeFluorescenceUnit
B5
Day 0.1
Day 5
Day 162
Day 164
Day 215
169.73 bp
183.31 bp
210.22 bp
0
20
40
60
80
100
0.1 4.7 161.9 164.4 215.2
Time (days)
Portionofpeakareaof
T-RFL(%)
169.73 bp 183.31 bp 210.22 bp 276.95 bp
279.81 bp 281.71 bp others

25. Bioaugmented microcosms B2, B3 and B4Bioaugmented microcosms B2, B3 and B4
: pre-exposed to 1,1-DCE: pre-exposed to 1,1-DCE
0
20
40
60
80
0 20 40 60 80 100 120 140 160
Time (days)
Butanemass(µmol)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1,1-DCEmass(µmol)
Butane
1,1-DCE
B2

26. Bioaugmented microcosms B2, B3 and B4Bioaugmented microcosms B2, B3 and B4
: pre-exposed to 1,1-DCE: pre-exposed to 1,1-DCE
0
20
40
60
80
100
0.1 13.9 75.0 93.8 142.8
Time (days)
Portionofpeakareaof
T-RFL(%)
169.68 bp 179 bp 207.81 bp 210.02 bp
277.84 bp 279.85 bp 281.58 bp others
B2

27. Bioaugmented microcosms B2, B3 and B4Bioaugmented microcosms B2, B3 and B4
: pre-exposed 1,1-DCE: pre-exposed 1,1-DCE cont’dcont’d
T-RFLP (base pair)B2
RelativeFluorescenceUnit
169.68 bp
179 bp
Day 0.1
Day 14
Day 75
Day 94
Day 143
T-RFLP (base pair)B2
RelativeFluorescenceUnit
169.68 bp
179 bp
Day 0.1
Day 14
Day 75
Day 94
Day 143
T-RFL (base pair)
RelativeFluorescenceUnit
Day 14
Day 85
Day 94
Day 144
169.66 bp
277.82 bp
B3 T-RFL (base pair)
RelativeFluorescenceUnit
Day 14
Day 85
Day 94
Day 144
169.66 bp
277.82 bp
B3
T-RFL (base pair)
169.65 bp
RelativeFluorescenceUnit
179 bp
Day 14
Day 78
Day 94
Day 143
Day 151
B4 T-RFL (base pair)
169.65 bp
RelativeFluorescenceUnit
179 bp
Day 14
Day 78
Day 94
Day 143
Day 151
B4

28. Bioaugmented microcosm B8, B9 and B10Bioaugmented microcosm B8, B9 and B10
: simultaneously fed butane and 1,1-DCE: simultaneously fed butane and 1,1-DCE
0
20
40
60
80
0 20 40 60 80 100 120 140 160
Time (days)
Butanemass(µmol)
0
0.5
1
1.5
2
2.5
1,1-DCE(µmol)
Butane
1,1-DCE
B8

29. Bioaugmented microcosm B8, B9 and B10Bioaugmented microcosm B8, B9 and B10
: simultaneously fed butane and 1,1-DCE: simultaneously fed butane and 1,1-DCE cont’dcont’d
T-RFL (base pair)
RelativeFluorescenceUnit
B8
Day 0.1
Day 12
Day 33
Day 71
Day 89
Day 124
Day 126
Day 132
169.72 bp
183.31 bp
179.01 bp
T-RFL (base pair)
RelativeFluorescenceUnit
B8
Day 0.1
Day 12
Day 33
Day 71
Day 89
Day 124
Day 126
Day 132
169.72 bp
183.31 bp
179.01 bp
0
20
40
60
80
100
0.1 12.2 33.1 70.8 88.8 123.8 125.8 131.5
Time (days)Portionofpeakareaof
T-RFL(%)
169.72 bp 179.01 bp 183.31 bp 207.81 bp 208.63 bp 210.12 bp
276.82 bp 277.71 bp 279.74 bp 281.72 bp others

30. Results of modeling 1,1-DCEResults of modeling 1,1-DCE
cometabolic transformationcometabolic transformation

31. Consecutive feedings in the bioaugmentedConsecutive feedings in the bioaugmented
microcosm B8microcosm B8
feeding Time
(hous)
Butane
(µmol)
1,1-DCE
(µmol)
1’st 0.0 68.53 0.23
2’nd 703.8 70.31 0.25
Kinetic
test
955.3
963.5
1079.6
1081.0
7.07
66.99
-
71.98
-
-
0.17
-
3’rd 1653.7 69.84 0.56
4’th 2035.7 68.01 1.17
5’th 2974.9 58.00 1.85
0
10
20
30
40
50
60
70
80
90
0 500 1000 1500 2000 2500 3000 3500 4000
Time (hours)
Butanemass(µmol)
0
0.5
1
1.5
2
2.5
1,1-DCE(µmol)
Butane
1,1-DCE

32. Microcosm experiment data and model output in B8Microcosm experiment data and model output in B8
with b=0.0028 hrwith b=0.0028 hr-1-1
and Tand Tc,1,1-DCEc,1,1-DCE=0.517x0.6 µmol 1,1-=0.517x0.6 µmol 1,1-
DCE/mg cellDCE/mg cell
0
10
20
30
40
50
60
70
80
0 500 1000 1500 2000 2500 3000 3500
Time (hours)
Butanemass(µmol)
0
0.5
1
1.5
2
2.5
1,1-DCEmass(µmol)
B8 Butane B8 Butane model
B8 1,1-DCE B8 1,1-DCE model
0
10
20
30
40
50
60
70
80
90
0 500 1000 1500 2000 2500 3000 3500
Time (hours)
Cellmass(mg/L)
B8 Cell model

33. Microcosm experiment data and model output in B8Microcosm experiment data and model output in B8
with b=0.0028 hrwith b=0.0028 hr-1-1
and Tand Tc,1,1-DCEc,1,1-DCE=0.517x2.8 µmol 1,1-=0.517x2.8 µmol 1,1-
DCE/mg cellDCE/mg cell
0
10
20
30
40
50
60
70
80
90
100
0 500 1000 1500 2000 2500 3000 3500
Time (hours)
Cellmass(mg/L)
B8 Cell model
0
20
40
60
80
0 500 1000 1500 2000 2500 3000 3500
Time (hours)
Butanemass(µmol)
0.0
0.5
1.0
1.5
2.0
2.5
1,1-DCEmass(µmol)
B8 Butane B8 Butane model
B8 1,1-DCE B8 1,1-DCE Model

34. Standard Error of Estimate (SEE)Standard Error of Estimate (SEE)
2
1
1
2^














−






−∑
n
CC ii
SEE =
where,
SEE = standard error of estimate
Ci = model prediction
Ĉi = experimental data

35. Comparison of Standard Error of Estimate inComparison of Standard Error of Estimate in
modeling B8modeling B8
Tc
*
Feedings
0.517x
0.6
0.517x
0.7
0.517x
1.0
0.517x
1.6
0.517x
2.0
0.517x
2.8
STDV
1’st**
Butane
1,1-
DCE
16.52
0.03
16.71
0.01
26.95
0.04
31.76
0.06
32.64
0.06
33.92
0.06
7.95
0.02
2’nd**
Butane
1,1-
DCE
6.26
0.03
2.64
0.01
3.80
0.01
9.35
0.02
10.87
0.02
12.59
0.02
3.99
0.01
Kinetic test***
Butane
Butane
1,1-DCE
Butane
1.58
12.00
0.08
3.08
1.58
12.06
0.08
8.90
1.48
10.75
0.08
10.53
1.36
9.38
0.08
15.69
1.32
8.90
0.08
16.20
1.27
8.36
0.08
16.77
0.13
1.60
0.00
5.39
3’rd**
Butane
1,1-
DCE
27.56
0.15
21.17
0.13
12.20
0.09
6.82
0.06
5.79
0.05
5.80
0.04
9.18
0.05
4’th**
Butane
1,1-
35.65
0.66
34.48
0.58
27.91
0.26
16.11
0.07
19.96
0.07
24.01
0.08
7.38
0.27Orange-colored values indicate the best fit for that portion of the test

36. ConclusionsConclusions
• Bioaugmented butane utilizers showed a reduced lag periodBioaugmented butane utilizers showed a reduced lag period
compared to indigenous butane utilizers.compared to indigenous butane utilizers.
• There were differences in the lag time between bioaugmentedThere were differences in the lag time between bioaugmented
microcosm setsmicrocosm sets
-The microcosms fed butane only and not-exposed to 1,1-DCE-The microcosms fed butane only and not-exposed to 1,1-DCE
showed immediate butane utilization without significant lagshowed immediate butane utilization without significant lag
time (time to 50% of butane removal=3 days).time (time to 50% of butane removal=3 days).
-The microcosms simultaneously fed butane and 1,1-DCE had a-The microcosms simultaneously fed butane and 1,1-DCE had a
longer lag time (time to 50% of butane removal=11 days).longer lag time (time to 50% of butane removal=11 days).
-The microcosms pre-exposed to 1,1-DCE for 29 days with no-The microcosms pre-exposed to 1,1-DCE for 29 days with no
butane presence showed a much longer lag time (time to 50%butane presence showed a much longer lag time (time to 50%
of butane removal=48 days).of butane removal=48 days).

37. ConclusionsConclusions cont’dcont’d
• The T-RFLP method showed good consistency with theThe T-RFLP method showed good consistency with the
microcosm study results. It was possible to characterizemicrocosm study results. It was possible to characterize
microbial community structure and track microbialmicrobial community structure and track microbial
community shifts.community shifts.
• There were differences in the microbial profiles between theThere were differences in the microbial profiles between the
indigenous microcoam and the bioaugmented microcosmindigenous microcoam and the bioaugmented microcosm
sets. The difference was existed within the indigenoussets. The difference was existed within the indigenous
microcosms fed in different patterns.microcosms fed in different patterns.
-The indigenous microcosms exposed to butane with no 1,1--The indigenous microcosms exposed to butane with no 1,1-
DCE presence, microorganisms corresponding to the T-RFLDCE presence, microorganisms corresponding to the T-RFL
of 179 base pair was predominant.of 179 base pair was predominant.
-The indigenous microcosms fed butane and 1,1-DCE-The indigenous microcosms fed butane and 1,1-DCE
simultaneously, a T-RFL of 207.5 base pair was dominant.simultaneously, a T-RFL of 207.5 base pair was dominant.

38. ConclusionsConclusions cont’dcont’d
• There were differences in microbial community profileThere were differences in microbial community profile
between bioaugmented microcosm sets.between bioaugmented microcosm sets.
- The T-RFL of 183 bp dominated the microcosms- The T-RFL of 183 bp dominated the microcosms
fed butane only and not-exposed to 1,1-DCE andfed butane only and not-exposed to 1,1-DCE and
microcosms simultaneously fed butane and 1,1-DCE. Themicrocosms simultaneously fed butane and 1,1-DCE. The
T-RFL of 183 bp was present in the source culture andT-RFL of 183 bp was present in the source culture and
bioaugmented culture but not in the indigenous culture.bioaugmented culture but not in the indigenous culture.
- The T-RFL of 179 or 277 bp dominated the microcosms- The T-RFL of 179 or 277 bp dominated the microcosms
pre-exposed to 1,1-DCE for 29 days with no butane presentpre-exposed to 1,1-DCE for 29 days with no butane present
These results indicates that the toxicity pressure ofThese results indicates that the toxicity pressure of
1,1-DCE transformation cause the change in the microbial1,1-DCE transformation cause the change in the microbial
community.community.

39. ConclusionsConclusions cont’dcont’d
• The model predicted the general trends of biotransformationThe model predicted the general trends of biotransformation
when using the kinetic, inhibition, and product toxicitywhen using the kinetic, inhibition, and product toxicity
values determined from independent experiments. Althoughvalues determined from independent experiments. Although
the model did not distinguish between the different microbesthe model did not distinguish between the different microbes
present, it reasonably simulated the microcosm performance.present, it reasonably simulated the microcosm performance.
• The model sensitivity analysis focused on TThe model sensitivity analysis focused on Tc,1,1-DCEc,1,1-DCE value. Bothvalue. Both
butane utilization and 1,1-DCE transformation werebutane utilization and 1,1-DCE transformation were
sensitive to this value, especially at high dose of 1,1-DCEsensitive to this value, especially at high dose of 1,1-DCE
exposure.exposure.

40. Engineering significance of this workEngineering significance of this work
• A significant contribution of this study was developing theA significant contribution of this study was developing the
laboratory methods to evaluate the microbial abilities tolaboratory methods to evaluate the microbial abilities to
cometabolize 1,1-DCE, extremely toxic CAH, and determiningcometabolize 1,1-DCE, extremely toxic CAH, and determining
microorganisms correlated with those biotransformationmicroorganisms correlated with those biotransformation
activities.activities.
• This study demonstrated that a culture could be added andThis study demonstrated that a culture could be added and
performed well under conditions that mimic groundwaterperformed well under conditions that mimic groundwater
remediation.remediation.
• Since T-RFLP was successfully used to track certainSince T-RFLP was successfully used to track certain
bioaugmented microorganisms, it may be possible to determinebioaugmented microorganisms, it may be possible to determine
the fate of bioaugmented microorganism in the field.the fate of bioaugmented microorganism in the field.
• The model comparison to experimental data indicated that thereThe model comparison to experimental data indicated that there
was a potential in using the existing model to predict andwas a potential in using the existing model to predict and
improve bioremediation strategies.improve bioremediation strategies.

41. AcknowledgementsAcknowledgements
• Dr. Lewis SempriniDr. Lewis Semprini
• Dr. Mark E. DolanDr. Mark E. Dolan
• Dr. Stephen J. GiovannoniDr. Stephen J. Giovannoni
• Dr. Robert W. CollierDr. Robert W. Collier
• Past and present colleagues in MerryfieldPast and present colleagues in Merryfield
• Family in KoreaFamily in Korea
• My husband, Jae-Hyuk Lee and my almost-one year daughter,My husband, Jae-Hyuk Lee and my almost-one year daughter,
HannahHannah

42. AppendicesAppendices

43. Commonly used molecular biological analysisCommonly used molecular biological analysis
• without-PCR method: Fluorescence in situwithout-PCR method: Fluorescence in situ
hybridization (FISH)hybridization (FISH)
--using 16S rRNA probesusing 16S rRNA probes
-without polymerase chain reaction (PCR).-without polymerase chain reaction (PCR).
-since the probes target labile rRNA, not DNA,-since the probes target labile rRNA, not DNA,
provides definitive confirmation of theprovides definitive confirmation of the
presence of active species in a consortium.presence of active species in a consortium.

44. Commonly used molecular biological analysisCommonly used molecular biological analysis
cont’dcont’d
• PCR-based method-PCR-based method- techniques for communitytechniques for community
fingerprintingfingerprinting
-D-Denaturing Gradient Gel Electrophoresis (DGGE)enaturing Gradient Gel Electrophoresis (DGGE)
-Temperature Gradient Gel Electrophoresis (TGGE)-Temperature Gradient Gel Electrophoresis (TGGE)
-Single-Strand-Conformation Polymorphism (SSCP)-Single-Strand-Conformation Polymorphism (SSCP)
--Terminal- Restriction Fragment LengthTerminal- Restriction Fragment Length
Polymorphism (T-RFLP)Polymorphism (T-RFLP)

45. Commonly used molecular biologicalCommonly used molecular biological
analysis-analysis-limitationslimitations
--data analysis as with all methods, there are importantdata analysis as with all methods, there are important
limitations relate tolimitations relate to
-sample collection-sample collection
-nucleic acids extraction from environmental samples-nucleic acids extraction from environmental samples
-kinetic biases-kinetic biases
-artifacts associated with enzymatic amplification-artifacts associated with enzymatic amplification

46. Commonly used molecular biologicalCommonly used molecular biological
analysis-analysis- limitationslimitations cont’dcont’d
• A major limitation-quantitative recovery of nucleic acids fromA major limitation-quantitative recovery of nucleic acids from
environmental samples :Usually spores and gram-positive cellenvironmental samples :Usually spores and gram-positive cell
are more resistant than vegetative and gram-negative cell.are more resistant than vegetative and gram-negative cell.
• problems during PCR amplificationproblems during PCR amplification
-Humic acids or humic substances co-extracted with nucleic-Humic acids or humic substances co-extracted with nucleic
acids strongly inhibit enzymatic function for DNAacids strongly inhibit enzymatic function for DNA
modification.modification.
-Differential PCR amplification has many limitations.-Differential PCR amplification has many limitations.
Amplified DNA can reflect quantitative abundance of speciesAmplified DNA can reflect quantitative abundance of species
if the amplification efficiencies are same for all molecules.if the amplification efficiencies are same for all molecules.
-the appearance of PCR artifacts-a potential risk in the PCR--the appearance of PCR artifacts-a potential risk in the PCR-
mediated analysis: it suggests the existences of organisms thatmediated analysis: it suggests the existences of organisms that
do not actually exist in the sample investigated. PCR artifactsdo not actually exist in the sample investigated. PCR artifacts
include formation of chimeric molecules, deletion mutants,include formation of chimeric molecules, deletion mutants,
and point mutants.and point mutants.

47. T-RFLP results for clone libraryT-RFLP results for clone library
GeneBank database
comparison
Predicted
MnlI T-RFL
Actual
MnlI T-RFL
Hydrogenophaga
palleronii
209 207.3-207.7
Acidovorax 277 277.1-277.3
Rhodococcus 179 178.9, 179.0
Unidentified bacterium
(deep clay)
168 167.0, 167.2
Ferribacterium 237.2
Ultramicrobacterium
(proteobacterium)
100.4
Hydrogenophaga
palleronii ???
208.7
Adapted from results of clone library with the source culture, Dolan (2002).

48. Comparison of T-RFLs in the source,Comparison of T-RFLs in the source,
bioaugmentation and no-manipulatedbioaugmentation and no-manipulated
indigenous cultureindigenous cultureculture
T-RFL (base pair)
Source
culture (%)
Bioaugmentation
culture (%)
Indigenous
culture (%)
40.79 - 6.26 -
41.60 - 4.68 -
42.67 - 5.01 -
46.40 - 0.77 -
47.43 1.14 - -
48.21 - - 3.21
51.44 - 4.78 -
63.72 - 3.48
67.34 - 0.92 -
99.16 1.86 - -
100.46 - 2.27 -
131.79 - 0.85 -
167.22 4.67 10.21 2.35
168.63 - - 0.85
169.73 - - 40.52
173.10 - - 1.65
178.97 35.13 1.58 -
183.24 1.37 14.86 -
184.36 1.71 - -
206.51 - 0.88 -
207.60 0.49 26.34 -
210.60 2.8 4.61 -
211.23 - - 2.44
212.78 - - 0.90
213.43 - - 2.85
237.55 - - 2.39
239.61 - - 2.26
251.00 - - 1.75
253.69 - - 2.84
255.21 - - 2.06
275.55 - 1.08 -
276.61 42.98 6.44 -
277.28 - - 6.24
278.49 - - 2.26
279.46 - 3.84 13.45
281.53 - 1.14 12.81
283.51 - - 4.01

49. Starting parameter values and units used inStarting parameter values and units used in
modeling bioaugmented microcosm resultsmodeling bioaugmented microcosm results
Parameter Value Unit
Butane concentration*
approx. 7, 70 µmol
1,1-DCE concentration*
0.2-1.9 µmol
Initial cell concentration in
bioaugmented microcosm (X0)*
1.385 mg/L
Cell yield (Y)**
0.0406 mg cell/µmol butane
Decay (b)**
0.00625 hr-1
Hcc,Butane
38 -
Hcc,1,1-DCE
0.8576 -
Kic,DCEBUT
***
8.7 µmol/L
Kic,BUTDCE
***
0.33 µmol/L
Kiu,BUTDCE
***
6.9 µmol/L
kmax,Butane
**
1.261 µmol/mg cell-hr
kmax,1,1-DCE
***
1.3 µmol/mg cell-hr
Ks,Butane
**
1.897 µmol butane/L
Ks,1,1-DCE
***
1.48 µmol 1,1-DCE/L
Transformation capacity (Tc)***
0.517 µmol 1,1-DCE/mg cell
Volume of gas phase (VG
) 0.076 L

50. Required future workRequired future work
• Set up the method to prepare appropriateSet up the method to prepare appropriate
control microcosm which can functioncontrol microcosm which can function
reasonablyreasonably
• Search the potential microorganisms correlatedSearch the potential microorganisms correlated
with concerned T-RFL using web-basedwith concerned T-RFL using web-based
databasedatabase