AN ABSTRACT OF THE THESIS OF
Darin Rungkamol for the degree of Master of Science in Civil Engineering
presented on March 15, 2001. Title: Aerobic Cometabolism of 1,1.1
Trichioroethane and Other Chlorinated Aliphatic Hydrocarbons by Indigenous and
Redacted for privacy
This thesis has focused on developing a culture of butane-utilizing
microorganics to be bioaugmented into the subsurface for the cometabolie
treatment of 1,1,1 -TCA, TCE and 1,1 -DCE. The culture was tested in groundwater
and aquifer solid microcosms that mimic conditions where a field test is to be
performed. The butane mixed culture was selected from the existing microcosms
(Jitnuyanont 1998) by comparing 1,1,1 -TCA transformation abilities among these
microcosms. Butane-utilizers that showed rapid 1,1,1 -TCA transformation kinetics
and long-term (10 - 44 days) 1,1,l-TCA transformation ability in the absence of
butane utilization were selected. Modeling studies were performed and compared
to the results of microcosm test to generate kinetic parameters for the microbial
processes under conditions of the field.
Microcosms bioaugmented with the selected culture maintained effective
transformation of 1,1,1 -TCA, and mixtures of 1,1,1 -TCA, TCE, and 1,1 -DCE.
Little 1,1,1 -TCA transformation was found in microcosms where indigenous
microorganisms were stimulated. The transformation of 1,1 -DCE in the
bioaugmented microcosms was the fastest, followed by 1,1,1 -TCA and TCE. The
limited transformation of chlorinated solvents and the cessation in butane
utilization occurred after the transformation of the CAH mixtures.
The culture having the best 1,1,1 -TCA transformation ability was developed
for bioaugmentation studies for both the laboratory and the field tests. The culture
was grown on mineral salts growth media, harvested, placed in 1 mL vials with
7%DMSO (Dimethyl Sulfoxide), and stored in 80 °C liquid nitrogen for future
studies. The frozen cells were thawed, washed with growth media to rinse away
DMSO and then grown in growth media. The microcosms bioaugmented with cell
grown from the very frozen culture had reproducible performance, maintaining the
long-term (13 days) l,1,l-TCA transformation in the absence of butane utilization
and transforming mixtures of 1,1,1 -TCA, TCE, and 1,1 -DCE. The transformation
rates gradually decreased with continued additions of 1,1,1 -TCA. The cell decay
rates (b) determined with an analytical regression method were also similar to those
obtained prior to using the frozen culture.
Half-saturation constants for butane and 1,1,1 -TCA (Ks,But and KS,TCA) of
bioaugmented cultures under microcosm conditions were determined using an
analytical regression method that was developed. The bioaugmented
microorganisms had KS,Bt and
KS,TCA
values of 0.11 mg/L and of 0.37 mg/L,
respectively.
Kinetic parameters including maximum specific rate for butane utilization
(kmax,But) and 1,1,1 -TCA transformation
(kmaxTCA),
cell decay rate (b), and
transformation capacity (Ta) for indigenous and bioaugmented microcosms were
determined through simulations using a non-steady-state model. Reasonable fits
between the model simulation and microcosm data at both low and high
concentrations of both butane and 1,1,1 -TCA were obtained. The values of kinetic
parameters in all bioauginented microcosms, which were inoculated with the
media-culture from batches of cells grown at different times, were similar. This
indicates the reproducibility of bioaugmentation process. The bioaugmented
microorganisms had high transformation capacity (0.1 0.3 mg 1,1,1-TCA/mg
cells) and achieved ratios Of km,But and kmX,TCA, ranging from 0.05 to 0.09. The
indigenous microorganisms potentially had lower transformation capacity
(Ta),
higher decay rate (b), higher butane and 1,1,1 -TCA half-saturation constants (Ks,But
and KS,TCA), and lower butane and 1,1,1 -TCA maximum specific rates (kmax,But and
kmax,TCA) than the bioaugmented microorganisms.
Different butane-utilizing cultures are likely present in the indigenous and
bioaugmented microcosms. A slower rate of 1,1,1 -TCA transformation was
probably due to the indigenous microorganism being less effective towards 1,1,1 -
TCA transformation. Unlike the bioaugmented mierocosms, the enzyme present in
the indigenous microcosms was not active over long periods of time.
Augmentation with proven contaminant-degrading microorganisms having
consistent transformation abilities will likely lead to improved bioremediation
treatment processes.
Aerobic Cometabolism of 1,1,1 -Trichioroethane and Other Chlorinated Aliphatic
Hydrocarbons by Indigenous and Bioaugmented Butane-Utilizers in Moffett Field
Microcosms
by
Darin Rungkamol
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented March 15, 2001
Commencement June 2001
Master of Science thesis of Darin Rungkamol presented on March 15, 2001
APPROVED:
Redacted for privacy
MajrP'rofessor, representii Civil
Redacted for privacy
Head of Department of Civil, Construction, and Environmental Engineering
Redacted for privacy
Dean of tl{ejllraduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to
any reader upon request.
Redacted for privacy
Darin
ACKNOWLEDGMENTS
First of all, I would like to take this opportunity to thank Dr. Lewis
Semprini, my major professor, for giving me an opportunity to work with him. He
has considerably provided a new valuable knowledge and guidance beyond all
expectations during my pursuit of this degree. I would also like to express my
sincere appreciation to Dr. Mark E. Dolan for all his help and patience during the
course of this research and my stay at Oregon State University. My work would
did have gone as well without his suggestions. I also appreciate the assistance of
my committee members, Dr. Peter 0. Nelson, and my graduated representative, Dr.
James A. Coakley, Jr. I greatly appreciate all my colleagues, especially Dr. Young
Kim, and Adisorn (Merf) Tovanabootr, for their laboratory assistance as I started
my research. Merf, your kindheartedness will be in my memory for the rest of my
life. I would also like to thank Brian Timmins for helping me review my thesis
draft. I am most thankful to my parents, Kamol and Vilavan Rungkamol, who have
provided endless love, support, and encouragement to my life.
TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION ......................................................... 1
1.1 PROBLEM DEFINITION ...................................................
1
1.2 OBJECTIVES .................................................................. 3
1.2.1 Culture development for bioaugmentation studies ............. 3
1.2.2 Microcosm studies with groundwater and soil from the
original microcosms of part 1 .................................... 4
1.2.3 Microcosm studies with the frozen butane-utilizing culture. .4
CHAPTER 2: LITERATURE REVIEW................................................. 6
2.1 PROPERTIES OF 1,l,1-TCA ............................................... 6
2.2 TRANSFORMATION of 1,1,1-TCA....................................... 7
2.2.1 Cometabolism by methane-utilizers ............................. 8
2.2.2 Cometabolism by butane-utilizers ............................... 10
2.2.3 Cometabolism by propane-utilizers .............................. 12
2.3 KINETICS AND MODEL DEVELOPMENT OF CAH
COMETABOLIC TRANSFORMATION ................................. 13
2.4 BIOAUGMENTATION ...................................................... 16
TABLE OF CONTENTS (CONTINUED)
Page
CHAPTER 3: MATERIALS AND METHODS ....................................... 18
3.1 CHEMICAL SOURCES ...................................................... 18
3.2 MICROCOSM PREPARATION AND OPERATION .................. 18
3.2.1 Preparation of CAH saturated aqueous stock solution ......... 19
3.2.2 Utilization of butane and propane ............................... 19
3.2.3 Transformation of 1,1,1-TCA .................................... 19
3.2.4 Transformation of CAll mixture ................................. 20
3.2.5 Rate measurement of 1,1,1-TCA transformation ............... 20
3.2.6 Butane and 1,1,1 -TCA kinetic experiments ..................... 21
3.2.7 Groundwater amendment .......................................... 21
3.3 GROWTH REACTORS ...................................................... 22
3.3.1 Growth reactor inoculated with groundwater/soil slurry ...... 22
3.3.2 Growth reactor inoculated with frozen culture ................. 23
3.4 BIOAUGMENTATION ...................................................... 24
3.4.1 Bioaugmentation with groundwater/soil slurry ................ 27
3.4.2 Bioaugmentation with media-culture ............................ 27
3.5 ANALYTICAL PROCEDURES ............................................. 27
3.6 MODEL DEVELOPMENT ................................................. 28
TABLE OF CONTENTS (CONTINUED)
CHAPTER 4: RESULTS AND DISSCUSSION
4.1 CULTURE DEVELOPMENT FOR BIOAUGMENTATION
STUDIES .......................................................................
.32
32
4.2 MICROCOSM STUDIES WITH GROUNDWATER AND SOIL
FROM MICROCOSM B6 ................................................... 42
4.2.1 Transformation of l,1,1-TCA..................................... 42
4.2.2 Transformation of CAH mixture ................................. 61
4.2.3 Selecting microcosm for bioaugmentation studies ............ 65
4.3 MICROCOSM STUDIES WITH THE FROZEN BUTANEUTILIZiNG CULTURE ...................................................... 66
4.3.1 Transformation of chlorinated solvents ......................... 66
4.3.2 Butane and l,1,1-TCA kinetic experiments ..................... 76
4.3.3 Model development ................................................ 93
4.3.3.1 The simulation of butane utilization and 1,1,1TCA transformation in the BR3 microcosm set.... 94
4.3.3.2 The simulation of butane utilization and 1,1,1TCA transformation in the BR4 microcosm set... 108
4.3.3.3 The simulation of butane utilization and 1,1,1TCA transformation in the B4 microcosm set ...... 117
4.3.3.4 The simulation of butane utilization and 1,1,1TCA transformation in the BRC microcosm set.. .126
CHAPTER 5: SUMMARY AND ENGINEERING SIGNIFICANE .............. 145
5.1 SUMMARY ................................................................
145
5.2 ENGINEERING SIGNIFICANCE ....................................... 153
TABLE OF CONTENTS (CONTINUED)
Pge
BIBLIOGRAPHY
155
APPENDICES .............................................................................
161
LIST OF FIGURES
Figure
Pge
2.1
Abiotie transformation pathway of 1,1,1-TCA
9
2.2
Biotic transformation pathway of 1,1,1 -TCA
9
3.1
Continuously fed 2-L growth reactor
23
3.2
Growth curve of the continuously fed batch reactor preparing the
culture for inoculation
25
Percentage of butane and oxygen in the inlet and outlet measured
in the continuously fed batch reactor
25
Growth curve of the other continuously fed batch reactors
preparing the culture for inoculation
26
4.1
Control bottles for all the microcosms
33
4.2
Butane utilization and 1,1,1-TCA transformation achieved in
indigenous microcosm Bi
35
Butane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm B6
36
Butane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm B7
37
Propane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm P6
39
4.6
1,1,1-TCA transformation yield achieved in the microcosms
41
4.7
Butane utilization and 1,1,l-TCA transformation achieved in the
indigenous microcosms constructed with groundwater and aquifer
solids
44
3.3
3.4
4.3
4.4
4.5
LIST OF FIGURES (CONTINUED)
Figure
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
Page
Butane utilization and 1,1,1 -TCA transformation achieved in the
Slurry (microcosm B6) inoculated microcosms constructed with
groundwater and aquifer solids
46
The competitive inhibition of 1,1,1 -TCA transformation by butane
in the slurry (microcosm B6) inoculated microcosms constructed
with groundwater and aquifer solids
47
Butane utilization and 1,1,1 -TCA transformation achieved in the
media-culture inoculated microcosms constructed with
groundwater and aquifer solids
48
The competitive inhibition of l,1,l-TCA transformation by butane
in the media-culture inoculated microcosms constructed with
groundwater and aquifer solids
49
Butane utilization and 1,1,1 -TCA transformation achieved in the
slurry (microcosm B6) inoculated microcosms constructed with
mineral salts growth media
50
Transformation yield for 1,1,1 -TCA achieved in the inoculated
microcosms during the first 50 days of incubation
51
1,1,1 -TCA transformation rate data for microcosm B2- 1 during
day3o-35
53
l,1,1-TCA transformation rate data for microcosm B2-1 during
day 30 - 35 plotted on a semi-logarithmic scale
54
The decrease in pseudo-first-order rate coefficient (P) as a
function of time during day 30-35
55
1,1,1-TCA transformation rate data for microcosm B2-1 during
day38-45
57
1,1,1 -TCA transformation rate data for microcosm B2- 1 during
day 38 45 plotted on a semi-logarithmic scale
58
LIST OF FIGURES (CONTINUED)
Figure
4.19
4.20
4.21
4.22
4.23
4.24
4.25
4.26
4.27
4.28
The decrease in pseudo-first-order rate coefficient (P) as a
function of time during day 38 45
59
The decrease in pseudo-first-order rate coefficient (P) as a
function of transformed 1,1,1-TCA mass during day 30 - 35 and
day38-45
60
Butane utilization and chlorinated compound transformation
achieved in slurry-inoculated microcosm B2- 1
63
Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM2-2
64
Growth curve of one of the batch reactors providing the culture fo
bioaugmentation study
66
Butane utilization and l,l,l-TCA transformation achieved in
indigenous microcosm BR2-C3
68
Butane utilization and 1,1,1 -TCA transformation achieved in
media-culture inoculated microcosm BR2-2
69
Butane utilization and 1,1,1 -TCA transformation achieved in
media-culture inoculated microcosm BR2-3
70
1,1,1-TCA transformation rate data for microcosm BR2-2 during
day28-37
71
1,1,1-TCA transformation rate data for microcosm BR2-2 during
day 28 - 37 plotted on a logarithmic scale
72
4.29
The decrease in pseudo-first-order rate coefficient (P) as a function
of time during day 28 37
73
4.30
Butane utilization and chlorinated compound transformation achieved
in the media-culture inoculated microcosm BR2-2
75
LIST OF FIGURES (CONTINUED)
Figure
4.31
4.32
4.33
4.34
4.35
4.36
4.37
4.38
4.39
4.40
Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-2
77
Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 on day 5
78
Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 on day 6
79
Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 from day 13 to 14
80
Overall results from the 1,1,1 -TCA kinetic experiment conducted
in bioaugmented microcosm, BR3-2
82
Experimental data from the 1,1,1 -TCA kinetic experiment
conducted in microcosm BR3-2 on day 15
83
Experimental data from the 1,1,1 -TCA kinetic experiment
conducted in microcosm BR3-2 from day 15 to 16
84
Overall results from the butane and 1,1,1 -TCA kinetic experiment
conducted in bioaugmented microcosm BR4-5
86
Experimental data from the butane kinetic experiment conducted
in microcosm BR4-5 on day 4
87
Experimental data from the butane kinetic experiment conducted
in microcosm BR4-5 on day 13
88
4.41
Experimental data from the 1,1,1-TCA kinetic experiment conducted
in microcosm BR4-5 on day 5
90
4.42
Experimental data from the 1,1,1-TCA kinetic experiment conducted
in microcosm BR4-5 from day 13 to 14
91
LIST OF FIGURES (CONTINUED)
ig
Figure
4.43
Butane utilization data from microcosm BR3-5
94
4.44
Simulation of butane utilization for microcosm BR3-5
95
4.45
l,1,l-TCA transformation data from microcosm BR3-5
98
4.46
Simulation of 1,l,1-TCA transformation for microcosm BR3-5
99
4.47
Simulations of butane utilization and 1,l,1-TCA transformation for
microcosm BR3-1 without the term for transformation capacity
101
4.48
Simulations of butane utilization and 1,l,l-TCA transformation for
microcosm BR3-2 without the term for transformation capacity
102
4.49
Simulations of butane utilization and l,l,1-TCA transformation for
microcosm BR3-.3 without the term for transformation capacity
103
4.50
Simulations of butane utilization and l,1,l-TCA transformation for
microcosm BR3-4 without the term for transformation capacity
104
4.51
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BR3-5 without the term for transformation capacity
105
4.52
Simulations of l,1,l-TCA transformation for microcosm BR3-5
over a range of transformation capacities
106
4.53
Simulations of butane utilization and 1,l,l-TCA transformation for
microcosm BR4-M without the term for transformation capacity
110
4.54
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-4 without the term for transformation capacity
111
4.55
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-5 over a range transformation capacities capacity 112
4.56
Butane utilization and 1,1,1-TCA transformation achieved in
microcosm B4-3
118
LIST OF FIGURES (CONTINUED)
ig
Figure
4.57
4.58
Simulations of butane utilization for microcosm B4-3 without the
term for transformation capacity
119
Simulations of 1,1,1-TCA transformation for microcosm B4-3
without the term for transformation capacity
121
4.59
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm B4-3 over a range transformation capacities
122
4.60
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BRC-1 without the term for transformation capacity
127
4.61
Simulations of butane utilization and 1,l,l-TCA transformation in
each section for microcosm BRC-1 without the term for
transformation capacity
128
4.62
Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of transformation capacities
133
Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of decay rate
136
Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of 1,1,1 -TCA maximum specific rates
138
Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of 1,1,1 -TCA half-saturation constants
140
4.63
4.64
4.65
LIST OF TABLES
Table
4.1
Conditions of the existing microcosms (Jitnuyanont 1998)
32
4.2
The maximum transformation period in the absence of substrate
utilization, the average and final 1,1,1 -TCA transformation yield
achieved in the microcosms
40
4.3
Microcosms used for the experiment of l,l,l-TCA transformation
43
4.4
Transformation yield for 1,1,1-TCA achieved in the microcosms
during the first 50 days of incubation
52
4.5
The pseudo-first-order rate parameter (kmaxXolKs) and cell decay
rate (b) for microcosm sets B2 and B4 during day 30 35
56
Microcosms used for the experiment of CAll mixture
transformation
61
4.7
Microcosms used for the experiment of CAH transformation
67
4.8
The pseudo-first-order rate parameter (kmXIKs) and cell decay rate
(b) for the B2 and B4 microcosm sets during 28 37
74
4.9
Microcosms used for the kinetic experiments
76
4.10
Butane half-saturation constant
microcosm set
81
4.6
4.11
4.12
4.13
4.14
(Ks) achieved in the BR3
1,1,1 -TCA half-saturation constant
microcosm set
Butane half-saturation constant
microcosm set
(Ks) achieved in the BR3
85
(Ks) achieved in the BR4
1,1,1 -TCA half-saturation constant
microcosm set
89
(Ks) achieved in the BR4
Average values of butane and 1,1,1-TCA half-saturation constant
achieved in all microcosms
92
92
LIST OF TABLES (CONTINUED)
Table
4.15
Measured parameters for model development
93
4.16
Butane maximum specific rate (kmax,But) and cell decay rate (b)
achieved in the BR3 microcosm set
97
4.17
1,1,1 -TCA maximum specific rate (kmax,TCA) achieved in the BR3
microcosm set
100
Transformation capacity ranges (Ta) achieved in the BR3
microcosm set
108
4.19
Parameters for model development in the BR3 microcosm set
108
4.20
Butane and 1,1,1 -TCA maximum specific rate (km,But and kmax,TCA)
4.18
4.21
and transformation capacity (Ta) values achieved in the BR4
microcosm set
115
Parameters for model development in microcosm BR4-M and the
BR4 microcosm set
116
4.22
Butane maximum specific rate (km,But) values achieved in the B4
microcosm set
120
4,23
1,1,1-TCA maximum specific rate (kmax,TCA) and transformation
capacity (Ta) values achieved in the B4 microcosm set
124
4.24
The pseudo-first-order rate parameter (kmaxXolKs) and cell decay
rate (b) for the B4 microcosm set from the rate measurement during
day 28 37 and from the model simulation
125
4.25
Parameters for model development in the B4 microcosm set
125
4.26
Initial biomass (X0) for the BRC microcosm set
131
4.27
Initial biomass (Xo) in section E for the BRC microcosm set
obtained from the adjustment and the model simulation
131
LIST OF TABLES (CONTINUED)
Table
4.28
Transformation capacity (Ta) values achieved in the BRC
microcosm set
134
Transformation capacity (Ta) values used in the simulation of
1,1,1 -TCA transformation for the BRC microcosm set
135
4.30
Decay rate (b) values achieved in the BRC microcosm set
137
4.31
1,1,1 -TCA maximum specific rates
the BRC microcosm set
139
4.29
(km,TCA)
values achieved in
4.32
1,1,1 -TCA half-saturation constant (KS,TCA) values achieved in the
BRC microcosm set
141
4.33
Parameters for model development in the BRC microcosm set
141
4.34
Parameters used in the simulations of butane utilization and
1,1,1 -TCA transformation
142
LIST OF APPENDICES
APPENDIX A: MICROCOSM PREPARATION AND OPERATION ........... 162
A.1 AQUIFER MATERIAL ................................................... 162
A.2 GROUNDWATER ......................................................... 162
A.3 MINERAL SALT GROWTH MEDIA .................................. 163
APPENDIX B: CAH ANAYSIS ........................................................ 165
B.1 PREPARATION OF CAH STOCK SOLUTION ...................... 165
B.2 STANDARD CURVE PREPARATION ................................. 165
APPEND1X C: PROPANE AND BUTANE ANAYSIS ............................ 169
C.1 STANDARD CURVE PREPARATION ................................. 169
APPENDIX D: THE TRANSFORMATION RATE OF 1,1,1-TCA .............. 171
D.1 DERIVATION OF MONOD KINETICS ................................ 171
D.2 EXAMPLE OF CALCULATION ........................................ 174
LIST OF APPENDICES (CONTINUED)
APPENDIX E: THE KINEITC EXPERIMENT OF BUTANE AND
1,l,l-TCA .................................................................................
178
E.1 DERIVATION OF MONOD KINETICS ................................ 178
E. 1.1 Psuedo-first order model ........................................ 179
E.1.2 Zero-order model ................................................ 180
E.2 EXAMPLE OF CALCULATION........................................ 181
APPENDIX F: MODEL DEVELOPMENT .......................................... 184
F.1 BUTANE UTILZATION AND 1,1,1-TCA TRANSFORMATION. 184
F.2 MICROBIAL GROWTH ................................................... 186
APPENDIX G: LABORATORY DATA.............................................. 192
G. 1 CULTURE DEVELOPMENT FOR BIOAUGMENTATION
STUDIES .....................................................................
192
G.2 MICROCOSM STUDIES WITH GROUNDWATER AND SOIL
FROM MICROCOSM B6 .................................................. 196
G.3 MICROCOSM STUDIES WITH THE FROZEN BUTANEUTILIZING CULTURE ................................................... 203
LIST OF APPENDIX FIGURES
Figure
Bi
Chromatograms for 1,1-DCE, 1,1,1-TCA and TCE
166
C.1
Chromatograms for butane
170
D.1
1,1,1-TCA transformation data for microcosm B2-1
174
D.2
1,1,1-TCA transformation rate data plotted on a semi-logarithmic
scale
175
D.3
The decrease in pseudo-first-order rate coefficient (P) as a function 177
E. 1
Experimental data for the kinetic experiment conducted in
microcosm BR3-2
181
E.2
Experimental data at high concentration plotted against time to test
for zero-order kinetics
183
E.3
Experimental data at low concentration plotted on a logarithmic
scale to test for first-order kinetics
183
F. 1
Map for model simulation in STELLA®
188
F.2
Diagram for model simulation in STELLA®
189
F.3
Equations for model simulation in STELLA®
190
G. 1
Propane utilization and 1,1,1 -TCA transformation achieved in
indigenous microcosm P1
192
Propane utilization and 1,1,1 -TCA transformation achieved in
bioaugmented microcosm P2
193
Propane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm P7
194
Butane utilization and 1,1,1 -TCA transformation achieved in
bioaugmented microcosm B2
195
G.2
G.3
G.4
LIST OF APPENDIX FIGURES (CONTINUED)
Figure
G.5
Control bottles of microcosm sets Bi, B2 and B4
196
G.6
Butane utilization and chlorinated compound transformation
achieved in slurry-inoculated microcosm B2-2
200
Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM1-2
201
Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM3-2
202
Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BR2-3
204
Overall results from the butane kinetic experiments conducted
in the bioaugmented microcosm, BR3-1
205
Overall results from the butane kinetic experiments conducted
in the bioaugmented microcosm, BR3-3
205
Overall results from the butane kinetic experiments conducted
in the bioaugmented microcosm, BR3-4
206
Overall results from the butane kinetic experiments conducted
in the bioaugmented microcosm, BR3-5
206
G.7
G.8
G.9
G. 10
G. 11
G. 12
G. 13
G. 14
Overall results from the 1,1,1 -TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-1
208
G. 15
Overall results from the 1,1,1 -TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-3
208
G. 16
Overall results from the 1,1,1 -TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-4
209
G. 17
Overall results from the 1,1,1 -TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-5
209
LIST OF APPENDIX FIGURES (CONTINUED)
Figure
G. 18
G. 19
G.20
G.2 1
G.22
Overall results from the butane and 1,1,1 -TCA kinetic experiments
conducted in bioaugmented microcosm BR4-M
211
Overall results from the butane and 1,1,1 -TCA kinetic experiments
conducted in bioaugmented microcosm BR4- 1
212
Overall results from the butane and 1,1,1 -TCA kinetic experiments
conducted in bioaugmented microcosm BR4-2
213
Overall results from the butane and 1,1,1 -TCA kinetic experiments
conducted in bioaugmented microcosm BR4-3
214
Overall results from the butane and 1,1,1 -TCA kinetic experiments
conducted in bioaugmented microcosm BR4-4
215
G.23
Simulations of 1,1,1-TCA transformation for microcosm BR3-1 over a
range transformation capacities
220
G.24
Simulations of 1,1,1-TCA transformation for microcosm BR3-2 over a
range transformation capacities
220
G.25
Simulations of 1,1,1 -TCA transformation for microcosm BR3-3 over a
range transformation capacities
223
G.26
Simulations of 1,1,1-TCA transformation for microcosm BR3-4 over a
range transformation capacities
223
G.27
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-1 without the term for transformation capacity
225
Simulations of butane utilization and l,1,1-TCA transformation for
microcosm BR4-2 without the term for transformation capacity
227
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BR4-3 without the term for transformation capacity
229
G.28
G.29
LIST OF APPENDIX FIGURES (CONTINUED)
Figure
G.30
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-5 without the term for transformation capacity
231
G.3 1
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BR4-M over a range transformation capacities
235
G.32
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-1 over a range transformation capacities
241
G.3 3
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BR4-2 over a range transformation capacities
247
G.34
Simulations of butane utilization and 1,1,1-TCA transformation for
microcosm BR4-3 over a range transformation capacities
253
G.35
Simulations of butane utilization and 1,l,l-TCA transformation for
microcosm BR4-4 over a range transformation capacities
259
G.36
Simulations of butane utilization for microcosm B4-lwithout the
term for transformation capacity
263
Simulations of 1,1,1-TCA transformation for microcosm B4-1
without the term for transformation capacity
263
G.37
G.38
Simulations of butane utilization and 1,l,l-TCA transformation for
microcosm B4-1 over a range transformation capacities
264
G.39
Simulations of butane utilization for microcosm B4-2 without the
term for transformation capacity
267
G.40
Simulations of l,1,1-TCA transformation for microcosm B4-2
without the term for transformation capacity
GAl
267
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm B4-2 over a range transformation capacities
268
LIST OF APPENDIX FIGURES (CONTINUED)
Figure
G.42
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BRC-2 without the term for transformation capacity
271
G.43
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BRC-3 without the term for transformation capacity
273
G.44
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BRC-4 without the term for transformation capacity
275
G.45
Simulations of butane utilization and 1,1,1 -TCA transformation for
microcosm BRC-5 without the term for transformation capacity
277
G.46
Simulations of 1,1,1 -TCA transformation for microcosm BRC-2
over a range of transformation capacities
279
Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of transformation capacities
281
Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of transformation capacities
283
Simulations of 1,1,1 -TCA transformation for microcosm BRC-2
over a range of decay rates
285
Simulations of 1,1,1 -TCA transformation for microcosm BRC-3
over a range of decay rates
287
Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of decay rates
289
Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of 1,1,1 -TCA maximum specific rates
291
Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of 1,1,1 -TCA maximum specific rates
293
Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of 1,1,1 -TCA maximum specific rates
295
G.47
G.48
G.49
G.50
G.51
G.52
G.53
G.54
LIST OF APPENDIX FIGURES (CONTINUED)
Figure
G.55
G.56
G.57
Page
Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of 1,1,1 -TCA half-saturation constants
297
Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of 1,1,1 -TCA half-saturation constants
299
Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of 1,1,1-TCA half-saturation constants
301
LIST OF APPENDIX TABLES
Table
A. 1
List of core material used in microcosm fabrication
162
D. 1
The values of the pseudo first-order rate coefficient (P) and time
176
0.1
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B2-1
196
G.2
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B2-2
197
G.3
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B2-3
197
0.4
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B4-1
198
G. 5
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B4-2
199
G.6
The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement for
microcosm B4-3
199
0.7
The values of pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement in
microcosm BR2-2
203
LIST OF APPENDIX TABLES (CONTINUED)
ig
Table
G.8
The values of pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1 -TCA transformation rate measurement in
microcosm BR2-3
203
G.9
The results from the butane kinetic experiments conducted in the
BR3 microcosm set
207
G. 10
The results from the 1,1,1 -TCA kinetic experiments conducted in
the BR3 microcosm set
210
G. 11
The results from the butane kinetic experiments conducted in the
BR4 microcosm set
216
G. 12
The results from the 1,1,1 -TCA kinetic experiments conducted in
the BR4 microcosm set
217
G. 13
Parameters for model simulations in Figure G.23
218
G.14
Parameters for model simulations in Figure G.24
219
G. 15
Parameters for model simulations in Figure G.25
221
G.16
Parameters for model simulations in Figure G.26
222
G. 17
Parameters for model simulations in Figure G.27
224
G. 18
Parameters for model simulations in Figure G.28
226
G. 19
Parameters for model simulations in Figure G.29
228
G.20
Parameters for model simulations in Figure G.30
230
G.21
Parameters for model simulations in Figure G.31
232
G.22
Parameters for model simulations in Figure G.32
238
LIST OF APPENDIX TABLES (CONTINUED)
Table
G.23
Parameters for model simulations in Figure G.33
244
G.24
Parameters for model simulations in Figure G.34
250
G.25
Parameters for model simulations in Figure G.35
256
G.26
Parameters for model simulations in Figures G.36 and G.37
262
G.27
Parameters for model simulations in Figure G.38
262
G.28
Parameters for model simulations in Figures G.39 and G.40
266
G.29
Parameters for model simulations in Figure G.41
266
G.30
Parameters for model simulations in Figure G.42
270
G.3 I
Parameters for model simulations in Figure G.43
272
G.32
Parameters for model simulations in Figure G.44
274
G.33
Parameters for model simulations in Figure G.45
276
0.34
Parameters for model simulations in Figure G.46
278
G.35
Parameters for model simulations in Figure G.47
280
G.36
Parameters for model simulations in Figure G.48
282
G.37
Parameters for model simulations in Figure G.49
284
0.38
Parameters for model simulations in Figure G.S0
286
G.39
Parameters for model simulations in Figure G.5 1
288
0.40
Parameters for model simulations in Figure G.52
290
LIST OF APPENDIX TABLES (CONTINUED)
Table
Page
G.41
Parameters for model simulations in Figure G.53
292
G.42
Parameters for model simulations in Figure G.54
294
G.43
Parameters for model simulations in Figure G.55
296
G.44
Parameters for model simulations in Figure G.56
298
G.45
Parameters for model simulations in Figure G.57
300
Aerobic Cometabolism of 1,1,1-Trichioroethane and Other
Chlorinated Aliphatic Hydrocarbons by Indigenous and
Bioaugmented Butane-Utilizers in Moffett Field Microcosms
CHAPTER 1
INTRODUCTION
1.1 PROBLEM DEFINITION
Groundwater contamination is a major environmental problem encountered
in the United States since groundwater is used as a source of drinking water supply.
An assessment of the extent and severity of contamination is further complicated
by the almost exponential growth of the synthetic organic chemistry industry in the
U.S. since the early '40s (Barcelona et al., 1990). 1,1,1-trichioroethane (1,1,1TCA), one of chlorinated aliphatic hydrocarbons (CAHs), is a synthetic organic
chemical widely used as an industrial solvent, and commonly enters groundwater
because of leaking storage tanks or improper disposal. As the result, it and its
transformation products, such as 1,1 -dichroloethane (1,1 -DCA), 1,1 -dichloroethene
(1,1-DCE), and vinyl chloride (VC) are frequently reported contaminants in
groundwater and soils.
1,1,1 -TCA and other CAHs are probably the most serious groundwater
contaminants today. They and their transformation products are known or
suspected carcinogens. This has lead to development of treatment methods
including pump-and-treat, soil vapor extraction, air stripping, and activated carbon
adsorption, for their removal. These treatment processes are mostly operating at
the surface, however, and carmot complete contaminant destruction, but only
transfer contaminants from one medium to another. In addition, CAll persistence
in the subsurface environment entails the great expense of these conventional
2
remediation technologies (AlvarezCohen and McCarty, 1991a). Biological
processes occurring in situ can aerobically or anaerobically transform most CAHs,
and hence have become a potentially low-cost alternative approach for the
remediation of contaminated soils and groundwater. Microbial processes have now
gained popularity for the transformation of CAHs in groundwater.
/ Moffett Naval Air Station (Moffett Field), Mountain View, California, has
been used to evaluate in-situ transformation processes through aerobic
cometabolism for several CAHs by indigenous microorganisms that use methane,
phenol, or toluene as primary substrates. The initial concentration of target CAHs
including trichioroethene (TCE), 1,1,1 -TCA, 1,1 -DCE, cis- 1 ,2-dichloroethene (c-
DCE), tran-1,2-dichlroethene (t-DCE) and VC ranged from 60 to 250 .xg/L. The
aquifer is confined and consists of silt, fine- to coarse-grained sand and gravel. The
major contaminant found in site groundwater is 1,1,1-TCA present at an average
concentration on the order of 100 ig/L, while the other contaminants such as 1,1 DCA and TCE are present in trace amounts (Robert et al., 1990).
'
In one of the field tests, methane-oxidizing bacteria, using methane as a
primary substrate and cometabolically transforming CAHs with a methane
monooxygenase enzyme (MMO), achieved significant transformation of VC and tDCE, but exhibited limited transformation of 1,1,1 -TCA, TCE and c-DCE
(Semprini et al., 1990; Semprini et al., 1991). In other field demonstrations,
Hopkins et al. (1993) and Hopkins and McCarty (1995) found that phenol- and
toluene-oxidizing bacteria, having a toluene oxygenase enzyme responsible for the
CAHs cometabolism, were equally effective in removing c-DCE and TCE, while t-
DCE was the least transformed of the three contaminants. Hopkins and McCarty
(1995) also observed great efficiency in VC removal and moderate efficiency in
1,1 -DCE removal by phenol utilizers. In microcosm studies conducted by Hopkins
et al. (1993) and Hopkins and McCarty (1995), none of the microcosms effectively
degraded 1,1,1 -TCA, when fed phenol, toluene, methane or ammonia.
The results of these tests indicated that further studies were required to
explore 1,1,1 -TCA degradation efficiency by microorganisms having different
oxygenase systems. Jitnuyanont (1998) found that propane- and butane-oxidizing
bacteria had a very high 1,1,1 -TCA transformation capacity, representing the
maximum mass of CAH that can be transformed per unit mass of resting cell, and
that bioaugmentation was a good strategy to reduce the long lag time required to
stimulate the indigenous microorganisms in microcosms containing Moffett Field
sediments and groundwater. The enrichments used for bioaugmentation were
obtained from the Hanford DOE site, Washington (Kim 1996), which transformed
1,1,1 -TCA effectively. The augmented propane- and butane-oxidizing bacteria also
had a great efficiency for 1,1,1 -TCA removal. However, both indigenous and
augmented propane and butane utilizers needed further studies for 1,1,1 -TCA
transformation along with other CAHs including TCE, and l,1-DCE.
1.2 OBJECTIVES
Previous work indicated the effective removal of 1,1,1 -TCA by propane-
and butane-oxidizing bacteria (Jitnuyanont, 1998). My study has focused on
exploiting aerobic microbial metabolism in microcosms to achieve the
transformation of CAHs appearing in Moffett site, including 1,1,1 -TCA, TCE and
1,1 -DCE. The main focus was to compare 1,1,1 -TCA transformation abilities of
indigenous and bioaugmented butane-utilizers in groundwater and aquifer solids
microcosms. The kinetic parameters for butane utilization and 1,1,1 -TCA
transformation were evaluated through modeling the results of microcosm studies.
The thesis is divided into three parts performed to fulfill these objectives as
follows:
1.2.1 Culture development for bioaugmentation studies
Objective 1 was to develop a butane mixed culture for bio augmentation
studies. The culture for bioaugmentation was developed from the existing
microcosm cultures (Jitnuyanont 1998) of butane-utilizers and propane-utilizers.
The culture was selected by comparing 1,1,1 -TCA transformation abilities among
the microcosms.
1.2.2 Microcosm studies with groundwater and soil from the
original microcosms
Objective 2 was to conduct bioaugmentation microcosm studies with
groundwater and soil from the original microcosms of part 1. These studies were
performed to compare the 1,1,1 -TCA transformation ability between indigenous
and bioaugmented butane-utilizers, and to study the long term transformation of
1,1,1 -TCA as well as the transformation ability of CAH mixtures of 1,1,1 -TCA,
TCE, and 1,l-DCE. The culture having the best transformation ability was chosen
for growing a bioaugmentation culture. The grown culture was then frozen and
kept in the 196 °C liquid nitrogen for the next study.
1.2.3 Microcosm studies with the frozen butane-utilizing culture
Objective 3 was to perform bioaugmentation microcosm studies with the
frozen butane-utilizing culture. Microcosm studies were conducted in order to
compare the 1,1,1 -TCA transformation ability between indigenous and
bioaugmented butane-utilizers. The transformation of 1,1,1 -TCA, TCE, and 1,1 -
DCE was also evaluated in bioaugmented microcosms. Studies were conducted
over a range of butane and 1,1,1 -TCA concentrations to determine half-saturation
constant
(Ks) of indigenous and bioaugmented cultures under microcosm
conditions. Additional, kinetic parameters including maximum specific rate
(kmax)
of butane and 1,1,1 -TCA co-oxidation, cell decay rate (b), and/or transformation
capacity
(Ta) for indigenous and bioaugmented microcosms under microcosm
conditions were determined through simulations of butane utilization and 1,1,1 -
TCA transformation, using a non-steady-state model. The equations were solved
using STELLA® software (High Performance System, Inc. Hanover, NH).
CHAPTER 2
LITERATURE REVIEW
1,1,1 -trichioroethane (1,1,1 -TCA) is among the most frequently
encountered chlorinated aliphatic compounds in contaminated groundwater
resulting from its common use as industrial solvent. Its improper storage and
disposal resulted in the contamination of groundwater at many federal and private
facilities. It is of particular concern due to its toxicity, and potential
carcinogenicity. The maximum contaminant level (MCL) of 1,1,1-TCA in drinking
water is 200 tg/L (Federal Register, 1989).
2.1 PROPERTIES OF 1,1,1-TCA
l,1,l-TCA is relatively soluble in water with a solubility of 730
gum3
at 20
°C. It can be rapidly transported with groundwater. Thus, extensive plumes can
exist in the subsurface. Due to high vapor pressure (13.2 kPa at 20 °C), 1,1,l-TCA
can evaporate quickly. With a high vapor pressure, 1,1,1 -TCA can be effectively
removed from the vadose zone by soil vapor extraction (SVE). The Henry's
coefficient of 1,1,1-TCA is 3.06 kPa m3/mol. The formula of 1,1,1-TCA is
CC13CH3, having a molecular weight of 133.4 g/mole. The melting point and the
boiling points of 1,1,l-TCA are 30.4 °C and 74.1 °C, respectively (Mackay and
Shiu, 1982).
7
2.2 TRANSFORMATION OF 1,1,1-TCA
1,1,1 -TCA can be abiotically transformed via dehalogenation as well as
hydrolysis. 1,1,1 -TCA hydrolysis produces acetic acid that can be mineralized
rapidly by microorganisms, while dehalogenation produces 1,1 -dichlroethene (1,1 -
DCE) that also can be transformed further by biological reductive dehalogenation
to vinyl chloride (VC) (Vogel et al.,1987a; Vogel et al., 1987b; Vogel and
McCarty, 1987). 1,1,1 -TCA can also be reduced by transition metal complexes
providing ethane, ethanol, ethene, chloroethene, and 1,1 -dichlroethane (1,1 -DCA)
as products (Vogel et al., 1 987a). The pathways are shown in Figures 2.1 and 2.2.
Under anaerobic conditions, 1,1,1 -TCA can be biologically reduced to 1,1
DCA, an intermediate product that can be either reduced further to chloroethane
(CA) and VC, or transformed abiotically by hydrolysis to ethanol. Ethanol can be
mineralized easily by microorganisms (Vogel and McCarty, 1 987a; Vogel and
McCarty, 1987b; Vogel et al., 1987). Acetic acid and methanol are common
substrates to drive the anaerobic transformation of 1,1,1 -TCA. Doong and Wu
(1996) found that the increase of substrate addition could enhance the
transformation of 1,1 ,1-TCA. The growth of anaerobic microcosms was inhibited
by high concentrations of 1,1,1-TCA (Galli, 1989; Doong and Wu, 1996).
1,1,1 -TCA can also be biodegraded through aerobic cometabolism. The
1,1,1 -TCA transformation is carried out by a non-specific oxygenase enzyme that is
produced by microorganisms when oxidizing primary substrates for energy and
growth. The oxygenase enzyme oxidizes 1,1,1 -TCA to form 2,2,2-trichioroethanol
(Oldenliuis et al., 1989; Hommes et al., 1998). However, the overall 1,l,1-TCA
oxidation pathways via microbially mediated reactions under aerobic condition are
still unknown. The transformation of 1,1,1 -TCA can be achieved by the methaneoxidizing bacterium Methylosinus trichosporium OB3b (Oldenhuis et al., 1989),
the ethane-oxidizing bacteria Mycobacterium spp. (Yagi et al., 1999), the propane-
oxidizing bacteria Rhodococcus rhodochrous and Rhodococcus sp. Strain Sm-i
(Malachowsky et al., 1994), and the ammonia-oxidizing bacteria Nitrosomonas
europaea (Hommes et al., 1998).
2.2.1 Cometabolism by methane-utilizers
Methane-oxidizing bacteria (Methanotrophs) utilize methane as carbon and
energy source. The oxidation of methane to methanol is catalyzed by methane
monooxygenase (MMO) enzyme by inserting an oxygen atom into a methane
molecule and initiating the production of methanol. The reaction requires the
presence of both oxygen and NADH. MMO is broadly nonspecific enzyme, and
hence can catalyze a wide variety of oxidative reactions including the cometabolic
transformation of CAHs including 1,1,1-TCA (Strand et al., 1990; Arvin, 1991;
Broholm et al., 1990). A methane-oxidizing bacteria culture that degraded CAHs
was able to grow on other primary substrates such as methanol and propane (Arvin,
1991). The MMOs can be divided into two groups, the soluble form (sMMO) and
the particulate form (pMMO). Under copper limitations, some microorganisms can
produce sMMO, which has a much broader substrate range and is more effective in
transformation of CAHs than the pMMO form (Jansen et al., 1991; Oldenhuis et
al., 1989).
Strand et al. (1990) reported that the transformation of 1,1,1-TCA by
methane-oxidizing bacteria followed first-order kinetics with rate constants of 8.8
* i05
L/mg VSS-hr for aqueous concentration of TCA less than 7770 tg/L.
Methane in excess of 0.25 mg/L aqueous concentration inhibited the transformation
of 1,1,1 -TCA. The transformation rate was lower when treating a mixture of 1,1,1 TCA and TCE. Met hylosinus trichosporium OB3b incompletely transformed 1,1,1
-
TCA, and yielded 2,2,2-trichioroethanol as a chlorinated intermediate (Oldenhuis et
al., 1989). Compared to other CAHs, the transformation rate of TCA by
Methylosinus trichosporium OB3b was the lowest (Oldenhuis et al., 1991).
a) Hydrolysis
CH3COOH + 3H + 3Cr
CH3CC13 + 2H20
(1,1,1 -TCA)
(Acetic Acid)
b) Dehalogenation
CH2=CC1 + H + cr
CH3C13
(l,1,1-TCA)
(1,l-DCE)
Figure 2.1 Abiotic transformation pathway of 1,1,1-TCA.
a) Anaerobic condition
CH3CC13 -, CH3CHC12
H2
(l,1,1-TCA)
H+ Cl
H2
(1,1-DCA)
CHCHCl - CH3CH2OH -.- CO2
H+Cl
(CA)
(Ethanol)
b) Aerobic condition
CH3CC13
02+ NH + H
(1,1,1 -TCA)
CC13CH2OH
3C1 + other products
NAD +1120
(2,2,2-trichioroethanol)
Figure 2.2 Biotic transformation pathway of 1,1,1-TCA.
10
Increasing concentrations of 1,1,1 -TCA resulted in a decreased rate of methane
consumption (Brohoim et al., 1990).
There are several methane-oxidizing bacteria that were able to transform
CAHs, but not 1,1,1-TCA (Speitel et al., 1993; Chang and Alvarez-Cohen, 1995a;
Chang and Criddle, 1997). In Moffett field, methane-oxidizing bacteria exhibited
significant cometabolic transformation of VC and trans-dichioroethene (t-DCE),
and limited transformation of TCE and cis-1 ,2-dichloroethene (c-DCE), however
1,1,1-TCA was not transformed (Semprini et al, 1990; Semprini et al., 1991).
Jitnuyanont (1998) found that propane- and butane-oxidizing bacteria had very
high efficiency to transform 1,1,1 -TCA. As a result, the cometabolic
transformation of 1,1,1 -TCA by propane- and butane-utilizing bacteria was
performed in this study.
2.2.2 Cometabolism by butane-utilizers
In contrast to the significant studies with methane-utilizing bacteria, little
work has been performed with butane-utilizing bacteria. Due to the relatively low
solubility of methane, the production rate of biomass is limited by the transfer rate
of methane to the culture. To increase the production rate of biomass, normal
alkanes, such as propane and, n-butane, have been used instead since the transfer
rate and solubility limits of those alkanes are higher than those of methane.
Propane and butane were expected to provide 1.4 times higher biomass yield than
methane (MeLee et al., 1972). The advantages of using butane as a substrate have
led to the isolation of several microorganisms (Davis, 1964; McLee et al., 1972;
Phillips and Perry, 1979; Van-Ginkel et al., 1987).
McLee et al. (1972) found that all isolates of bacteria strains and molds
grown on n-butane were able to grown on ethane, propane, isobutane, n-
hexadecane, sugar, and peptides but not methane. In 1987, a nonspecific butane
monooxygenase was found in Norcadia TB1, which could grow on butane and
11
several saturated straight-chain hydrocarbons but not on 1 -alkenes (Van-Ginkel et
al., 1987). Similar to MMO, this nonspecific enzyme driving butane oxidation
might be capable of CAHs oxidation.
Kim (1996) reported the effective cometabolic transformation of chloroform
induced by the oxygenase enzymes of butane-utilizing bacteria from the Hanford
DOE site, Washington. In addition, these butane-utilizing microorganisms also had
abilities to degrade a broad range of CAHs including 1,1,1 -TCA, 1,1 -DCE and
their abiotic transformation products (Kim et al., 1997). The complete
transformation of 1,1,1 -TCA concentrations as high as 2400 j.ig/L in aqueous
solution was observed. Resting cells were not significantly affected upon exposer
to 1,1,1 -TCA. Compared to 1,1 -DCE and other chlorinated ethanes, the
transformation of 1,1,1 -TCA was relatively slow. However, the potential of 1,1,1
TCA transformation was still attractive.
As a result, the butane-utilizing enrichments from the Hanford DOE site
were selected for a bioaugmentation study. The cometabolism of 1,1,1 -TCA was
evaluated in microcosms constructed with groundwater and aquifer solids from the
Moffett site, California (Jitnuyanont, 1998). A maximum 1,1,1-TCA concentration
of 8310 tg/L was completely transformed by bioaugmented butane-utilizing
bacteria. The final transformation yield (the amount of cometabolic substrate
degraded prior to cell inactivation divided by the amount of primary substrate
consumed) obtained for the butane-utilizing bacteria was 0.04 mg 1,1,1-TCA!mg
butane. Nutrient addition was also studied in order to enhance the performance of
inoculated strains. The microcosms with the highest amount of mineral salt
medium most effectively consumed butane and transformed 1,1,1-TCA.
12
2.2.3 Cometabolism by propane-utilizers
Propane-oxidizing bacteria oxidize propane for energy and growth, using a
propane monooxygenase enzyme (PMO) to start the oxidation of propane (Wackett
et al., 1989). PMO is nonspecific enough to oxidize short-chain alkenes and other
aliphatic hydrocarbons (Hou et al., 1983; Wackett et al., 1989; Tovanabootr, 1997).
Propane oxidation is carried out by inserting an oxygen molecule, converting
propane to 2-propanol, which is further oxidized to acetone (Perry, 1980). Propane
can also undergo terminal oxidation and be oxidized to 1 -propanol (Stephens and
Dalton, 1986).
Owing to the non-specification, PMO is also responsible for CAHs
transformation. PMO in Mycobacterium vaccae JOB5 was able to transform TCE,
VC, cis- and tran-DCE, but not 1,1,1-TCA and PCE (Wackett et al., 1989).
Propane-oxidizing bacterium Mycobacterium sp. TCE28 could utilize ethane,
butane, and n-butanol as well as propane, and degrade TCE, cis- 1 ,2-DCE, 1,1 DCE, 1 ,2-DCA, dichioromethane, and CF cometabolically (Imano et aL, 1999).
The degradation of TCE, CF and 1 ,2-DCA by a propane-utilizing enrichment
resulted in cell inactivation due to product toxicity (Imano, et al. 1999).
Transformation could be enhanced by the addition of low concentration of growth
substrate, presumably, due to the regeneration of reducing energy (Chang and
Alvarez-Cohen, 1 995b).
The cometabolic transformation of 1,1,1 -TCA by propane-oxidizing
enrichments was first demonstrated by Keenan et al. (1993). Propane-oxidizing
bacterium Rhodococcus species and R. rhodochrous ATCC2 1197 was able to
degrade 1,1,1-TCA but not very effectively (Malachowsky et al., 1994). Highly
efficient removal of 1,1,1 -TCA by propane-utilizers enriched from McClellan Air
Force Base (California) aquifer material was observed in soil microcosm studies
with a transformation yield of 0.088 to 0.106 g 1,1,1 -TCA/g propane (Tovanabootr,
1997). 1,1,1 -TCA transformation was strongly inhibited by propane (Keenan et al.,
1993; Tovanabootr, 1997). Transformation was slow in the presence of high
13
propane concentrations and then accelerated after propane was nearly consumed
(Tovanabootr, 1997). However, the presence of propane helped maintain the
transformation capacity of 1,1,1-TCA in an extended study (Keenan et al., 1993,
Tovanabootr, 1997). Tovanabootr (1997) also found that the high concentration of
1,1,1 -TCA could slow the propane degradation rate.
Jitnuyanont (1998) evaluated the cometabolism of 1,1,1-TCA by propane-
and butane-utilizing bacteria from the Moffett site, California. She found that the
highest transformation yield obtained with propane-utilizers was 0.07 mg 1,1,1
TCA/mg propane. 1,1,1 -TCA transformation was competitively inhibited by both
butane and propane. Propane-utilizers also appeared to be more stable than butane
utilizers with prolonged stimulation.
2.3 KINETICS AND MODEL DEVELOPMENT OF CAH
COMETABOLIC TRANSFORMATION
The kinetics of cometabolism is an important factor for application of
bioremediation. Predictive models for the cometabolic process can be useful for
engineering design to plan and monitor site remediation, to conduct risk and
exposure assessments and also to evaluate the project cost and duration. (Alvarez-
Cohen and McCarty, 1991a; Semprini et al., 1998). However, the kinetics of
cometabolic transformation is often complicated by several influencing factors. A
number of different models, ranging from simple first-order reaction models to
complex multi-substrate mixed models, have been developed to describe the
cometabolic degradation process (Alvarez-Cohen and Speitel, in press).
Aerobic cometabolism is a biological process where microorganisms do not
gain any energy or carbon for cell growth from CAH transformation (McCarty,
1997). So, the presence of a compound as energy and carbon source and oxygen is
also required. The transformation rate of CAHs can be affected by many different
14
factors including competitive inhibition, reducing power availability, and
transformation product toxicity.
Competitive inhibition can occur when the primary substrate is present
along with a CAH since both primary substrate metabolism and CAll
cometabolism are catalyzed by the same nonspecific oxygenase enzyme. This
results in a decrease in both primary substrate and CAlls transformation rate
(Broholm et al., 1990; Speitel et al., 1993; Hommes et al., 1998).
In addition to oxygen, the oxidation of primary substrate and the CAH also
requires energy or reducing power in form of nicotinamide adenine dinucleotide
(NADH) which can be regenerated via the oxidation pathway of primary substrate.
This reducing power can be provided by internal or external sources. An internal
source of reducing power can be energy reserves stored in cells in form of
polyhydroxybutyrate (PUB) (Henry and Grbic-Galic, 1991). In order to increase
the transformation rate and transformation capacity, but avoid competitive
inhibition, an external source of reducing power can also be provided. For
example, a nongrowth-inducing and noncompetitive substrate such as formate can
be used (Alvarez-Cohen and McCarty, 1991a; Alvarez-Cohen and McCarty, 1991b;
Alvarez-Cohen and McCarty, 1991c). However, microorganisms likely require a
primary substrate to maintain the oxygenase enzyme levels. The depletion of
reducing power source could be another factor that hinders the transformation of
CAHs.
Transformation product toxicity is another significant factor that can cause
decreased rates of CAH transformation. Toxic intermediates and products formed
by the cometabolic transformation of CAHs, rather than CAHs themselves,
inactivate oxygenase enzymes, as well as other cellular components (Alvarez-
Cohen and McCarty, 199lb; Alvarez-Cohen and McCarty, 1991c; Oldenhuis et aL,
1991; Hommes et al., 1998). Transformation product toxicity can be measured in
terms of transformation yield (Tv), representing the maximum mass of CAH that
can be transformed per unit mass of primary substrate consumed, or transformation
15
capacity
(Ta),
representing the maximum mass of CAH that can be transformed per
unit mass of resting cell (Alvarez-Cohen and McCarty, 1991a).
The cometabolic transformation of CAHs has been modeled using
Michaelis-Menten and Monod kinetics, with an active microbial concentration that
is constant (Alvarez-Cohen and McCarty, 1991b) or decays over time (Galli and
McCarty, 1989), or with incorporation of competitive inhibition (Brohoim et al.,
1990). For relatively low concentrations of chlorinated compounds, the
biodegradation of chlorinated compounds could be described by pseudo first-order
rate expression (Wackett and Gibson, 1988; Oldenhuis et al., 1989; Strand et al.,
1990; Arvin, 1991; Speitel et al., 1993).
The transformation capacity
(Ta) was introduced to incorporate the effects
of product toxicity and reductant supply for the cometabolic transformation of
halo genated aliphatics by resting cells of a mixed methanotrophic culture (Alvarez-
Cohen and McCarty, 1991a). Alvarez-Cohen and McCarty (1991c) proposed a
cometabolic transformation model incorporating competitive inhibition in addition
to the transfonnation capacity. The reasonable fit between model predictions and
experimental observations was supportive of the model formulation.
Chang and Alvarez-Cohen (1995a) proposed a mechanistic model
integrating the effects of product toxicity, reducing energy limitation, and
competitive inhibition, together with cell growth and decay, to describe the kinetics
of the cometabolic degradation of chlorinated organics by oxygenase-expressing
cultures. Various conditions were examined: resting cells, cells with reducing
energy substrate, and cells with growth substrate. Assuming that cometabolic
transformation rates are enhanced by reducing power obtained from oxidation of
growth substrates, Chang and Criddle (1997) developed the model accounting for
the effects of cell growth, endogenous cell decay, product toxicity, and competitive
inhibition. The model successfully predicts TCE transformation and methane
utilization for a wide range of concentrations of TCE (0.5 to 9 mg/L) and methane
(0.O5to6mg/L).
f'1
2.4 BIOAUGMENTATION
Bioaugmentation, the introduction of microorganisms, is another strategy
for enhancing the bioremediation when an immediate remediation of a chemical is
required, or indigenous microorganism is unable to degrade the existing
compounds (Baud-Grasset and Vogel,
1995).
The inability of indigenous
microorganisms to transform target compounds is probably due to the lack of the
appropriate enzyme(s), low population of microorganisms (biomass), and
contaminant toxicity. Augmentation with proven contaminant-degrading
microorganisms leads to a higher degree of confidence in remediation success, and
also eliminates long lag periods to stimulate indigenous microorganisms. This
process has been practiced in various fields including soil and groundwater
remediation (Mayotte et al., 1996; Munakatta-Marr et al., 1996; Duba et al., 1996;
Dybas et al., 1998; Salanitro et al., 2000; Ellis et al., 2000).
The most common factors considered in bioaugmentation are pollutant
characteristics (bioavailability, concentration, microbial toxicity), soil physicochemical characteristics (humidity, organic matter content, clay content and pH),
microbiology (enzyme stability and activity) and methodology (method of
inoculation) (Vogel, 1996). However, a major concern with bioaugmentation is the
survival of the inoculated microorganism. Nutrient limitations (P, N, and possibly
other elements), suppression by predators and parasites, inability of bacteria to
move appreciably through soil, concentration of organic substrate too low to
support multiplication, improper pH, temperature and salinity, and toxins are
factors causing bioaugmentation to fail (Martin, 1994).
Using small-column microcosms, Munakatta-Marr et al. (1996) evaluated
the TCE degradation potential with bioaugmentation of both wild type and
genetically altered Burkholderia (Psedomonas) cepacia G4 and PR1301, phenolutilizing microorganisms. B. cepacia strain PR1301 was a nonrecombinant NTG-
induced mutant of B. cepacia strain G4 capable of uninduced constitutive
degradation of TCE in the absence of phenol or toluene. It was found that phenol-
17
utilizing microorganisms augmented with either B. cepacia strain G4 or PR1301
transformed twice as much TCE as the indigenous phenol-utilizing microcosm
stimulated from Moffett groundwater and soil. They were also able to transform
TCE in the absence of phenol. In addition, immediate activity towards phenol and
TCE by augmented microorganisms was observed, eliminating long startup
periods.
The significant removal of Cd4 from the aquifer solids in a bench-scale
study was achieved by the inoculation with Pseudomonci
sp.
strain KC
accompanied by the niche adjustment with base, acetate, and phosphorus under
denitrifying conditions (Mayotte et al., 1996). The in-situ remediation of CCI4 at
Schoolcraft, Michigan, was enhanced by bioaugmentation with strain KC and
substrate pH adjustment (Dybas et al., 1998). There also are several
bioaugmentation studies on in-situ bioremediation: TCE transformation by restingstate methane-utilizing bacteria Methylosinus trichosporium OB3b (Duba et al.,
1996), MTBE bioremediation through bioaugmentation and oxygenation (Salanitro
et al., 2000), and anaerobic TCE transformation in the aerobic aquifer prereduced
by lactate addition before inoculation (Ellis et al., 2000).
The effective transformation of 1,1,1 -TCA by propane-and butane-utilizing
cultures and successful bioremediation enhanced by bioaugmentation have led to
this study of the transformation of CAHs, including 1,1,1 -TCA, TCE and 1,1 -DCE,
in bioaugmented microcosms constructed with Moffett Field groundwater and
aquifer solids. Butane-utilizing cultures were studied in the most detail. The
kinetic parameters for butane utilization and 1,1,1 -TCA transformation were also
evaluated by modeling the results of microcosm studies. The work was performed
in support of a pilot scale field demonstration of bioaugmentation that will be
conducted at the Moffett Field test site.
18
CHAPTER 3
MATERIALS AND METHODS
3.1 CHEMICAL SOURCES
Butane (N-butane; CP grade) was purchased from Airgas Co. (Corvallis,
OR). Propane (98%) was purchased from Aldrich Chemical Co. (Milwaukee, WI).
1,1,1 -trichloroethane (1,1,1 -TCA; 99.9% {GC } grade), trichioroethylene (TCE;
>99%), and 1,1-dichioroethylene (1,1-DCE; >99%) were also purchased from
Aldrich Chemical Co (Milwaukee, WI).
3.2 MICROCOSM PREPARATION AND OPERATION
Microcosms were set up in either 125 mL amber serum bottles with PTFE/
red rubber aluminum seal or 125 mL media bottles with gray butyl rubber septa
(Wheaton Glass Co., Millville, NJ). They were constructed under a laminar flow
hood (The Baker Company, Sanford, MA) to avoid microbial contamination. All
tools for microcosm fabrication were autoclaved. The microcosms were
constructed with aquifer material and groundwater from the Stanford Test Facility
at Moffett field, or, in some studies, containing mineral salt growth media. The
remaining was air filled headspace. The composition of mineral salt growth media
is given in Appendix A.3. Various ratios of groundwater and aquifer material were
used, as will be described for specific microcosm tests. The core materials and
groundwater were kept in cold storage prior to use. The background aqueous
concentrations of 1,1,1 -TCA in the Moffett groundwater ranged from 2.51 to 3.73
tgIL. The nitrate concentration ranged from 40 to 43 mg/L as NO3. The core
samples consisted of particles sizes ranging from silts to large cobbles. The core
materials were unpacked under a laminar flow hood and wet sieved through a no. 8
sieve (2.38 mm opening) to remove the large particles. The core materials used in
the microcosm fabrication are listed in Appendix A. 1.
3.2.1 Preparation of CAH saturated aqueous stock solution
1,1,1 -TCA, ICE or 1,1 -DCE was added to a 125 mL serum bottle filled
with demineralized water to achieve concentrations at the solubility limit in water.
The bottle was crimped sealed, and, prior to use, shaken vigorously, and allowed to
settle overnight.
3.2.2 Utilization of butane and propane
Butane or propane gas was added into each microcosm as a substrate, using
a 100 pL gas tight syringe (Hamilton, NEV). The bottles were vigorously shaken
by hand for 10 s to allow the equilibration of substrate before initial headspace
samples were taken. During the course of the experiments, the microcosms were
incubated at 20 °C temperature on a shaker table at 200 rpm. Gas samples, 100 tL,
were withdrawn periodically for analysis. Vacuums created by the consumption of
butane or propane were re-equilibrated by injecting air or pure oxygen into the
headspace.
3.2.3 Transformation of 1,1,1-TCA
The 1,1,1 -TCA experiments were conducted on microcosms stimulated on
butane or propane through successive additions of 1,1,1-TCA in order to test the
20
continuity of transformation of 1,l,1-TCA in the absence of butane utilization. The
experiments were performed similarly to the butane and propane experiments. The
saturated 1,1,1 -TCA solution was injected by a 100 p1 liquid microsyringe
(Hamilton, NEV) into the microcosms through the rubber septa to obtain an
aqueous concentration of around 350 ig/L. A control bottle contained the same
amount of 1,1,1 -TCA as the microcosms. The concentration of 1,1,1 -TCA in
microcosm headspace was monitored periodically.
3.2.4 Transformation of CAH mixture
A mixture of saturated 1,1,1 -TCA, TCE, and 1,1 -DCE stock solutions was
added into microcosms stimulated on butane to study the transformation ability of
this mixture of compounds. Similar to the TCA experiments, the saturated aqueous
stock solution of CAHs was added by a 100 j.iL liquid microsyringe into the
microcosms through the rubber septa to obtain the desired concentration. The
aqueous concentration of 1,1,1-TCA, TCE, and 1,1-DCE ranged from 300 to 600
tg/L, 500 to 900 tg/L, and 100 to 500 tg/L, respectively. The concentration of
CAHs in the microcosm headspace was monitored periodically.
3.2.5 Rate measurement of 1,1,1-TCA transformation
Transformation rate of 1,1,1 -TCA was measured in microcosms stimulated
on butane, through successive additions of 1,1,1 -TCA. The 1,1,1 -TCA-saturated
aqueous stock solution was injected by a 100 iL liquid microsyringe into the
microcosms through the rubber septa to obtain the aqueous concentration of around
350 tg/L. The concentration of 1,1,1-TCA in the microcosm headspace was
monitored to obtain at least 3 data points for a rate determination. During the
21
course of the experiments, the microcosms were incubated at 20°C temperature on
a shaker table at 200 rpm. The derivation of the equations and the calculation
method to determine the transformation rate of 1,1,1 -TCA are shown in Appendix
D.
3.2.6 Butane and 1,1,1-TCA kinetic experiments
The kinetic experiments were conducted similarly to the above butane and
TCA experiments. Butane and 1,1,1 -TCA-saturated aqueous stock solution were
added into microcosms, using a 100 .tL gas tight syringe and a 100 L liquid
microsyringe, respectively. The microcosms were incubated at 20°C temperature
on a shaker table at 200 rpm. The headspace gaseous concentration was monitored
during the experiment. The amounts of gaseous butane or saturated solution of
1,1,1 -TCA added resulted in aqueous concentration much more than the value of
the half-saturation constant
(Ks). This provided concentrations that covered both
zero-order rates for the high concentration range and first-order rates for the low
concentration range. In the case of the butane kinetic experiments, the increase of
biomass from butane utilization in each test was less than 5% of total biomass so
that the biomass can be assumed to be constant. The derivation of the rate
equations and the calculation method to evaluate butane and 1,1,1 -TCA K values
are shown in Appendix E.
3.2.7 Groundwater amendment
The microcosms were maintained for a long period of time, with periodic
exchanges of groundwater and with growth substrate readdition. Prior to adding
the growth substrate and chlorinated solvents, the groundwater was exchanged to
22
resupply nutrients and prevent the accumulation oftransformation by-products.
The microcosms were centrifuged for 20 minutes at 1000 rpm to settle and keep the
microorganisms in the microcosms. The caps were then removed under a laminar
flow hood. Sixty percent of the groundwater was replaced with new groundwater
and the microcosms were then resealed. When nitrogen was found to be limiting,
the groundwater was amended with nitrate to 30 mg/L.
3.3 GROWTH REACTORS
3.3.1 Growth reactor inoculated with groundwater/soil slurry
To create the mixed culture for bioaugmentation studies, a batch growth
reactor was set up in a 500 mL bottle sealed with a Teflon fluorocarbon resin-lined
cap. One mL of suspended liquid taken from the soil microcosms (Jitnuyanont
1998) was added to inoculate the growth reactor, which was filled with 250 mL
mineral salt growth media (Appendix A.3). Butane or propane was added by
gaseous addition to each bottle to yield 10% vol/vol in the headspace. The growth
reactor was placed on a shaker table at 200 rpm in a 20 °C constant temperature
room. Pressures were equilibrated daily with pure oxygen or air. After about 90%
of the butane was consumed, all liquid from each bottle was combined and
centrifuged to concentrate the biomass. The concentrated biomass was either used
for bioaugmentation, or placed in 1 mL vials with 7%DMSO (Dimethyl Sulfoxide)
and stored in 196 °C liquid nitrogen for future studies.
23
3.3.2 Growth reactor inoculated with frozen culture
To create a bioaugmentation culture, a second growth reactor with low
headspace was set up in a 2000 mL flask filled with 1800 mL of mineral salt
growth media at room temperature. The reactor is shown in Figure 3.1.
Figure 3.1 Continuously fed 2-L growth reactor.
The reactor was completely mixed, using a star-shaped stirring bar that was
rotated at 600 rpm to facilitate butane and oxygen transfer. To provide for
continuous feed of substrate and oxygen, butane and air gas tanks were connected
to the reactor through a flowmeter equipped with an inline filter device and glass
24
dispersion tubes with fitted cylinders. The air flowrate was approximately 50
mL/min and the butane flowrate was adjusted to achieve the headspace partial
pressure ranging from 2 % to 4%. The vent gas was exhausted to a fume hood. To
inoculate the growth reactor, one mL of the frozen culture (-196 °C) was left in the
freezer overnight and then at room temperature until it was completely thawed.
The culture was washed with mineral salt growth media to rinse away the DMSO
prior to inoculation into the growth reactor. The optical density (0D600) and dry
mass density were measured periodically.
Figure 3.2 shows the growth curve, presenting the optical density and dry
density of biomass of one of the culture growth tests. Both 0D600 and dry density
are in the lag phase during the first 3 days and then increase with time, resulting
from the microbial growth in the exponential phase. The percentage of butane and
oxygen in the inlet and outlet were measured in the growth reactor, and are shown
in Figure 3.3. The measured percentage of inlet and outlet gases was similar,
indicating neither oxygen and butane became limited.
The growth curves for other tests are shown in Figure 3.4. All growth tests
provided similar results. The lag phase for microbial growth was around 3 days.
The enrichment was harvested from the growth reactor in the exponential phase,
between day 4 and 5. The measured optical density and dry mass density ranged
from 0.2 to 0.3 and from 0.09 to 0.12 gIL, respectively. After day 5, the color of
culture changed from milky to yellow.
3.4 BIOAUGMENTATION
Bioaugmentation microcosm studies were conducted to compare the 1,1,1
TCA transformation ability between indigenous and bioaugmented butane-utilizers,
and to study the long term transformation of 1,1,1 .-TCA. The transformation of
CAH mixtures of l,l,1-TCA, TCE, and l,1-DCE was also studied.
25
0.50
0.12
1-0.1
0.40
-0.084
0.30
c
,
0
0.20
0.04
0.10
0.02
+0
0.00
0
1
2
3
4
5
Time, day
Figure 3.2 Growth curve of the continuously fed batch reactor preparing the
culture for inoculation.
3
25
20
C
C
15
Butane in
-10 0
Butane Out
..A..Oxygen In
..x..Oxygen Out
i
0
1
2
3
4
5
Time, day
Figure 3.3 Percentage of butane and oxygen in the inlet and outlet measured in
the continuously fed batch reactor.
a) Growth Reactor 1
1.20
1.2
..-.-OD
1.00
1
-..- Density
C,
r'o
>
I_p.o
0.80
.
U,
0.60
0.6
0.40
\__ Color starts
0.4
0.20
changing to
yellow
0.2
0.00
C)
0
0
2
4
6
8
10
Time, day
b) Growth Reactor 2
0.60
0.25
-.-- OD
0.50
0.2
.- Density
0.40
-J
0.15
0.30
0.1
0.20
U)
Cu
0.05
0.10
0.00
0
1
2
3
4
5
I
Time, day
Figure 3.4 Growth curve of the other continuously fed batch reactors
preparing the culture for inoculation.
27
3.4.1 Bioaugmentation with groundwater/soil slurry
To conduct initial bioaugmentation microcosm studies, one mL of
suspended liquid was taken from selected soil microcosms (Jitnuyanont 1998) and
added to inoculate the microcosm. The suspended liquid was composed of
groundwater and aquifer material.
3.4.2 Bioaugmentation with media-culture
The bacterial culture for bioaugmentation was acquired from the enrichment
obtained from the growth reactor grown in the mineral salts growth medium. In an
earlier study, the culture was obtained from the growth reactor inoculated with
groundwater/soil slurry and was washed with groundwater to rinse away the media
before adding to microcosms. In this case the culture was obtained from the
continuously fed 2 L growth reactor and was directly added to microcosms without
washing.
3.5 ANALYTICAL PROCEDURES
1,1,1 -TCA, TCE and 1,1 -DCE were measured by taking
a 100 L
headspace sample and injecting it into Hewlett Packard (Wilmington, DE) 5890 gas
chromatograph equipped with a 63Ni electron capture detector (ECD). Separation
was obtained by an HP-624 30 m x 0.25 mm x 1.4 mm film thickness stainless steel
packed column (Hewlett Packard, Wilmington, DE). The column was operated
isothermally at 80 °C. An argon/methane (95%:5%) mixture at a head pressure of
80 psi was used as the carrier gas (60 mL/min). The gaseous concentrations were
determined by comparing peak areas of samples with those of external standards
curves as shown in Appendix B.
Butane and propane sampling was also measured by taking a 100 j.xL
headspace sample and injecting it into Hewlett Packard (Wilmington, DE) 5890 gas
chromatograph equipped with a flame ionization detector (FID) and a GS-Q 30 m x
0.53 mm stainless steel packed column (J&W Scientific, Folsom, CA). The
column was operated isothermally at 100 °C. Nitrogen gas at a head pressure of
60 psi was used as the carrier gas (25 mL/min). The method was calibrated using
an external standard (Appendix C).
Oxygen was analyzed by taking a 100 tL headspace sample and injecting it
into a gas chromatograph on an HP 5890 Series II GC equipped with a thermal
conductivity detector (TCD) and a Supelco 15 fix 1/8 inch 60/80 CardoxenTM 1000
packed column (Supelco, Bellefonte, PA). Argon gas at a head pressure of 40 psi
was used as the carrier gas. The method was calibrated using an external standard.
The culture dry mass density was measured as a total suspended solid (TSS)
analysis (American Public Health Association, 1985). The density was determined
by filtering a specific volume of suspended culture through a 0.1 -tL-membrane
filter (Micro Separation Inc., Westboro, MA), drying overnight at 60 °C, cooling in
desiccator for 30 mm and weighing. Before being used, the filter was kept in the
desiccator overnight and weighed. The dry mass density was determined from the
weight change of the filter. The optical density (0D600) of the culture was
measured at 600 urn using an HP 8453 UV-Visible spectrophotometer.
3.6 MODEL DEVELOPMENT
Predictive models for the cometabolic process are useful for engineering
design to plan and monitor site remediation, to conduct risk and exposure
assessments and also to evaluate the project cost and duration (Alvarez-Cohen and
29
McCarty, 1991a; Semprini et al., 1998). In this study, the kinetic parameters for
indigenous and bioaugmented microcosms under microcosm conditions were
determined through modeling the results of the microcosm studies. The simulation
of butane utilization and 1,1,1 -TCA transformation was conducted, using partial
differential equations that were solved using STELLA® software (High
Performance System, Inc. Hanover, NH).
The rates of butane utilization and 1,1,1 -TCA transformation were
described by Monod kinetics that relates the transformation rate to concentration in
aqueous phase. To simplify the kinetics expressions, the experiments were
designed to avoid the effects of competitive inhibition. The effect of reducing
power limitation was also assumed to be negligible. Since the experiments were
conducted for long periods of time, equilibrium two-phase (air and liquid) partition
was assumed based on Henry's Law. The dimensionless Henry partition
coefficient (J-J) was used to describe the distribution of butane and 1,1,1 -TCA
between phases. Equations 3.1 and 3.2 expressed the rates of butane utilization and
1,1,1 -TCA transformation in terms of total mass of butane or 1,1,1 -TCA
transformed in the microcosms. Both equations assume oxygen does not limit
butane utilization or 1,1,1 -TCA reaction rates, and all transformations occur only in
the liquid phase.
km< But
dMBUf
dt
(
'vBut
MBUt
I
K
x,TCA
V + HcctVg J
(3.1)
+ HCcButVg)
ktnaTCA XJ
dMTCA
cit
+1
'But
+1
MTCA
+ HCCTCA Vg)
MT(
T7
Y'e
cc,TCA'g
(3.2)
30
where,
MBUt
=
Total mass of butane, mg
MTCA
=
Total mass of 1,1,1 -TCA, mg
kmax,But
=
Maximum specific rate of butane, mg/mg-day
Maximum specific rate of butane or 1,1,1 -TCA, mg/mg-day
kmax,FCA
KS,BUt
=
Half-saturation constant for butane, mg/L
KS,TCA
=
Half-saturation constant for butane or 1,1,1 -TCA, mg/L
HCC,BUt
Henry partition coefficient of butane, mg/L/mg/L
HCC,TCA
Henry partition coefficient of 1,1,1 -TCA, mg/L/mg/L
Vg
=
Gas volume,L
Vi
=
Liquid volume, L
X
t
Active microbial concentration in the aqueous phase, mg/L
Time, day
=
Similar to the rate of butane utilization and 1,1,1 -TCA transformation,
Monod kinetics was also used to express the rate of microbial growth as a function
of growth and cell decay:
kmaxButX
= Y
dt
MBU
KSBU, +
[
[
MBU,
bX
(3.3)
Ve + HCcB11tVg
+HccButVgj
where,
=
Y
b
Net specific cellular growth rate, day'
Cellular yield of butane, mg cell/ mg butane
=
Cell decay rate, day'
Since the transformation product toxicity is another factor effecting cell
decay, it was also incorporated into the model. The transformation capacity term
31
(Ta) was incorporated into the model to include the effect of transformation product
toxicity on cell activity:
I(max,ButX
4i =
dt
MBUt
KcBUt +
MBU
e
+ N BuYg J
+ Ht Vg)
M
kmaxqriX
_--
MTCA
Ks,TCA +1
Ve
+ HccTCAVg)
bX
(3.4)
+HcCTCAVg
where,
T
= Transformation capacity for
1,1,1 -TCA,
mg TCA / mg cells
To develop the model, all parameters were introduced into the above
equations. The known parameters, including initial biomass (Xo) in bioaugmented
microcosms, butane and 1,1,1 -TCA mass added to the microcosms (MBut and
MTCA), butane and 1,1,1 -TCA half-saturation constant (Ks,But, KS,TCA), and cellular
yield (Y), were fixed in the equations. The remaining kmax,But, kmax,TCA, b or/and T
values were varied in a heuristic approach to obtain a good fit between model
predictions and the experimental observations. The errors of unknown parameters
were expressed in terms of standard deviation based on modeling of the five
replicate microcosms. The differential equations 3.1,
3.2,
and 3.3 or 3.4 were
solved in STELLA®. Numerical integration in time was performed in STELLA®
using a fourth-order Runge-Kutta method. The derivation of equations
illustrated in Appendix F.
3.1
to
3.4
is
32
CHAPTER 4
RESULTS AND DISCUSSION
4.1 CULTURE DEVELOPMENT FOR BIOAUGMENTATION
STUDIES
The objective of this work was to develop a mixed culture for
bioaugmentation. The culture for bioaugmentation was obtained from the existing
microcosms of butane-utilizers and propane-utilizers (Jitnuyanont, 1998). The
existing microcosms were constructed in 125 mL amber serum bottles with PTFE/
red rubber aluminum seals. The bottles contained 65 mL of groundwater and
aquifer material from Moffett field, leaving 60 mL of headspace. The conditions of
all microcosms are presented in Table 4.1.
Table 4.1 Conditions of the existing microcosms (Jitnuyanont, 1998).
Microcosm
Contents
Substrate
Description
P1
P2
GW/Soil
GW!Soil
Propane
Propane
P6
P7
GW/Soil
GW/Soil
B1
GW/S oil
B2
GW/Soil
Propane
Propane
Butane
Butane
B6
B7
GW/Soil
GW/Soil
Butane
Butane
Indigenous Moffett field microcosm
Inoculated with the enrichments obtained
from the Hanford DOE site.
Microcosm augmented with propane culture
Microcosm augmented with propane culture
Indigenous Moffett field microcosm
Inoculated with the enrichments obtained
from the Hanford DOE site.
Microcosm augmented with butane culture
Microcosm augmented with butane culture
33
All microcosms had been previous operated, but were inactive for about 1
year before being re-stimulated by adding 4-5 mg of gaseous propane or butane.
After the microcosms were stimulated through substrate addition, 1,1,1 -TCA tests
were performed to determine the long-term transformation ability of 1,1,1 -TCA.
The aqueous concentration of l,1,l-TCA ranged from 200 to 1300 gIL. To
resupply nutrients and prevent the accumulation of transformation by-products,
groundwater exchange was performed before each new 1,1,1 -TCA experiment.
No 1,1,1 -TCA transformation was observed in the controls, as shown in
Figure 4.1. The control bottles contained 65 mL of demineralized water and 1,1,1 TCA at the same concentration as the microcosms.
0.1
-.- Controll
a)
0.08
-a-- Control2
E
'I)
0 0.06
0
0.04
I-
-
0.02
0
50
100
150
200
250
Time, day
Figure 4.1 Control bottles for all the microcosms.
Figure 4.2 presents butane utilization and 1,1,1-TCA transformation by
indigenous butane-utilizers in microcosm B 1. The first addition of butane was
consumed within 7 days. On day 19, more butane and l,l,1-TCA (1300 j.tg/L)
were added. The rates of butane utilization and TCA transformation were very
slow. Butane was consumed in 11 days and only 50% of the TCA was
34
transformed. On day 48, the microcosm was purged under a laminar flow hood to
remove 1,1,1-TCA. More butane and 1,1,1-TCA at lower concentration (500 tg/L)
were added to repeat the experiment. TCA transformation ceased after 40% of the
TCA was transformed. On day 70, butane was added to enhance TCA
transformation. The transformation of successive 1,1,1 -TCA additions was
maintained for 12 days in the absence of butane utilization. The transformation rate
decreased with continued addition of 1,1,1-TCA. The total mass of TCA
transformed was 0.11 mg. On day 92, the experiment was repeated with the
addition of a lower butane mass (1.42 mg). The transformation of 1,1,1-TCA
ceased after 0.04 mg of TCA was transformed. As a result, 5.5 mg of butane was
added on day 131 to increase the transformation ability. More 1,1,1 -TCA (0.09
mg) was transformed.
Butane utilization and 1,1,1 -TCA transformation by butane-utilizers in
microcosm B6 are shown in Figure 4.3. Successive l,l,1-TCA additions of 200 to
600 tg/L were started after eight additions of butane, and were rapidly
transformed. The transformation rate of 1,1,1 -TCA decreased with continued
additions of 1,1,1-TCA. Microcosm B6 showed long-term transformation ability of
1,1,1 -TCA in the absence of butane utilization. For example, on day 70, 1,1,1 -
TCA transformation ability was maintained for 44 days after butane was consumed.
The total amount of 1,1,1-TCA transformed was 0.44 mg. The successive
additions of 1,1,l-TCA were also transformed after butane additions on days 115,
131 and 144. The transformation continued from 10 to 42 days in the absence of
butane utilization. The amount of 1,1,1-TCA transformed ranged from 0.12 to 0.18
mg, which was less than that occurred during the first transformation period.
Figure 4.4 presents the butane utilization and 1,1,1-TCA transformation by
the butane-utilizers in microcosm B7. Five additions of butane were made to the
microcosm in order to re-stimulate the butane-utilizers. On day 51, 1,1,1 -TCA was
then added along with another addition of butane. The transformation of 1,1,1TCA was much slower than that in microcosm B6. Approximately 90% of 1,1,1TCA was transformed in 9 days. More butane and TCA were added on day 67.
35
I
s---- B 1
E
4
Co
U)
C)
C
o
4-.
2
1
I
50
100
150
200
250
150
200
250
Time, day
0.12
0.1
E
0.08
U)
0)
CU
0.06
002
S
Ij
50
100
Time, day
Figure 4.2 Butane utilization and 1,1,1-TCA transformation achieved in
indigenous microcosm Bi.
36
5
0)
E
U)
Co
CD
4
3
C)
2
m
1
0
0
50
100
150
200
250
150
200
250
Time, day
0.05
0.04
U)
Co
'5
0.02
I-;.
001
50
100
Time, day
Figure 4.3 Butane utilization and 1,1,1-TCA transformation achieved in
bioauginented microcosm B6.
37
0
C
.
4
3
2
0
50
100
150
200
Time, day
0.05
0.04
E
(0
U)
0
0.02
'. 0 01
0
50
100
150
200
250
Time, day
Figure 4.4 Butane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm B7.
38
TCA transformation was still slow and by day 92 only 0.05 mg of l,1,1-TCA was
transformed. Before starting another experiment, the microcosm was purged under
a laminar flow hood to remove 1,1,1 -TCA. After the addition of 2 mg butane, slow
long-term TCA transformation was observed. Approximately 0.05 mg of 1,1,1-
TCA was transformed in 42 days. On day 134, successive additions of 1,l,1-TCA
were conducted after the addition of 7.5 mg/L butane. TCA transformation rates
increased and transformation continued 59 days in the absence of butane. The total
mass of 1,l,1-TCA transformed was 0.25 mg. The improvement of transformation
ability in microcosm B7 was possibly due to an increase of organisms responsible
for the transformation of 1,1,1 -TCA. This implies that the microbial community of
the mixed butane-utilizing culture probably changed over time.
Figure 4.5 presents propane utilization and 1,1,1-TCA transformation by
propane-utilizers in microcosm P6. 1,1,1-TCA at a concentration of 1000 igIL was
added after two additions of propane, and was transformed within 12 days.
Competitive inhibition between propane and 1,1,1 -TCA was observed. Successive
additions of 1,1,1-TCA at lower concentration were performed on days 35, 51, 92,
115 and 131. The concentrations ranged from 400 to 750 ig/L. The
transformation rate decreased with continued additions of 1,1,1 -TCA. Similar to
butane-utilizers in microcosm B6, propane-utilizers in microcosm P6 were able to
transform 1,1,1 -TCA over long periods. The transformation continued from 13 to
22 days after propane was consumed. The amount of 1,1,1 -TCA transformed
ranged from 0.07 to 0.45 mg. After 130 days of incubation, propane utilization
became slower, even in the absence of 1,1,1 -TCA. This probably resulted from the
transformation product toxicity of 1,1,1 -TCA.
The substrate utilization and 1,1,1 -TCA transformation achieved in the
other propane and butane-utilizers in microcosms P1, P2, P7, and B2 are shown in
appendix Figures G. 1 to G.4. All microorganisms were capable of transforming
1,1,1-TCA. The indigenous propane and butane-utilizers in microcosm P1 and Bi
transformed 1,1,1 -TCA more slowly and had a lower transformation capacity of
1,1,1 -TCA than the bioaugmented microorganisms. Similar to microcosms B6 and
39
7
6
E5
0
2
0
0
50
100
150
200
250
Time, day
[II
0.08
U)
2
C)
i- 0.04
'- 0.02
[I]
0
50
100
150
200
250
Time, day
Figure 4.5 Propane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm P6.
P6, the bioaugmented microcosms P2, P7, B2 and B7 had long-term transformation
ability of 1,1,1 -TCA in the absence of substrate utilization. The maximum
transformation periods in the absence of substrate utilization are shown in Table 4.2
along with the transformation yields. The bioaugmented butane-utilizers showed
longer transformation periods than the propane-utilizers. This may have resulted
from a greater amount of substrate addition. The long-term transformation ability
of 1,1,1 -TCA observed in the microcosms indicates that the enzyme responsible for
transformation was present and active long after the substrate was utilized.
Table 4.2 The maximum transformation period in the absence of substrate
utilization, the average and final 1,1,1-TCA transformation yield achieved in
the microcosms.
Microcosm
Maximum period
(day)
P1
22
27
22
28
P2
P6
P7
Bi
B2
B6
B7
14
43
44
50
Transformation yield
(mg 1,1,1 -TCA/mg substrate)
Average
On day 200
0.007
0.014±0.011
0.019
0.031±0.022
0.019
0.046±0.044
0.003
0.029 ± 0.023
0.019
0.013±0.006
0.031
0.021±0.015
0.036
0.024±0.010
0.035
0.019 ± 0.016
The transformation yield for 1,1,1 -TCA at aqueous concentrations ranging
from 220 to 530 tg/L was determined by dividing the total amount of 1,1,1 -TCA
transformed by the amount of butane utilized. The comparison of 1,1,1 -TCA
transformation yield among the microcosms is shown in Figure 4.6. The
microcosms containing only indigenous microorganisms (P1 and Bi) had lower
transformation yields than the inoculated microcosms P2, P6, P7, B2, B6 and B7.
The microcosms that were fed propane (P2, P6 and P7) had much high
41
transformation yields during the period of 25
50 days; however, the yield
decreased considerably at later time, Unlike propane-utilizers, butane fed
microcosms B2, B6 and B7 had lower transformation yields, but maintained higher
yield values over the study period. The transformation yield for 1,1,1 -TCA relates
to the amount of active biomass in the microcosms and energy reserves to drive the
transformation. Similar to the transformation rate of 1,1,1 -TCA, the transformation
yield decreases with the additions of 1,1,1 -TCA. This probably results from the
transformation product toxicity of 1,1,1 -TCA, decreasing the amount of active
microbial cells and/or oxygenase enzyme responsible for the 1,1,1 -TCA
transformation.
0.12
- - .--
P1
0.1
a)
--.---- P2
>.2
- -
0.08
--A--
II
I
P7
x
0.06
-x--- BI
C.)
I,.
P6
0) 0.04
..'"
--.------ B2
/
'
S
-
U
002
B6
S
I ---
4 --
B7
x
[II
0
50
100
150
200
250
Time, day
Figure 4.6 1,1,1-TCA transformation yield achieved in the microcosms.
As shown in Table 4.2, the average and final (on day 200) transformation
yields achieved in indigenous microcosms P1 and Bi were lower than those in the
other inoculated microcosms except microcosm P7, which had the lowest final
42
yield. The final transformation yields of inoculated propane-utilizers were lower
than the average yields. The average and final yields of inoculated butane-utilizers
were similar. Propane-utilizers had higher average transformation yields, but lower
final transformation than butane-utilizers. To ensure that there was a sufficient
amount of active biomass, the final yield was used to determine which microcosm
was suitable for the bioaugmentation of additional microcosms. As a result, butane
fed microcosm B6 was chosen to be the parent microcosm for bioaugmentation
studies. Microorganisms in microcosm B6 also exhibited very rapid transformation
of 1,1,1 -TCA, which was also a desirable characteristic for the bioaugmentation
culture.
4.2 MICROCOSM STUDIES WITH GROUNDWATER AND
SOIL FROM MICROCOSM B6
4.2.1 Transformation of 1,1,1-TCA
Microcosm studies were conducted to compare the 1,1,1 -TCA
transformation ability between indigenous and bioaugmented butane-utilizers, and
to study long-term 1,1,1 -TCA transformation. Microcosms were constructed in
125 mL media bottles sealed with Teflon fluorocarbon resin-lined caps. They were
filled with either 75 mL of Moffett groundwater and aquifer material or 75 mL of
mineral salt growth media. Each microcosm was constructed in triplicate. Since
microcosm B6 showed the best 1,1,1 -TCA transformation performance, it was
selected to be the parent culture for bioaugmentation. A slurry, composed of
groundwater and aquifer material, was taken from microcosm B6 on day 141
(Figure 4.3) and used to inoculate microcosms. The microcosm conditions are
shown in Table 4.3. Butane gas was added as the primary substrate for each
microcosm. Successive additions of saturated 1,1,1 -TCA solution were made to
achieve an aqueous concentration of 350 tg/L. Control microcosms contained the
same amount of TCA (appendix Figure G.5). Before starting the new 1,1,1-TCA
experiment, groundwater was exchanged to resupply nutrients and prevent the
accumulation of transformation by-products.
Table 4.3 Microcosms used for the experiment of 1,1,1-TCA transformation.
Microcosm
Contents
Substrate
CAHs
Description
Bl-1 to B1-3
GW/Soil
GW/Soil
Butane
Butane
l,1,1-TCA
B2- 1 to B2-3
B3-1 to B3-3
Growth media
Butane
1,1,1-TCA
B4- 1 to B4-3
GW/Soil
Butane
1,1,1 -TCA
Indigenous microcosm
Inoculated with 1 ml of
GW/soil slurry
transferred from
microcosm B6
Inoculated with 1 ml of
GW/soil slurry
transferred from
microcosm B6
Inoculated with 100 tl
of washed media-culture
harvested from the
growth reactor having 1
mL of GW/soil slurry
transferred from
microcosm B6 as a
culture seed.
1,1,1 -TCA
The butane utilization and 1,1,1 -TCA transformation achieved in the
triplicate indigenous microcosms (no bioaugmentation) B 1-1, B 1-2 and B 1-3 are
shown in Figure 4.7. The first addition of butane was completely consumed on day
35, 25, and 45, respectively. l,1,1-TCA was then added to microcosms along with
the second addition of butane. All indigenous microorganisms utilized butane, but
little 1,1,1 -TCA transformation occurred.
44
5
4
C)
E
Co
cu
a)
2
4..
0
50
100
150
200
Time, day
0.06
0.05
E
0.04
U)
Cu
0.03
'B1-3
0
50
100
150
200
Time, day
Figure 4.7 Butane utilization and 1,1,1-TCA transformation achieved in the
indigenous microcosms constructed with groundwater and aquifer solids.
45
Figure 4.8 presents butane utilization and 1,l,1-TCA transformation
achieved in slurry, transferred from microcosm B6, inoculated microcosms B2- 1,
B2-2, and B2-3 during the first 50 days of incubation. The microcosms were
constructed with Moffett field groundwater and aquifer solids. Butane was
completely consumed within 10 days of incubation. With the second addition of
butane, l,1,1-TCA was rapidly consumed. Repeated additions of 1,1,1-TCA (350
j.ig/L) were transformed in the absence of butane, although the transformation rate
decreased after each addition. Butane was again added on day 29 and was rapidly
consumed, and 1,1,1 -.TCA transformation accelerated as butane concentration was
reduced, as shown in Figure 4.9. This indicates the competitive inhibition of 1,1,1 TCA transformation by butane. The high concentration of 1,1,1 -TCA (2 mg/L) that
microcosms experienced on day 40 (Figure 4.8) was also rapidly transformed. The
triplicate microcosms all showed similar results. The bioaugmented microcosms,
thus, had very reproducible performance towards butane utilization and 1,1,1 -TCA
transformation. The average transformation yield achieved in the B2 microcosm
set during the first 50 days of incubation was 0.079 ± 0.005 mg 1,1,1-TCAImg
butane.
The butane utilization and 1,1,1 -TCA transformation achieved in the media-
grown culture bioaugmented microcosms are presented in Figure 4.10. Butane in
microcosms B4- 1, B4-2 and B4-3 was completely consumed within 6 to 8 days.
Similar to the B2 microcosm set, the second addition of butane and 1,1,1 -TCA was
rapidly consumed. The competitive inhibition of butane on 1,1,1 -TCA
transformation was also observed, as shown in Figure 4.11. Successive additions
of 1,1,1-TCA (350 tgIL) were also transformed in the absence of butane, and the
transformation rate decreased after each addition. While 1,1,1 -TCA in microcosms
B4-1 and B4-3 was completely transformed, microcosm B4-2 behaved differently.
The transformation of 1,1,1-TCA in microcosm B4-2 began to slow down at day
34. The limited transformation of 1,1,1-TCA in microcosm B4-2 appears to be
correlated with a slower rate of butane uptake. The average transformation yield
46
5
-I
0)
E
có
w
3
CD
'
C
2
ml
[ii
0
10
20
30
Time, day
40
50
20
30
Time, day
40
50
0.35
0.3
0.25
B2-2
0.2
-a--
B2-3
0c:
0.05
0
0
10
Figure 4.8 Butane utilization and 1,1,1-TCA transformation achieved in the
slurry (microcosm B6) inoculated microcosms constructed with groundwater
and aquifer solids.
47
0)
E
2
U)
(0
(0
0)
29
30
31
32
33
34
35
33
34
35
Time, day
0.1
0.08
E
U)
U)
Cu
0.04
0.02
29
30
31
32
Time, day
Figure 4.9 The competitive inhibition (day 29 to 31) of 1,1,1-TCA
transformation by butane in the slurry (microcosm B6) inoculated microcosms
constructed with groundwater and aquifer solids.
48
5
4
E
u3
C
ml
[11
0
10
20
30
40
50
Time, day
0.35
0.3
0.25
0.2
0.15
C.)
I-
0.05
n
U
0
10
20
30
40
50
Time, day
Figure 4.10 Butane utilization and 1,1,1-TCA transformation achieved in the
media-culture inoculated microcosms constructed with groundwater and
aquifer solids.
3
E2
(a)
CO
(5
22
24
26
28
30
Time, day
:iisi
-u-- B4-2
E
-h-- B4-3
0.06
CO
5
< 0.04
0
I0.02
0
22
24
26
28
30
Time, day
Figure 4.11 The competitive inhibition (day 23 to 25) of 1,1,1-TCA
transformation by butane in the media-culture inoculated microcosms
constructed with groundwater and aquifer solids.
50
5
4
E
Lii
0
10
20
30
40
50
40
50
Time, day
0.07
I,.
0.05
.04
0.03
I 0.02
:: 0.01
0
0
10
20
30
Time, day
Figure 4.12 Butane utilization and 1,1,1-TCA transformation achieved in the
slurry (microcosm B6) inoculated microcosms containing mineral salts growth
media.
51
for the B4 microcosm set during the first 50 days of incubation was 0.061 ± 0.012
mg 1,1,1 -TCA/mg butane, which was slightly lower than that achieved in the B2
microcosm set.
Figure 4.12 presents the butane utilization and 1,1,1-TCA transformation in
microcosms B3-1, B3-2, and B3-3. The microcosms were constructed with mineral
salts growth media and bioaugmented with slurry from microcosm B6. The first
addition of butane was consumed by day four. Transformation of 1,1,1 -TCA
achieved in the B3 microcosm set was lower than that achieved in the B2 and B4
microcosm sets. By day 28, 1,1,1-TCA transformation had essentially ceased after
24 days of active transformation. More butane was then added, and with its
utilization 1,1,1 -TCA was again transformed. The average transformation yield in
the B3 microcosm set during the first 50 days of incubation was 0.024 ± 0.001 mg
1,1,1-TCA/mg butane.
0.12
C)
>-:
CC)
0.1
OC
0.08
E.Q
I-
OD)
0.06
C -f
c.)
'-p- 0.04
OE
'-;
0.02
-. .
. B3-3
0
0
10
20
30
40
50
Time, day
Figure 4.13 Transformation yield for 1,1,1-TCA achieved in the inoculated
microcosms during the first 50 days of incubation
52
The transformation yields achieved in the microcosms during the first 50
days of incubation are presented in Figure 4.13 and Table 4.4. The measured
amount of TCA in the indigenous micro cosms fluctuated, yet little decrease in
concentration was observed; thus, the transformation yield could not be accurately
determined. Transformation yields in the B2 and B4 bioaugmented microcosm sets
were similar to each other. Jitnuyanont (1998) reported a similar transformation
yield of butane-utilizers (0.07mg 1,l,1-TCA/mg butane), which was determined
during the first 190 days of incubation. In this study, the transformation yield
increased with time. This is likely due to more frequent additions of TCA and
exposure to higher TCA concentrations. Although all bioaugmented microcosms
behaved similarly during the first 10 days of incubation, the B3 bioaugmented
microcosm set, which contained mineral salts growth media, showed significantly
lower transformation ability. The transformation yield for the B3 microcosm set
was two to three times lower than the other microcosm sets. The culture
characteristics in the B3 microcosm set may have changed when grown in mineral
salts growth media for longer than 20 days.
Table 4.4 Transformation yield for 1,1,1-TCA achieved in the microcosms
during the first 50 days of incubation.
Microcosm
Transformation yield
(mg 1,1,1 -TCA/mg butane)
B 1 set: indigenous microcosms constructed with
groundwater and aquifer solids
B2 set: slurry-inoculated microcosms constructed with
groundwater and aquifer solids
B3 set: slurry-inoculated microcosms constructed with
mineral salts growth media
B4 set: media-culture inoculated microcosms
constructed with groundwater and aquifer solids
0
0.079 ± 0.005
0.024 ± 0.001
0.06 1 ± 0.0 12
53
The rates of 1,1,1 -TCA transformation in the B2 and B4 microcosm sets
were measured during day 30 35, and day 38 - 45. To exclude competitive
inhibition effects between butane and 1,1,1 -TCA, the rates were measured in the
absence of butane. Thus, these represent resting cell transformation rate values.
Figure 4.14 presents the 1,1,1 -TCA transformation rate data for microcosm
B2-1 during day 30
35. A gradual slowing in the transformation rate is shown.
The experimental data was plotted on a semi-logarithmic scale to test for first-order
transformation, as shown in Figure 4.15. The slope of the linear regression line in
each graph represented the pseudo-first-order rate coefficient (P), the combination
term of kinetic parameters and biomass and equilibrium distribution of 1,1,1 -TCA,
which is illustrated in equation D.5, Appendix D. The values of pseudo-first-order
rate coefficient (P) obtained from the rate tests in all microcosms are presented in
appendix Tables G. 1 to G.6.
Ii
'I,
C.)
0.02
0
30
31
32
33
34
35
Time, day
Figure 4.14 1,1,1-TCA transformation rate data for microcosm B2-1 during
day 30-35.
I
54
b) The 2nd addition of 1,1,1 -TCA
a) The 1St addition of 1,1,1 -TCA
U
0
-1
0
-2
0
-2
0 -3
=
-4
-3
-5
0
-4
0.2
0.4
0.6
0.8
0.2
0
0.4
0.6
Time, day
Time, day
c) The 3rd addition of 1,1,1 -TCA
d) The
4th
addition of 1,1,1 -TCA
U
o
-i
0
C
C
-3
0
0.2
0.4
0.6
0.8
Time, day
Time, day
e) The 5th addition of 1,1,1 -TCA
U
0
-2
C
3
-4
0
0.2
0.4
0.6
0,8
1
1.2
Time, day
Figure 4.15 1,1,1-TCA transformation rate data for microcosm B2-1 during
day 30 - 35 plotted on a semi-logarithmic scale.
0.8
55
Values of the pseudo-first-order rate coefficient (P) from each graph in
Figure 4.15 are plotted against time, as shown in Figure 4.16. The exponential
regression line in the graph was obtained from Microsoft Excel 2000. The slope
and the power of the exponential regression line provided the values of the pseudo
first-order rate parameter (kmaxXo/Ks) and cell decay rate (b), respectively. The
calculation of these two values is based on equation D.8, as illustrated in Appendix
D. The pseudo-first-order rate coefficient (P) decreased with time. This is
probably due to a decreased amount of active biomass or the gradual inactivation of
oxygenase enzyme or a loss in energy, or the combination of all three processes.
10
8
B2-1
= 5.4817e2lB4x
R2 = 0.956
0
0
1
2
3
4
5
Time, day
Figure 416 The decrease in pseudo-first-order rate coefficient (P) as a
function of time during day 30 - 35.
Table 4.5 presents the values of pseudo-first-order rate parameters
(kmaxXo/Ks) and cell decay rates (b) obtained from the rate tests for all the
microcosms. The average decay rates of both microcosm sets were similar, ranging
from 0.15 to 0.28 day1. These values are in the range for cell decay by aerobic
microorganisms. Similar values of the decay rates between both microcosm sets
56
indicate that growing the culture on growth media before bioaugmentation may not
change the microbial community. The pseudo-first-order rate parameters
(kmaxXø/Ks) were within a factor of two of each other. This possibly resulted from
the different amount of active biomass in the microcosms.
Table 4.5 The pseudo-first-order rate parameter (kmaxXüIKs) and cell decay
rate (b) for the B2 and B4 microcosm sets during day 30 - 35.
Microcosm
b
kmaxXo/Ks
(day')
B2-1
B2-2
B2-3
Average
B4-1
B4-2
B4-3
Average
(day1)
8.77
10.66
10.49
0.22
0.23
9.97± 1.04
0.22±0.013
6.62
3.38
4.99
4.99 ± 1.62
0.28
0.20
0.15
0.21 ± 0.064
0.21
Experimental data for microcosm B2-1 during day 38 45 are shown in
Figure 4.17. Similar to the former rate test, the data are plotted on a semilogarithmic scale, as shown in Figure 4.18, in order to determine the value of
pseudo-first-order rate coefficient (P) from the slope of the linear regression line in
each graph. Figure 4.19 presents the decrease in pseudo-first-order rate coefficient
(P) as a function of time. The data did not fit the exponential decay model well.
The high 1,1,1 -TCA concentration in the second TCA addition data maybe out of
the pseudo-first-order range and may partly be causing the poor fit.
The pseudo-first-order rate coefficients (P) from the experimental data
during both day 30 35 and day 38 45 were plotted against the accumulation
mass of 1,1,1 -TCA transformed, to examine if there was a correlation. As shown
in Figure 4.20, the transformation rate decreases with the increasing mass of 1,1,1 -
TCA transformed, Similar values of the coefficient of determination
(R2)
indicate
57
that the data at low concentration during day 30 35 are fit equally well by time
(Figure 4.16) or mass (Figure 4.20a) regression lines. When in a high
concentration range, during day 38
45, the coefficient of determination (R2) is
smaller for the time fit than the mass fit as shown in Figures 4.19 and 4.20b. Thus
the decrease in pseudo-first-order coefficients (P) tends to be more strongly
correlated with the transformed mass of 1,1,1 -TCA rather than time.
0.3
0.25
E
ui
0.2
-
0.05
0-i-
38
40
42
44
46
Time, day
Figure 4.17 1,1,1-TCA transformation rate data for microcosm B2-1 during
day 38-45.
Similar to the microorganisms in parent microcosm B6, the microorganisms
in all bioaugmented microcosms were able to transform l,1,l-TCA over long
periods in the absence of butane utilization. The indigenous microorganisms in the
B 1 microcosm set showed less ability to transform 1,1,1 -TCA than those in Figure
4.2. Thus the bioaugmented microcosms had more reproducible performance than
the non-augmented microcosms. Growing the culture on growth media before
bioaugmentation did not change 1,1,1 -TCA transformation abilities. However, the
mixed cultures probably changed when growing in mineral salts growth media for
longer than 20 days, as indicated by the differences in transformation yields (Figure
4.13).
58
a) The
1st
addition of 1,1,1 -TCA
0
-2
o
I
0.1
0.2
0.3
0.4
0.5
Time, day
b) The
2nd
addition of 1,1,1 -TCA
0
-1
0
-2
-3
0
0.2
0.4
0.6
0.8
Time, day
e) The 3rd addition of 1,1,1-TCA
Ii]
o -1
-3
0
0.2
0.4
0.6
0.8
Time, day
Figure 4.18 1,1,1-TCA transformation rate data for microcosm B2-1 during
day 38 - 45 plotted on a semi-logarithmic scale
59
16
. B2-1
12
y = 6.5596e°1629><
R2 = 0.4063
0
4
I
0
0
2
4
6
8
Time, day
Figure 4.19 The decrease in pseudo-first-order rate coefficient (P) as a
function of time during day 38 45
The long-term transformation ability of 1,1,1 -TCA observed in the
microcosms indicates that the enzyme responsible for transformation was present
and active long after butane was utilized. The decrease in transformation rate at
high TCA concentration tends to correlate better with the mass of 1,1,1 -TCA
transformed than with time, while that at low concentration transformation rate
decreases correlate with both time and mass transformed. It may be that repeated
transformation of low amounts of 1,1,1 -TCA did not significantly affect the
microorganisms, but served to induce enzyme activity. Limited and slow
transformation of 1,1,1 -TCA appeared to be correlated with a slow rate of butane
uptake. 1,1,1 -TCA transformation product toxicity, a drain of energy reserves and
cell decay may all be contributing to a loss in activity.
a) During day 30
35
10
y = 6.3808e341x
8
R2
B2-1
0.9764
I
2
0
0
0.05
0.1
0.15
0.2
0.25
1,1,1-TCA Mass mg
b) During day 38 - 45
16
TY = 12.56e'47312"
12
B2-1
R2 = 0.9421
4
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
1,1,1-TCA Mass mg
Figure 4.20 The decrease in pseudo-first-order rate coefficient (P) as a
function of transformed 1,1,1-TCA mass during day 30 -35 and day 38 45.
61
4.2.2 Transformation of CAB mixture
The transformation of CAH mixtures of 1,1,1 -TCA, TCE, and 1,1 -DCE was
also studied in microcosms stimulated on butane. The microcosms were
constructed in the same fashion as in the previous experiment. Microcosms B2-1
and B2-2 were used in the past study of 1,1,1 -TCA transformation. The BM
microcosm set was stimulated by one addition of butane, and was inactive for about
80 days before starting this experiment. The microcosm conditions are presented in
Table 4.6.
Table 4.6 Microcosms used for the experiment of CAl! mixture
transformation
Microcosm
Contents
Substrate
CAlls
Description
B2- 1
GW/Soil
Butane
1,1,1 -TCA,
1,1-DCE,
Inoculated with 1 ml of
GW/soil slurry
transferring from
microcosm B6
Inoculated with 100 p1 of
washed media-culture
harvested from the
growth reactor having 1
mL of groundwater/soil
slurry transferring from
microcosm B6 as a
culture seed
B2-2
TCE
BM 1-2
BM2-2
BM3-2
Growth media
Butane
1,1,1 -TCA,
1,1 -DCE,
TCE
Figure 4.21 presents the utilization of butane and the transformation of the
CAH mixture in microcosm B2-1. Microcosm B2-1 was inactive for 43 days after
the previous experiment of 1,1,1 -TCA transformation. On day 97, butane was
added to re-stimulate the microcosm and was consumed within three days. After
the second addition of butane was consumed, 390 pg/L of 1,1,i-TCA, 540 pg/L of
62
TCE, and 280 tg/L of 1,1-DCE were added to the microcosm. The transformation
rate of 1,1-DCE was so fast that all 1,1-DCE was transformed within 4 hours, while
1,1,1 -TCA, butane and TCE were transformed within 20 hours, 1 day, and 5 days,
respectively. The experiment was repeated on day 105 but at a higher
concentration of the CAlls. Butane utilization occurred within 2 days.
Approximately 730 tg/L of l,1-DCE, and 590 tg/L of 1,l,l-TCA were
transformed within 3 hours and 7 days, respectively. However, the transformation
of TCE (1400 tg/L) ceased after 80% of TCE was transformed. The re-addition of
butane on day 123 enhanced TCE transformation. On day 139, the experiment was
repeated. However, the transformation capacity decreased dramatically. Only 6%
of butane, 13 % of TCA, 17% of TCE, and 72% of 1,1 -DCE were transformed.
The utilization of butane and the transformation of a CAH mixture in
microcosm BM2-2 are presented in Figure 4.22. Microcosm BM2-2 was
stimulated once by one addition of butane, and was inactive for about 78 days. On
day 81, the microcosm was re-stimulated by two additions of butane. After butane
was consumed, 1,1,1 -TCA, TCE and 1,1 -DCE were added to the microcosm. 1,1 DCE (100 gIl), 1,1,1-TCA (550 tg/1) and TCE (630 tg/l) were transformed
within 1 day, 6 days, and 8 days, respectively. The inhibition of 1,1,1 -TCA and
TCE transformation by 1,1 -DCE was observed. The transformation rates of all
compounds including butane decreased in the second experiment on day 92 and 95.
1,1 -DCE (420 j.ig/l) was completely transformed within 3 days, while only 36% of
l,l,1-TCA (550 .ig/1) and 12% of TCE (840 tg/l) were transformed. More butane
was then added to enhance 1,1,1 .-TCA and TCE transformation on day 102. With
the third addition of CAH mixture, no butane utilization and limited chlorinated
solvent transformation occurred. The utilization of butane and the transformation
of mixtures of 1,1,1 -TCA, TCE and 1,1 -DCE in the other microcosms were similar,
as shown in appendix Figures G.6 to G.8.
In addition to 1,1,1 -TCA, the bioaugmented butane-utilizers were able to
transform other chlorinated solvents such as 1,1 -DCE and TCE. The
63
ri
C)3
E
U)
U)
C)
C
U)
4-.
110
130
Time, day
150
170
0.12
0.1
0.08
C)
E
j 0.06
0)
U)
0.04
0.02
90
110
130
150
170
Time, day
Figure 4.21 Butane utilization and chlorinated compound transformation
achieved in slurry-inoculated microcosm B2-1.
5
4
E
90
100
110
120
Time, day
130
140
150
90
100
110
130
140
150
0.14
0.12
0.1
0.08
0.06
0.04
0
'
0.02
120
Time, day
Figure 4.22 Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM2-2.
65
transformation of 1,1 -DCE was very fast, while the transformation rate of 1,1,1 -
TCA and TCE were slower. The transformation product toxicity from chlorinated
solvents, especially from 1,1 -DCE and possibly TCE at high concentrations, was
suspected to be the maj or cause of the limited transformation of CAlls and the
cessation in butane utilization.
4.2.3 Selecting microcosm for bioaugmentation studies
Based on the good performance of 1,1,1 -TCA transformation, microcosm
B2-1 was chosen to be the culture seed to obtain a uniform culture for
bioaugmentaion studies both in the laboratory and the field tests. After the
experiment of 1,l,1-TCA transformation (Figure 4.8), on day 53, butane was added
to enrich the culture before transferring one milliliter of the groundwater/soil slurry
for growth in each often batch reactors. A batch growth reactor was set up in a
500 mL bottle sealed with a grey butyl rubber lined cap, and filled with 250 mL
mineral salt growth media (Appendix A.3). Butane was added by gaseous addition
to each bottle to yield a 10% vol/vol in the headspace. The growth reactor was
placed on a shaker table at 200 rpm in a 20°C constant temperature room. Pressures
were equilibrated daily with pure oxygen or air. The butane mass and the optical
density were monitored during the incubation. Figure 4.23 presents the growth
curve of one of the batch reactors. The lag phase of the culture was around 4-5
days. The culture was harvested on day 7, at an 0D600 of approximate 0.36. The
liquids from all the bottles were combined and centrifuged to concentrate the
biomass. The concentrated biomass was placed in 1 mL vials with 7%DMSO
(Dimethyl Sulfoxide) and stored in 196 °C liquid nitrogen for future studies. The
calculated yield was around 0.7 mg cell / mg butane, and the density of the frozen
culture was 2500 mg TSS IL.
125
,
0.4
100
0.3
E
U)
U)
02
U)
4-
0.1
25
0
iJ
0
2
4
6
8
Time, day
Figure 4.23 Growth curve of one of the batch reactors providing the culture
for bioaugmentation study.
4.3 MICROCOSM STUDIES WITH FROZEN BUTANEUTILIZING CULTURE
4.3.1 Transformation of chlorinated solvents
The objective of these experiments was to compare the 1,1,1 -TCA
transformation ability between indigenous microorganisms from Moffett test site
and butane-utilizers bioaugmented to Moffett Field microcosms. The
transformation of CAll mixtures of 1,1,1 -TCA, TCE, and 1,1 -DCE was also
evaluated in bioaugmented microcosms. Microcosms were constructed in 125 mL
media bottles sealed with grey butyl rubber lined caps. They were filled with 100
mL of Moffett groundwater and aquifer material. The conditions of the
microcosms are shown in Table 4.7. A media-culture for bioaugmentation was
obtained from a growth reactor inoculated with the frozen butane-utilizing culture.
The enrichment was harvested from the batch growth reactor at the measured
optical density (0D600) of 0.25 and density of 120 mg TSS IL. The cells were not
67
washed before bioaugmentation. Successive additions of saturated 1,1,1 -TCA
solution were made to the microcosms stimulated on butane. The aqueous 1,1,1TCA concentrations ranged from 350 to 1000 pgIL. In the CAH mixture study,
l,1,l-TCA (350 ig/L), TCE (440 tg/L), and 1,1-DCE (340 pg/L) were added to
the bioaugmented microcosms.
Table 4.7 Microcosms used for the experiment of CAH transformation.
Microcosm
BR2-C3
BR2-2
Contents Substrate
GW/Soil
GW/Soil
Butane
Butane
CAHs
1,1,1-TCA
1,1,1 -TCA,
1,1-DCE, TCE
BR2-3
GW/Soil
Butane
1,1,1 -TCA,
1,1-DCE, TCE
Indigenous microcosm
Inoculated with 100 p1 of
media-culture
Inoculated with 500 p1 of
media-culture
Figure 4.24 presents the butane utilization and 1,1,1 -TCA transformation
achieved in non bioaugmented microcosm BR2-C3. No 1,1,1-TCA transformation
was observed even after four additions of butane. With long-term exposure to
1,1,1 -TCA, the rate of butane utilization slowed.
The butane utilization and 1,1,1 -TCA transformation achieved in
microcosm BR2-2 is presented in Figure 4.25. 1,1 ,1-TCA was added to the
microcosm along with butane. Butane was consumed in 5 days, and 1,1,1 -TCA
transformation was then observed. On day 18, 1,1,1-TCA transformation ceased
after 0.2 mg of 1,1,1 -TCA was transformed. More butane was then added to
enhance the transformation. On day 26 and 44, the successive additions of TCA
were repeated. The total mass of TCA transformed was 0.36 and 0.25 mg,
respectively. On day 28, after butane was consumed, 1,1,1-TCA transformation
accelerated. This is most likely due to butane competitive inhibitory effect on
68
5
4
E
w
2
m
1
20
40
60
80
100
120
140
Time, day
BR2-C3
E
0.06
(0
0.04
0.02
0 .L
0
20
40
60
80
100
120
140
Time, day
Figure 4.24 Butane utilization and 1,1,1-TCA transformation achieved in
indigenous microcosm BR2-C3
5
BR2-2
E
U,
U)
C)
CU
4-.
I
0
10
20
30
40
50
Time, day
0.16
a)
E 0.12
U)
0.08
0.04
[ii
0
10
20
30
40
50
Time, day
Figure 4.25 Butane utilization and 1,1,1-TCA transformation achieved in
media-culture inoculated microcosm BR2-2
70
[.1
m
I
0
10
20
30
40
50
Time, day
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
10
20
30
40
50
Time, day
Figure 4.26 Butane utilization and 1,1,1-TCA transformation achieved in
media-culture inoculated microcosm BR2-3
71
1,1,1 -TCA transformation. The transformation rate decreased gradually with
continued additions of 1,1,1 -TCA. The butane utilization also slowed down around
day 37 after continued l,1,l-TCA.transformation.
Figure 4.26 presents the butane utilization and 1,1,1-TCA transformation
achieved in microcosm BR2-3. Microcosm BR2-3 had the five times amount of
inoculation as microcosm BR2-2. Unlike microcosm BR2-2, only butane was
initially added to microcosm BR2-3, and it was consumed in 5 days, the same as
microcosm BR2-2. After the butane was consumed, 1,1,1-TCA was added to the
microcosm. Similar to microcosm BR2-2, 1,1,1 -TCA transformation ceased on
day 18. The mass of transformed 1,1,1-TCA was 0.15 mg. The re-addition of
butane was conducted to enhance the transformation. The 1,1,1 -TCA experiments
were repeated day 26, and 42. The total mass of TCA transformed was 0.3 and
0.23 mg, respectively. After day 37, the butane uptake rate had slowed down.
Similar to microcosm BR2-2, the competitive inhibition of 1,1,1 -TCA
transformation by butane and a decrease in transformation rate with continued
addition of 1,1,1-TCA were also observed in microcosm BR2-3.
BR2-2
o.oa
Ca,
U,
0.04
0428
30
32
34
36
Time, day
Figure 4.27 1,1,1-TCA transformation rate data for microcosm BR2-2 during
day 28-37
72
b) The 2nd addition of 1,1,1-TCA
a) The 1st addition of 1,1,1-TCA
0
0.5
0
-1
-1.5
002040608112
Time, day
c) The
3rd
Time, day
addition of 1,1,1-TCA
d) The
------------------------.-------------
0
4th
addition of 1,1,1 -TCA
0
-0.5
-0.5
0
0
-1.5
o -1.5
-I
0
-2
0.2
0.4
-2.5
0.6
0.8
1
1.2
Time, dai
0
0.5
1
Time, day
e) The 5th addition of 1,1,1 -TCA
0 .------------.--------------- ....
o -1
-3 I,.F
-2
0
0.5
1
1.5
2
2.5
Time, day
Figure 4.28 1,1,1-TCA transformation rate data for microcosm BR2-2 during
day 28 - 37 plotted on a logarithmic scale
15
73
Figure 4.27 presents the experimental data during on day 28 to 37 for
microcosm BR2-2. The gradual slowing in 1,1,1-TCA transformation rates can be
seen. These data were plotted on a logarithmic scale to test for first-order
transformation behavior, and to determine the pseudo-first-order rate coefficient (P)
from the slope of regression line, as shown in Figure 4.28. The pseudo-first-order
rate coefficient (P) is a combination term including kinetic parameters and biomass
as illustrated in equation D.5. The values of this term (P) obtained from the rate
tests in both microcosms are shown in appendix Tables 0.7 and G.8.
The values of the pseudo-first-order rate coefficient (P) from each graph in
Figure 4.28 are plotted against time, as shown in Figure 4.29. The values of pseudo
first-order rate parameter (kmaxXo/Ks) and cell decay rate (b) were determined from
the slope and the power of an exponential regression line, respectively. An
example calculation is shown in Appendix D. A good fit to the exponential decay
model was observed.
5
4
.BR2-2
R2O.9765
3
0
2
I
0
0
1
2
3
4
5
Time, day
Figure 4.29 The decrease in pseudo-first-order rate coefficient (P) as a
function of time during day 28 - 37.
74
The results from all microcosm rate tests are presented in Table 4.8. The
pseudo first-order rate parameter (kmXø/Ks) for microcosm BR2-3 was slightly
greater than that of BR2-2. The initial inoculated biomass of BR2-3 was five times
greater than that of BR2-2. The result is consistent with the more rapid uptake of
the third addition of butane. The cell decay rates (b) in both microcosms were
similar to those in microcosms inoculated with either slurry or media-culture (Table
4.5). The similarities in 1,1,1 -TCA transformation indicates the mixed cultures in
the slurry survive growth in media and freezing at 196 °C.
Table 4.8 The pseudo-first-order rate parameter (kmaxXolKs) and cell decay
rate (b) for the B2 and B4 microcosm sets during day 28 - 37.
Microcosm
BR2-2
BR2-3
Initial inoculum
(mg/L)
0.12
0.6
kmaxXo/Ks
b
(day')
(day')
3.4
4.3
0.18
0.16
Figure 4.30 presents the utilization of butane and transformation of
chlorinated solvent mixtures of 1, 1,1 -TCA, TCE and 1,1 -DCE achieved in
microcosm BR2-2 after 52 days of incubation. On day 53, butane was added along
with 1,1,l-TCA, TCE, and 1,1-DCE. Butane utilization occurred within 3 days.
1,1 -DCE, and 1,1,1 -TCA were transformed within 2 and 4 days, respectively.
However, the TCE transformation ceased after 90% of the TCE was transformed.
On day 58, the microcosm was purged under a laminar flow hood to remove TCE,
and more butane was added to increase the amount of biomass. The experiment
was repeated on day 60. Butane was consumed within 2 days. 1,1-DCE, 1,1,1TCA and 70% of TCE were transformed within 13 hours, 3 days and 6 days,
respectively. After 66 days of incubation, the utilization of butane and the
transformation of chlorinated solvents slowed down. Butane utilization and 1,1 DCE transformation occurred within 4 days. 1,1,1 -TCA and 87% of TCE was
4
D)
E
a)
m
50
60
70
80
90
Time, day
('I
ion
transformed within 10 days. Inhibition of 1,l,1-TCA and TCE transformation by
butane and 1,1-DCE was observed. The behavior of microcosm BR2-3 was
similar, as shown in appendix Figure G.9.
The microcosms bioaugmented with the frozen butane-utilizing culture
showed the same performance as the microcosms inoculated with slurry. Longterm transformation of 1,1,1 -TCA in the absence of butane utilization was achieved
at rapid rates. The transformation of a mixture of 1,1,1 -TCA, 1,1 -DCE and TCE
also showed similar trends. The transformation rate of 1,1 -DCE was the fastest,
while the transformation rate of 1,1,1 -TCA and TCE were slower. After
transforming the mixture of these chlorinated solvents, the rates of butane
utilization and chlorinated solvent transformation slowed down. This is probably
due to the transformation product toxicity, especially that of 1,1 -DCE.
4.3.2 Butane and 1,1,1-TCA kinetic experiments
Table 4.9 Microcosms used for the kinetic experiments
Microcosm
Contents
Substrate
BRC-1 to BRC5
BR3-1 to BR3-5
GW/Soil
GW/Soil
Butane
Butane
BR4-1 to BR4-5
GW/Soil
Butane
BR4-M
Growth
media
Butane
CAHs
Description
1,1,1-TCA Indigenous microcosm
1,1,1-TCA Inoculated with 500 t1 of
media-culture, and having
initial biomass of 0.47 mg/L
1,1,1-TCA Inoculated with 500 1 of
media-culture, and having
initial biomass of 0.45 mg/L
l,1,l-TCA Inoculated with 500 tl of
media-culture, and having
initial biomass of 0.45 mg/L
77
Studies were conducted over a range of butane and 1,1,1 -TCA
concentrations to determine the half-saturation constants
(Ks) of indigenous and
bio augmented butane-utilizers under microcosm conditions. The microcosms
constructed for this experiment are presented Table 4.9. The microcosm
fabrication was the same as for the previous experiment. The bioaugmented
cultures of the BR3 and BR4 microcosm sets, and microcosm BR4-M were
obtained from reactors inoculated with the frozen butane-utilizing culture but at
different times. These studies help demonstrate the reproducibility of the
bioaugmentation process.
0
0*
Butane Mdition#5
0
0
E
0
0
0
(0
(0
oBR3-2
o
C)
In-.....
Butane Addition#3
'I-
0
0
/
Butane Addition#4
Li
0
5
10
15
20
Time, day
Figure 4.31 Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-2
Figure 4.31 presents the overall results from the butane kinetic experiments
conducted in inoculated microcosm BR3-2. Two additions of butane (4 mg) were
added to each microcosm to stimulate the microbial population. To determine a
butane K value, a kinetic experiment was conducted on day 5, starting at an
aqueous concentration of 40 j.igIL. Butane utilization followed first-order kinetics
78
a) The third addition of butane
0.1
1
0.08
o BR3-2
*
E
0
Cl)
Cl)
0
0
0.04
0
0
0.02
*0
0
5.10
5.15
5.20
5.25
5.30
5.35
5.40
Time, day
b) Butane utilization following first-order kinetics
0
0 BR3-2
0
-2
-
-4
C
y = -0.8895x + 0.1381
R2 = 0.996
0
-5
-6
0
1
2
3
4
5
6
Time, hr
Figure 4.32 Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 on day 5
a) The fourth addition of butane
'J. ..,
Rate
o BR3-2
0.4
E
U)
U)
C
C
0.2
.0
0.1
0
6.25
6.30
6.35
6.40
6.45
6.50
Time, day
b) Initial butane utilization following zero-order kinetics
(r
* BR3-2
0.4
C)
E
0.3
C
C)
c
0.2
.0
0.1
0
0
0.5
1.5
Time, hr
c) Butane utilization following first-order kinetics
0
0
-1
o BR3-2
0-2
0
C3
y = -O.8136x + 0.2951
R20.9967
-4
-5
1
o
i
2
3
4
5
6
Time, hr
Figure 4.33 Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 on day 6
EIC
a) The fifth addition of butane
4
C)
3
*
E
(
*
13.00
13.50
BR3-2
o
Zero-Order Rate
/
14.00
First-Order Rate
14.50
15.00
Time, day
b) Initial butane utilization following zero-order kinetics
4
C)
BR3-2
3
y = -0.18x + 3.5893
R2 = 0.9991
040
5
10
20
15
Time, hr
c) Butane utilization following first-order kinetics
0 ------------.-.-2
o BR3-2
0
-4
C
-6
-8
21
22
23
24
25
26
27
Time, hr
Figure 4.34 Experimental data from the butane kinetic experiment conducted
in microcosm BR3-2 from day 13 to 14
81
as shown in Figure 4.32. Another kinetic experiment was then conducted on day 6
at a higher butane concentration (200 tg/L). As shown in Figure 4.33, the first five
data points tend to follow zero-order kinetics and the remaining seem to follow
first-order kinetics. On day 13, another kinetic test was performed at an aqueous
concentration of 1800 tg/L. Butane transformation rates covered both zero-order
and first-order ranges, as shown in Figure 4.34, All microcosms behaved similarly
as shown in appendix Figures G. 10 to G. 13. As shown in appendix Table G.9, the
butane K values of the BR3 microcosm set were determined from the results of the
fourth and fifth additions of butane, following the procedure described in Appendix
E. Table 4.10 presents the K values for butane and the initial aqueous
concentration of butane. The K values obtained from both experiments were
similar. The average value for the BR3 microcosm set is 0.11 ± 0.0 14 mg/L.
Table 4.10 Butane half-saturation constant
microcosm set.
(Ks) achieved in the BR3
The 4th addition of butane
Microcosm
C1
(.ig/L)
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
Average
196
196
196
193
175
The
5th
addition of butane
C1
(mg/L)
0.119
0.104
0.109
(j.xg/L)
1514
1577
0.11
1711
1711
0.12
1709
0.11±0.007
(mgIL)
0.077
0.109
0.102
0.113
0.132
0.11±0.02
After the fifth addition of butane was completely consumed, a 1,1,1 -TCA
kinetic test was started on day 15. The 1,1,1 -TCA kinetic experiments were
conducted in a similar maimer as the butane experiments. Figure 4.35 presents the
overall results from the 1,1,1 -TCA kinetic experiments conducted in microcosm
BR3-2. 1,1,1-TCA transformation at an initial concentration of 100 tg/L followed
82
first-order kinetics, as shown in Figure 4.36. Thus the K value could not be
determined from this 1,1,1 -TCA addition. Another kinetic experiment was
performed at a higher aqueous concentration. As shown in Figure 4.37, the
transformation of 1,1,1 -TCA at 700 .ig/L followed zero-order kinetics initially, and
then followed first-order kinetic after the 1,1,1 -TCA was lower than 250 j.tgIL.
This indicates that the K value is between 250 and 700 tg/L. Results from the
other microcosms were similar, as shown in appendix Figures G.14 to G.17. The
K values for 1,1,1 -TCA achieved in the BR3 microcosm set are determined from
the results of the second 1,l,l-TCA addition, as shown in appendix Table G.l0.
Table 4.11 presents the K values along with the initial aqueous concentration of
1,1,1 -TCA. All microcosms had similar K values. The average K value is
0.37 ± 0.05 1 mgIL. The results show that the bioaugmentation process is very
consistent.
0.12
0.1
LBR3-2
A
E
C,;'
U)
A
0.08
1,1,1 -TCA Addition#2
0.06
0.04
0.02
I
14.50
A/
A
1,1,1 -TCA Addition#1
AA1A
15.50
A
16.50
__________________
17.50
18.50
19.50
Time, day
Figure 4.35 Overall results from the 1,1,1-TCA kinetic experiment conducted
in bioaugmented microcosm, BR3-2
83
a) The first addition of 1,1,1 -TCA
0,02
BR3-2
E 0.015
£
0.01
': 0.005
::
0
0
2
,.Ar
:
4
6
Time, hr
8
10
12
b) 1,1,1 -TCA transformation following firstorder kinetics
0
-0.5
L BR3-2
y = -0.2472x - 0.0753
R2=0.988
0
2
4
6
8
10
12
Time, hr
Figure 4.36 Experimental data from the 1,1,1-TCA kinetic experiment
conducted in microcosm BR3-2 on day 15.
84
a) The second addition of 1,1,1 -TCA
1',
r.
U. It
Zero-Order Rate
A
0.1
)
E
BR3-2
AT
0.08
A
First-Order Rate
0.06
0.04
A
A
'
A
0.02
A
A
0
0
5
10
15
20
25
30
Time, hr
b) Initial 1,1,1 -TCA transformation following zero-order kinetics
n
0.1
0.08
0.06
0.04
0.02
0
0
1
2
3
6
5
4
Time, hr
c) 1,1,1 -TCA transformation following first-order kinetics
0 £
A
A
-0.5
A
BR3-2
0
-1.5
-2.5
-3
0
5
10
15
20
25
30
Time, hr
Figure 4.37 Experimental data from the 1,1,1-TCA kinetic experiment
conducted in microcosm BR3-2 from day 15 to 16
85
Table 4.11 1,1,1-TCA half-saturation constant (Ks) achieved in the BR3
microcosm set.
The
Microcosm
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
Average
2nd
addition of 1,1,1 -TCA
Ci
(jtg/L)
680
731
698
720
711
(mg/L)
0.400
0.443
0.324
0.335
0.345
0.37 ± 0.05 1
Kinetic experiments were also conducted in the BR4 microcosm set. The
overall results from the kinetic experiments for both butane and 1,1,1 -TCA in
microcosm BR4-5 are presented in Figure 4.38. The first two additions of butane
were performed to stimulate the microbial population. The butane kinetic
experiments were conducted with the third and fifth additions of butane. As shown
in Figure 4.39 and 4.40, the data from both experiments followed zero-order
kinetics initially and then first-order kinetics. The initial concentration ranged from
230 to 330 pgIL. The results from the kinetic experiments in all microcosms were
similar as shown in appendix Figures G. 18 to G.22 and appendix Table G. 11.
Table 4.12 presents the K values for butane from the BR4 microcosm set and the
initial concentrations of butane. The similar results from all microcosms indicate
the reproducibility of bioaugmentation process. The average K value for the BR4
microcosm set is 0.11 ± 0.012 mgIL. Microcosm BR4-M, which contained growth
media, had a slightly lower K value than the other microcosms.
After the third and fifth additions of butane were consumed, kinetic
experiments with 1,1,1 -TCA were performed on day 5 and 13. As shown in
Figures 4.41 and 4.42, 1,l,1-TCA transformation at low concentrations followed
first-order kinetics while transformation at high concentrations was approximated
by zero-order kinetics, On day 5, after six data points were collected, 1,1,1 -TCA in
['74
rSi
4
Butane Addition#4
I
0
E
oBR4-5
Butane Addition#3
1
r
Butane Mdition#5
0
0
5
10
20
15
Time, day
0.4
1,1,1-TCAAddthon#2
A BR4-5
E
0.3-
A
A
có
A
(0
1,1 ,1-TCA
0.2
17
Addition#4
1,1,1-TCAAddition#3
A
\f
-'i
11 1-TG Addition#1
/
A
\
A
\A
0
0
5
10
15
Time, day
Figure 4.38 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-5
20
87
a) The third addition of butane
0.6
0
0
(0
(0
Zero-Order Rate
o
BR4-5
0.4
0
0.2
First-Order Rate
0
4.30
00 0000
4.35
4.40
4.45
4.50
4.55
4.60
Time, day
b) Initial butane utilization following zero-order kinetics
xi
BR4-5
o
0.4
y = -0.2681x + 0.562
R2 = 0.9977
J3 0.2
0
0
0.2
0.4
0.6
Time, hr
0.8
1
c) Butane utilization following first-order kinetics
0
51
0
0
BR4-5
y = -1.042x + 2,1379
R2 = 0.9971
-2
-3
-4
4.4
4.6
4.8
5
5.2
5.4
5.6
Time, hr
Figure 4.39 Experimental data from the butane kinetic experiment conducted
in microcosm BR4-5 on day 4
['Z'J
a) The fifth addition of butane
0.8
o BR4-5
0.6
Zero-Order Rate
o
6,
C')
C')
5
0.4
0
w
0
0
0.2
___
0
12.50
12.60
000000
12.70
12.80
Time, day
b) Initial butane utilization following zero-order kinetics
0.8
o BR4-5
a)
U)
U)
0.4
0.2
y = -0.2485x 0.5567
R2 = 0.9836
0
r
0
0.2
0.4
0.6
0.8
1
1.2
Time, hr
c) Butane utilization following first-order kinetics
0
o BR4-5
y=-1.098x+2.9711
I
-
1
5
5.2
5.4
5.6
5.8
6
6.2
Time, hr
Figure 4.40 Experimental data from the butane kinetic experiment conducted
in microcosm BR4-5 on day 13
89
the microcosms was purged. The results from the experiments in all microcosms
were similar, as presented in appendix Table G. 12. The 1,1,1 -TCA K values
obtained from the BR4 microcosm set are presented in Table 4.13. The average K
value for the BR4 microcosm set is 0.35 ± 0.114 mg/L. Very reproducible
bioaugmentation was achieved in all the microcosms.
Table 4.12 Butane half-saturation constant
microcosm set.
The
3rd
(Ks) achieved in the BR4
addition of butane
The
5th
addition of butane
Microcosm
C1
(mg/L)
0.061
(.igIL)
242
(mg/L)
BR4-M
(jig/L)
326
271
253
0.095
0.108
0.129
0.127
0.113
267
233
232
248
254
0.105
0.093
0.116
0.117
0.100
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
Average
264
284
248
C1
0.12±0.014
0.071
0.11±0.01
Table 4.14 presents the average butane and 1,1,1-TCA K values achieved
in the BR3 and BR4 microcosm sets and microcosm BR4-M. The microcosms
were all bioaugmented with the same frozen culture, but from batches of cells
grown at different time and with different frozen cells. They all had the similar K
values for butane and 1,1,1 -TCA. The results indicate a very reproducibility of the
bioaugmentation process, using the procedures that were established.
a) The first and second additions of 1,1,1 -TCA
0.4
a)
BR4-5
.
0.3
_/
Zero-Order Rate
.
.
.
First-Order Rate
LI
4.70
4.80
4.90
5.00
b.]I.)
Time, day
b) 1,1,1 -TCA transformation following first-order kinetics
0
-0.5
0
-1
-1.5
-2
-2.5
0
2
1
4
3
5
6
Time, hr
c) 1,1,1 -TCA transformation following zero-order kinetics
0.4
C)
BR4-5
0.3
0.2
C-)
I
y = -0.0281x + 0.4868
= 0.979
01
0 -L
0
2
4
Time, hr
6
8
Figure 4.41 Experimental data from the 1,1,1-TCA kinetic experiment
conducted in microcosm BR4-5 on day 5
;,J1
a) The third and fourth additions of 1,1,1 -TCA
0.2
./-
0)
E 0.15
U)
U)
Zero-Order Rate
.
.
0.1
C)
F
.
'-:
0.05
BR4-5
First-Order Rate
0
12.50
13.00
13.50
14.00
14.50
Time, day
b) 1,1,1 -TCA transformation following first-order kinetics
-0.5
. BR4-5
0
C
-2
-2.5
-3
0
3
2
1
4
5
Time, hr
c) 1,1,1 -TCA transformation following zero-order kinetics
0.2
.
0)
E 0.15
BR4-5
U)
(0
0.1
C-)
I-
.-0.05
y = -0.0209x + 0.2788
= 0.9623
2
4
6
8
10
Time, hr
Figure 4.42 Experimental data from the 1,1,1-TCA kinetic experiment
conducted in microcosm BR4-5 from day 13 to 14
Table 4.13 1,1,1-TCA half-saturation constant
microcosm set.
(Ks) achieved in the BR4
a) The first and second additions of 1,1,1-TCA
Microcosm
Low C1
High C,
(.tgIL)
(ig/L)
BR4-M
112
2394
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
110
154
120
136
139
2633
2615
2399
2543
2557
Average
(mg/L)
0.306
0.519
0.480
0.259
0.464
0.427
0.430 ± 0.101
b) The third and fourth additions of 1,1,1 -TCA
Microcosm
Low C,
High C1
(g/L)
BR4-M
(jxg/L)
89
1386
(mg/L)
0.206
88
102
106
58
1663
1297
1326
1467
1381
0.365
0.263
0.186
0.289
0.276
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
121
Average
0.28 ±0.064
Table 4.14 Average values of butane and 1,1,1-TCA half-saturation constant
achieved in all microcosms
Microcosm
BR3 set
BR4 set
BR4-M
Butane K
(mg/L)
0.11 ± 0.014
0.11 ± 0.012
0.07 ± 0.007
1,1,1-TCA K
(mg/L)
0.37±0.051
0.35 ± 0114
0.26 ± 0.07
93
4.3.3 Model development
Kinetic parameters including the maximum specific rate of butane
utilization (km,But) and 1,1,1 -TCA transformation
(kmax,TCA),
cell decay rate (b),
and/or transformation capacity (Ta) for non-augmented and bioaugmented
microcosms for microcosm test were determined through simulations of butane
utilization and 1,1,1 -TCA transformation, using the non-steady-state model
described in Chapter
3.
The results of microcosm studies in the BR3, BR4 and B4
bioaugmented microcosm sets and the BRC indigenous microcosm set were
selected to conduct these simulations (Table 4.3 and
4.9).
Table 4.15 Measured parameters for model development
Parameter
BR3 set
Microcosm
BR4 set
B4 set
0.45
unknown
BRC set
unknown
Xo, mg/L
Y, mg/mg
0.47
0.7
0.7
0.7
0.7
KS BUt, mg/L
0.11
0.11
0.11
0.11
0.37
0.37
0.37
0.37
0.1
0.1
0.075
0.1
0.057
0.057
0.082
0.057
38.05
38.05
38.05
38.05
0.55
0.55
0.55
0.55
KSTCA,
VL,
Vg,
mg/L
L
L
HCC,BUt, mg-L/mg-L
HCC,TCA,
mg-L/mg-L
To run the model, all the parameters in equations 3.1,
3.2,
and
3.3
or 3.4
need to be supplied. Measured parameters including initial biomass (Xo) in the
bioaugmented microcosms, the mass of butane and 1,1,1 -TCA added, butane and
1,1,1 -TCA half-saturation constants
(K5 But
and KS,TCA), cellular yield (Y), volume
in liquid and gas phases (VL and Vg), and Henry partition coefficient for butane and
1,1,1 -TCA (Hcc,Bt and
are presented in Table
HCC,TCA)
4.15.
were not varied in the fitting proceed. These values
The values of KS,BUt and
KS,TCA
were obtained from the
kinetic experiments previously described, while the value of Y was determined
from growth studies with the frozen butane-utilizing culture. The unknown
parameter values for the maximum specific rate of butane utilization (km,ut) and
1,1,1 -TCA transformation (kmaxTCA), cell decay rate (b), and/or transformation
capacity (Ta) were varied in a heuristic approach to obtain a good fit between
model predictions and experimental observations. The errors of unknown
parameters were expressed in terms of standard deviation based on modeling of the
five replicate microcosms. Equations 3.1, 3.2, 3.3 and 3.4 were solved in
STELLA®. Numerical integration in time was performed in STELLA® using a
fourth-order Runge-Kutta method.
4.3.3.1 The simulation of butane utilization and 1,1,1 -TCA
transformation in the BR3 microcosm set
Figure 4.43 presents the butane utilization data from microcosm BR3-5 and
model simulations. The data was divided into 4 sections (A, B, C, and D). The A
section presented the data from the first and second additions of butane, while the
B, C, and D sections included the data from the third, fourth, and fifth additions of
butane, respectively. The values of kmax
But
and b were varied to obtain a good fit
between model predictions and experimental data shown in section A. The data of
the other sections were then modeled using these values, once they were fixed.
5
Section A
D)
0
4
SectionD
0
o
0
E
0
o
0
0
0
0
0
0
0
0
ection B
I
0
5
10
15
Time, day
Figure 4.43 Butane utilization data from microcosm BR3-5
20
95
Complete simulation of butane utilization for microcosm BR3-5
5
0
4
I
0
0
5
10
20
15
Time, day
Section A: The first and second additions of butane from day 0 to
5
5
D)
4
E
(I)
m
2
1
0
0
1
2
3
4
5
Time, day
Section B: The third addition of butane on day 5
U. I
,
BR3-5
0.08
E
Model
0.06
0.04
0.02
0
5.1
5.15
5.2
5.25
5.3
5.35
5.4
Time, day
Figure 4.44 Simulation of butane utilization for microcosm BR3-5
Section C: The fourth addition of butane on day 6
0.5
0) 0.4
E
CO
CO
Cu
S
C)
C
m01
0
6.25
6.3
6.35
6.4
6.45
6.5
6.55
Time, day
Section D: The fifth addition of butane from day 13.38 to 14.44
4
o
E3
0)
BR3-5
Model
(0
U)
Cu
2
2
C)
C
0
13.2
13.4
13.6
13.8
14
Time, day
Figure 4.44, Continued
14.2
14.4
14.6
97
The simulation of butane utilization for microcosm BR3-5 is presented in
Figure 4.44. The model fit data well at low concentration as well as high
concentration. The initial concentration ranged from 35 to 1800 tg/L. The
KS,BUt
values obtained from the kinetic experiments provided good matches of the
simulation to the microcosm results, as presented in section C. The model started
deviating from the experimental data in section D. This probably results from
fitting cell decay rate (b) with only early time data. Thus the value of cell decay
rate (b) may have been in error by a small amount. The results from the model
simulation for the other microcosms were similar, as presented in Figure 4.48 to
4.51. The values for the butane maximum specific rate (kmax,But) and cell decay rate
(b) of the BR3 bioaugmented microcosm setare shown in Table 4.16. Both values
obtained from all microcosms were similar. This indicates the reproducibility of
the bioaugmentation process. The average values for the butane maximum specific
utilization rate (kmax,But) and cell decay rate (b) were 1.76 ± 0.023 mg/mg-day and
0.15 ± 0.006 day1, respectively.
Table 4.16 Butane maximum specific rate (kmax,But) and cell decay rate (b)
achieved in the BR3 microcosm set.
Microcosm
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
Average
kmax,But
b
(mg/mg-day)
(day')
1.79
1.74
1.76
1.73
1.76
0.15
0.15
0.15
0.14
0.14
1.76±0.023
0.15 ±0.006
The 1,1,1-TCA transformation data from microcosm BR3-5 presented in
Figure 4.45 was also simulated. To simplify the modeling, the effect of
transformation product toxicity was not considered. Similar to butane utilization
98
modeling, the data were divided into 2 sections (E and F). The E and F sections
presented the data from the first and second additions of 1,1,1 -TCA. All
parameters previously determined for butane were used in the simulation. The only
unknown parameter was kmax.TCA. The variation of kmaX,TCA values was performed
until the model best fit the data in section E. This value was then used to model in
section F.
0.1
+
BR3-5
_____ Section F
0) 0.08
.1
0.06
.
0.04
.
4
'- 0.02
0 414.50
Section E
.
S
15.50
.
16.50
17.50
18.50
19.50
Time, day
Figure 4.45 1,1,1-TCA transformation data from microcosm BR3-5
Figure 4.46 presents the simulation of l,1,1-TCA transformation for
microcosm BR3-5. A good fit between the model simulation and data is achieved
in both sections of low and high concentrations. The initial concentration of data
ranged from 90 to 750 jtg/L. The l,1,1-TCA maximum specific rate (km,TCA)
values for microcosm set BR3 are shown in Table 4.17. All microcosms had
similar values, except microcosm BR3-1, which had the lowest l,1,1-TCA kmax.
The kmax,TCA values of microcosm set BR3 varied more than the kmax,But values.
The value of 1,l,1-TCA maximum specific rate (kmax,TCA) ranged from 0.05 to 0.12
mg/mg-day.
Complete simulation of 1,1,1-TCA transformation for microcosm BR3-5
0.1
BR3-5
Model
0.08
E
0.06
0.04
I-
::; 0.02
0
0
5
10
15
20
Time, day
Section E: The first addition of 1,1,1-TCA on day 15
0.015
BR3-5
C)
E
Model
0.01
.
0.005
0
15.00
15.10
15.20
15.30
15.40
1
,5U
Time, day
Section F: The second addition of 1,1,1-TCA from day 15 to 16
0.1
0.08
BR3-5
'I)
(0
0.06
Model
0.04
I-
0
15.00
15.50
16.00
16.50
17.00
Time, day
Figure 4.46 Simulation of 1,1,1-TCA transformation for microcosm BR3-5
100
Table 4.17 1,1,1-TCA maximum specific rate (kmax,TCA) achieved in the BR3
microcosm set.
Microcosm
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
Average
kmax,TCA
(mg/mg-day)
0.05
0.12
0.11
0.09
0.09
0.09 ± 0.027
The simulations of butane utilization and 1,1,1 -TCA transformation for
microcosms BR3-1 to BR3-5 are presented in Figure 4.47 to 4.51. A good fit
between model simulation and data at both low and high concentrations was
achieved in all microcosms. The deviation of model from the data occurred at late
time. This is probably due to the error is the cell decay rate (b) that were obtained
from fitting the data at early time. However, the reasonable fit between the model
and experimental data from all microcosms was still obtained. This supports the
model assumption that the effect of product transformation toxicity was negligible,
and the enzyme for 1,1,1 -TCA transformation remained active for long time
periods.
Simulations of 1,1,1 -TCA transformation were also conducted to determine
if inclusion of transformation capacity value (Ta) improved results. Instead of
equation 3.3, equation 3.4 was used to include the effect of a transformation
product toxicity in terms of a transformation capacity (Ta). The other values of
parameter values in equation 3.4 were maintained constant from the previous
model simulation. Values of T were varied to see if better fits were obtained.
101
Xo = 0.47 mg/L
4
H
0,
000
r
E
X=14.93mg/L
a)
0
0
5
10
15
20
15
20
Time, day
0.1
X= 28.14 mg/L
BR3-1
0)
0.08
Model
E
U)
0.06
H
C-)
I-
0.02
X= 30,51 mg/L
0
0
5
10
Time, day
Figure 4.47 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR3-1 without the term for transformation capacity
102
5
4
E
In
U)
(0
2
0
c
2
i
5
10
15
20
Time, day
0.12
BR3-2
0)0.1
X= 31.31 mgIL
Model
E
0.08
CU
2
<
0.06
()
I-
X= 33.55 mg/L
0.02
0
I
I
0
5
10
15
20
Time, day
Figure 4.48 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR3-2 without the term for transformation capacity
103
4
0)
E
a)
2
5
10
20
15
Time, day
0.1
X33.l9mg/L
.
0.08
Model
U,
U,
cu
BR3-3
0.06
0.04
.
X= 35.56 mg/L
0.02
0
5
10
15
20
Time, day
Figure 4.49 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR3-3 without the term for transformation capacity
104
5
4
C,
1
I
5
10
15
20
Time, day
0.1
BR3-4
X= 34.69 mg/L
Model
0.08
E
U)
C')
0.04
'-. 0.02
X= 35.56 mg/L
I
0
5
10
15
20
Time, day
Figure 4.50 Simulations of butane utilization and !,1,1-TCA transformation
for microcosm BR3-4 without the term for transformation capacity
105
5
4
0)
E
C)
0
5
10
15
20
Time, day
0.1
,0.08
0.06
0.04
I-
0.02
[II
0
5
10
15
20
Time, day
Figure 4.51 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR3-5 without the term for transformation capacity
106
0.1
Tc
0.08
0.01 mg/mg
0.06
.
BR3-5
0.01
0.04
No Tc term
TC = 0.1
\\
0.02
NoTc
S...
I
14.5
15.5
I
16.5
17.5
18.5
Time, day
Section
Biomass (X) at starting of model simulation
(m IL)
E
F
T=0.01
T=0.1
T=O.2
NoTterm
36.63
24.02
36.63
33.12
36.63
36.63
34.19
33.65
Figure 4.52 Simulations of 1,1,1-TCA transformation for microcosm BR3-5
over a range of transformation capacities
Figure 4.52 presents the results of model simulation over a range of
transformation capacities along with the biomass (X) at starting of model
simulation for microcosm BR3-5. The low T value of 0.01 mg 1,1,1- TCAImg cell
caused a significant decrease in biomass. Similar biomass values were obtained
from simulations with T
0.1 and those without a T term (T
= 00).
Little
improvement was obtained by including the transformation capacity term. The
results from the data at low concentration were not sensitive to the transformation
capacity term. Thus fits to transformation capacity model were obtained from the
data at high concentration. The maximum concentration of tested 1,1,1 -TCA was
approximately 700 igIL. Even at these high concentrations there was little
sensitivity to transformation capacity. Little sensitivity to transformation capacity
107
was also due to the amount of biomass available, as illustrated in following
calculations:
Initial biomass
= X0KV1
(36 mgIL)(0.1 L)
= 3.6 mg cells
Mass of transformed 1,1,1-TCA
= C1'Vi
= (0.7 mg/L)(0.1 L)
0.O7mgTCA
Destroyed cells
Mass of transformed 1,1,1 -TCA / T
= (0.07 mg TCA)/(0. 1 mg TCA!mg cells)
0.7 mg cells << 3.9 mg cells
Compared to the initial biomass, the amount of destroyed cells was small.
As a result, the simulation was not very sensitivity to the transformation capacity.
The results from the other microcosms were similar, as shown in appendix Figures
G.23 to G.26. A range of transformation capacity
(Ta) values obtained from
microcosm BR3 set are presented in Table 4.18 since there is little sensitivity to the
transformation capacity. The average value of transformation capacity was 0.11 ±
0.064 mg 1,1,1-TCAJmg cell. All parameters used in the model simulation for
microcosm set BR3 are presented in Table 4.19.
108
Table 4.18 Transformation capacity ranges (Tv) achieved in the BR3
microcosm set.
Microcosm
Maximum CI,TCA tested
(tg1L)
680
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
731
690
720
T
(mg TCA/mg cells)
0.1 0.2
0.05 0.07
0.04 - 0.06
0.1 0.2
0.1 -0.2
711
Average
0.11 ± 0.064
Table 4.19 Parameters for model development in the BR3 microcosm set
Parameter
Microcosm set BR3
Xo, mg/L
Y,mg/mg
b, day'
0.47
0.7
0.15 ± 0.006
KS,BUt,
mg/L
kmax,But, mg/mg-day
0.11
1.76 ± 0.02 3
KS,TCA, mg/L
0.37
0.09 ± 0.027
0.11 ± 0.064
kmax,TCA, mg/mg-day
T,mg/mg
4.3.3.2 The simulation of butane utilization and 1.1,1 -TCA
transformation in the BR4 microcosm set
The experimental data of butane utilization and 1,1,1 -TCA transformation
in microcosm set BR4 were modeled by using the same butane and 1,1,1 -TCA halfsaturation constant
(KS,BUt
and KS,TCA), cellular yield (Y), and cell decay rate (b)
values as were used in simulations of microcosm set BR3 (Table 4.19). Similar to
microcosm set BR3, the values of butane and 1,l,1-TCA maximum specific rates
(kmax,But
and kmax,TCA) were varied to obtain a good fit between the model and
experimental data. The model assumed that the effect of transformation product
toxicity was negligible. The simulation of butane utilization and 1,1,1 -TCA
transformation for microcosms BR4-M and BR4-4 are presented in Figure 4.53,
and Figure 4.54, respectively. Microcosm BR4-M contained growth media while
microcosm BR4-4 was constructed with groundwater and aquifer material.
Reasonable fits between the model and experimental data were obtained for both
sets of microcosm results. The model fit data well at low concentration as well as
high concentration. The initial concentration of tested butane and l,l,1-TCA
ranged from 230 to 2,600 ig/L and 60 to 1,500 tgIL, respectively. The model
simulations for the other microcosms are shown in appendix Figures G.27 to G.30.
A good fit was achieved in all microcosms. However, in some cases the simulation
deviated from the data. For example on day 12, as shown in appendix Figure G.27,
the model seems to overestimate the rate of butane utilization. The slower rate of
butane utilization may be due to the effect of transformation product toxicity,
resulting from the effect of the high concentration of TCA transformed at 7 days.
Model simulations including a transformation capacity
(Ta) term were
evaluated using the same procedure as the microcosm BR3set. Figure 4.55
presents the results of model simulation for microcosm BR4-5 over a range of
transformation capacities. The simulations for the other microcosms are presented
in appendix Figures G.3 1 to G.35. Although the effect of the transformation
product toxicity was included in the simulation, the simulations did not fit the data
better. Figures 4.55a and 4.55b present the transformation of the first and second
additions of 1,1,1 -TCA. A good fit was obtained from the data at low
concentration. However, the transformation at high concentration was a little faster
than the model prediction. The reason for this is not known. The model
overestimated the utilization rate of the fourth addition of butane, as shown in
Figure 4.55c. The model with a lower transformation capacity fit the data better,
but it underestimated the butane utilization and 1,1,1 -TCA transformation in later
time. As shown in Figures 4.55e and 4.55f, the third and fourth additions of 1,1,1TCA were transformed faster than the model prediction. The reason for the faster
transformation at both low and high concentration is not known. The effects of
110
4
a)
3
E
U)
U)
I
5
20
15
Tim
day
0.4
X= 42.43 mg/L
BR4..M
0)
Model
E Q3
U)
0)
X 33.25 mg/L
I-
'r
0.1
X43.92mg/L
00
X= 34.42 mgIL
I5
Tim
day
15
20
Figure 4.53 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-M without the term for transformation capacity
111
4
Xo = 0.45 mg/L
a,
E3
X14.O9mg/L
'I)
U)
Cu
o
BR4-4
Model
C
CU
4-.
X = 38 mg/L
X = 2914 mg/L
0+
0
5
10
20
15
Time, day
0.4
X
38.54 mglL
8R4-4
0)
E
Model
0.3
(0
U)
X
.r:
29.88 mg/L
0.1
X= 39.83 mg/L
X
30.79 mg/L
0+
0
5
10
15
20
Time, day
Figure 4.54 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-4 without the term for transformation capacity
112
a) The first addition of 1,1,1 -TCA on day 4
0.02
'a)
'I)
0
0.0c
0
I
4.7
4.75
4.8
4.85
4.9
5
4.95
Time, day
Biomass (X) at starting of model simulation
(mg/L)
= 0.1
39.45
T = 0.2
T = 0.3
No T term
39.45
39.45
39.45
b) The second addition of 1,1,1 -TCA on day 4
0.4
I
0)
E 0.3
.
(a,
BR4-5
-Tc = 0.1
Tc = 0.2
'
0.1
= 0.3
No Ic
4.9
4.95
5
5.05
5.1
5.15
5.2
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=O.2
T0.3
NoTterm
36.55
37.36
37.63
38.17
Figure 4.55 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-5 over a range transformation capacities capacity
113
c) The fourth addition of butane on day 11
o
2.5
r
Tc
BR4-5
0.1 mg/mg -Tc = 0.1
I::
11.2
11.4
11.6
12
11.8
12.2
12.4
12.6
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=O.2
T=0.3
NoTterm
10.40
12.28
12.97
14.48
d) The fifth addition of butane on day 12
0.8
8R4-5
o
-Tc=0.1
0.6
E
0.4
0.2
12.4
12.5
12.6
12.8
12.7
12.9
13
Time, day
Biomass (X) at starting of model simulation
(mg/L)
26.71
Figure 4.55, Continued
T=O.2
T=0.3
NoTterm
28.26
28.80
29.99
114
e) The third addition of 1,1,1-TCA on day 12
0.02
0. 016
E
0.012
0.008
0.004
0
12.95
13
13.05
i..i
Time, day
Biomass (X) at starting of model simulation
(mgIL)
0.1
28.80
T = 0.2
T = 0.3
No T term
30.24
30.78
31.86
f) The fourth addition of 1,1,1-TCA on day 13
0.2
0.15
(a,
<
0.1
C.)
I-
'
0.05
.
U
(
13
13.2
13.4
13.6
13.8
14
14.2
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=Q.1
T=O.2
T=O.3
NoTterm
26.72
28.72
29.45
30.92
Figure 4.55, Continued
115
enzyme induction or possible changes in microbial community that were not
included in the model simulations are possible factors resulting in the higher
transformation rates.
The results from the first and second additions of 1,1,1 -TCA were not
sensitive to the transformation capacity term, as shown in Figures 4.55a and 4.5 Sb.
As a result, the tests should have been performed for longer periods of time. The
simulation of 1,1,1 -TCA transformation in later time still had low sensitivity at low
concentration, but became more sensitivity at higher concentration, as shown in
Figures 4.55e and 4.55f. This is probably due to the high transformation capacity
of 1,1,1-TCA.
As shown in Figure 4.55c, the simulation of the fourth addition of butane
showed more sensitivity to transformation capacity values than that of 1,1,1 -TCA
transformation. A different result was observed in the simulation of the fifth
addition of butane, as shown in Figure 4.55d. No 1,1,1 -TCA was transformed
between these two additions of butane. The biomass in the microcosm was low
before the fourth addition of butane. Thus the high biomass at the fifth addition of
butane probably made the simulation less sensitive to the transformation capacity
values.
Table 4.20 Butane and 1,1,1-TCA maximum specific rate (kmax,But and
kmax,TCA) and transformation capacity (Tv) values achieved in the BR4
microcosm set
Microcosm
kmax,But
kmax,TCA
T
(mg/mg-day)
(mg TCA!mg cells)
BR4-M
(mg/mg-day)
2.2
0.11
0.3
BR4-1
BR4-2
BR4-3
BR4-4
BR4-S
Average
1.58
1.74
1.74
1.95
1.93
1.79 ± 0.154
0.1
0.3
0.3
0.3
0.3
0.3
0.3 ± 0
0.19
0.14
019
0.16
0.16 ± 0.038
116
The estimated values of butane and 1,1,1 -TCA maximum specific rates
(kmax,But and kmaXTCA) and transformation capacity (Ta) for microcosm BR4-M and
the BR4 microcosm set are shown in Table 4.20. All microcosms, either
constructed with groundwater and aquifer material or contained growth media, had
similar parameter values. This indicates the reproducibility of the bioaugmentation
process, and indicates a similar butane-utilizing microbial community was present
in both media and groundwater microcosms. Estimates of transformation capacity
were obtained through the simulation of 1,1,1 -TCA transformation at the fourth
addition, which showed the most sensitivity to the transformation capacity term.
All microcosms had a very high value of transformation capacity (0.3 mg/mg). The
results from the model simulation at the tested concentration showed little
difference between having no transformation term (T
= co)
and a T value of 0.3
mg/mg. Table 4.21 presents all parameters used in the model simulation for
microcosm BR4-M and the BR4 microcosm set. The use of the same parameters
with only small differences in kmax values also demonstrate the reproducibility of
the bioaugmentation process.
Table 4.21 Parameters for model development in microcosm BR4-M and the
BR4 microcosm set
Parameter
Xo, mg/L
Y,mg/mg
b,day'
B,mg/L
kmax,5ut, mg/mg-day
KSTCA,
mg/L
kmaxTcA, mg/mg-day
T, mg/mg
Microcosm BR4-M
Microcosm set BR4
0.45
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.45
0.7
0.15
0.11
1.79 ± 0.154
0.37
0.16 ± 0.378
0.3 ± 0
117
4.3.3.3 The simulation of butane utilization and 1,1,1 -TCA
transformation in the B4 microcosm set
The culture bioaugmented in the BR3 and BR4 microcosm sets was not
washed with groundwater to rinse away the mineral salts media prior to
inoculation. The nutrients were suspected to have an effect on the growth of the
culture and the values of the estimated kinetic parameters. For comparison
purposes, model simulations of butane utilization and 1,1,1 -TCA transformation
achieved in the B4 microcosm set, where cells were washed to remove the mineral
salts media was conducted. Estimates of butane and 1,1,1-TCA maximum specific
rates (km
BUt
and kmax,TCA) and the transformation capacity
(Ta) were obtained
through model simulations and compared with BR3 and BR4 microcosm results.
The values of cellular yield (0.7 mg/mg), cell decay rate (0.15 day'), butane halfsaturation constant (0.11 mg/L) and 1,1,1 -TCA half-saturation constant (0.37
mg/L) were the same as used in simulating the BR3 and BR4 microcosm sets.
Figure 4.56 presents the experimental data of butane utilization and 1,1,1-
TCA transformation for microcosm B4-3. The simulation of butane utilization was
performed by modeling the butane observations during the first 33 days of
incubation. The inhibition effect of 1,1,1 -TCA on butane utilization was ignored
since the concentration of 1,1,1 -TCA was much lower than the concentration of
butane. The augmented biomass of the B4 microcosm set was unknown, so initial
biomass of 0.5 mg/L was assumed in the simulation. The value Of
km,But was
varied until a good model fit was obtained. However, it was difficult to obtain a
good fit to all of the experimental data. As a result, the model simulation was
conducted by considering the data after day 20 since more data were collected
during this time, and initial conditions at early time were uncertain.
118
4
-o-- B4-3
E
U,
U,
CU
a,
I,
0
10
20
30
40
50
Time, day
0.3
-.-- B4-3
To avoid inhibition
effect, start modeling
at this data point.
0,
0.2
U,
U,
CU
0.1
0
0
10
20
30
40
50
Time, day
Figure 4.56 Butane utilization and 1,1,1-TCA transformation achieved in
microcosm B4-3
119
5
X= 15.73 mg/L
X= 15.73 mgJL
4
X=10.22mg/L
1
E
cic3
(I)
C
ml
o
0
0
Model
0
I
o
10
B4-3
20
30
40
50
Time, day
Figure 4.57 Simulations of butane utilization for microcosm B4-3 without the
term for transformation capacity
The model simulation of butane utilization for microcosm B4-3 is presented
in Figure 4.57. A good fit between the model and data was obtained after day 20.
The model did not fit the data well during the first 10 days of incubation. The rate
of butane utilization was slower than the model prediction. This implies that the
values of kmaxBut between two periods of time are different. Additionally, the
slower utilization rate of the second addition of butane was probably due to the
inhibition of 1,1,1-TCA, which was not considered in the model. The results from
the simulations of butane utilization for the other microcosms B4-1 and B4-2 were
similar, as shown in appendix Figures G.36 and G.39, respectively. Table 4.22
presents the values of butane maximum specific rate (kmax,But) for the B4
microcosm set. All microcosms had the same values (2.2 mg/mg-day). The kiax,But
values of all microcosms were the same, and also similar to those of microcosm
sets BR3 and BR4 (Tables 4.19 and 4.21). The results indicate a similar butane
utilization rate with and without minor nutrient amendments over a 1-month test
period.
120
Table 4.22 Butane maximum specific rate
microcosm set
(kmax,But)
values achieved in the B4
Microcosm
kmax,But
B4-1
B4-2
B4-3
(mg/mg-day)
2.2
2.2
2.2
2.2 ± 0
Average
Simulations of 1,1,1 -TCA transformation in the B4 microcosm set were
also conducted to evaluate the 1,1,1 -TCA maximum specific rate (kmax,TCA). Data
obtained in the absence of butane utilization were selected for simulation
comparisons, in order to avoid the effects of inhibition of butane on 1,1,1 -TCA
transformation. l,l,1-TCA data that was simulated is shown in Figure 4.56. For
initial simulations, the transformation product toxicity was assumed to be
negligible. Similar to the simulations for the BR3 and BR4 microcosm sets, a
heuristic fitting of kmax,TCA values was performed. Figure 4.58 presents the
simulation of 1,1,1 -TCA transformation for microcosm B4-3. Although the model
fit the data at early time well, it overestimated the 1,1,1 -TCA transformation at later
time. The slower transformation may have resulted from transformation product
toxicity or energy limitations or higher cell decay rates. The results from the
simulations for the other microcosms were similar, as shown in appendix Figures
G.37 and G.40, respectively
Additional simulations were conducted to incorporate the transformation
capacity (Ta) term. Figure 4.59 presents the simulation of butane utilization and
1,1,1 -TCA transformation for microcosm BR3 -5 over a range of transformation
capacities. The inclusion of transformation capacity term did not improve the
model fit of 1,1,1 -TCA transformation and butane utilization at early time, as
shown in Figures 4.59a and 4.59b. The model with high transformation capacity
(T = 0.4 mg 1,1,1-TCA/mg cells) fit the l,l,l-TCA transformation data in early
121
time. The deviation between model and data increased with time. The model
underpredicted 1,1,1-TCA transformation rates. The model with no transformation
capacity term (T
cc)
better simulated butane utilization also after low
concentration level 1,1,1 -TCA additions. However, a lower transformation
capacity fit the 1,1,1 -TCA transformation data at later time, as shown in Figure
4.59c, but it under predicted both butane utilization and 1,1,1-TCA transformation
in early time. The slower transformation of 1,1,l-TCA at later time could not be
completely explained by incorporating the transformation capacity term. The slow
deactivation of enzyme might be another factor, resulting in a decrease of kmax,TCA
as a function of the 1,1,1 -TCA mass transformed. Other factors such as a change
of microbial community, or nutrient limitations cannot be ruled out.
0.3
0)
E
0.2
C-)
24
29
34
39
44
49
Time, day
Figure 4.58 Simulations of 1,1,1-TCA transformation for microcosm B4-3
without the term for transformation capacity
122
a) 1,1,1-TCA transformation from day 25 to 32
0.2
B4-3
0)
E
Tc = 0.1 mg/mg
0.15
Tc
to
0.2 mg/mg
Tc = 0.4 mg/mg
0.1
No Tc term
0
I'-
0.05
24
26
28
30
32
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=0.4
NoTterm
24.44
24.44
24.44
24.44
b) Butane utilization from day 31 to 33
4
0)
E
U)
U)
C)
C
'"-
0 f__J_
30
32
v
34
36
38
40
42
44
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.1
T=O.2
T=0.4
NoTterm
0.09
2.37
6.26
10.26
Figure 4.59 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm B4-3 over a range transformation capacities
123
c) 1,1,1-TCA transformation from day 33 to 43
0.4
B4-3
E
0.3
Ic = 0.1 mg/mg
Ic = 0.2 mg/mg
0.2
- No Tc term
U)
Ic = 0.4 mg/mg
C)
I-
-
.
0.1
04r.
32
34
36
40
38
42
44
Time, day
Biomass (X) at starting of model simulation
T = 0.1
T = 0.2
T = 0.4
No T term
0.41
15.24
34.19
38.33
Figure 4.59, Continued
The results from the simulations for microcosms B4-1 and B4-2 are
presented in appendix Figures G.38 and G.41, respectively. The model with no or
high transformation capacity term (T
= cc
or T
0.4 mg/mg) better fit the data at
early time, while the model with lower transformation capacity better fit the data at
later time. For microcosms B4-2 and B4-3, it is difficult to point out the value of
transformation capacity since the model with only one value of transformation
capacity could not provide a good fit for all microcosm data. The values of 1,1,1
TCA maximum specific rate (kmTCA) and transformation capacity (Ta) for the B4
microcosm set are presented in Table 4.23. Values for all microcosms were similar
and also in the range obtained from the BR3 and BR4 microcosm sets (Tables 4.19
and 4.21).
124
Table 4.23 1,1,1-TCA maximum specific rate (kmax,TCA) and transformation
capacity ('[c) values achieved in the B4 microcosm set
Microcosm
B4-1
B4-2
B4-3
0.15
0.10
0.15
T
(mg TCA 1mg cells)
0.4
0.2 - 0.4
0.2 0.4
Average
0.13±0.024
0.33 ±0.103
kmax,TCA
(mg/mg-day)
The results obtained from the model simulations and the rate measurements
were compared to evaluate if the simplified analysis in the rate measurement could
provide reliable values of kinetic parameters. Table 4.24 presents the values of
pseudo-first-order rate parameter (kmaxXolKs) and cell decay rate (b) for microcosm
set B4 based on the rate measurements during day 30
35 and the model
simulations. The values of cell decay rate obtained from the rate measurement of
1,1,1 -TCA transformation (0.21 day1) were higher than those from the simulation
of butane utilization (0.15 day'). The higher b value probably represents both cell
and enzyme decay rate. The values of pseudo-first-order rate parameter
(kmaxXolKs) obtained from the rate measurements and the model simulations ranged
from 3.4 to 6.6 day1, and 6.6 to 10.6 day', respectively. The higher values of the
parameters obtained from the model simulation were probably due to the higher
value of initial biomass
(X0)
since the value of cell decay rate (b) from the model
simulation was lower than that obtained from the rate regression measurement.
The rate regression method provided fairly reasonable values for the pseudo-firstorder rate parameter (kmaxXIKs), within factor of 2. A good agreement between
the values of pseudo-first-order rate parameter (kmaxXo/Ks) and cell decay rate (b)
was also obtained. This indicates the practicality of the simplified analysis in the
rate regression measurement.
125
Table 4.24 The pseudo-first-order rate parameter (kmaxXofKs) and cell decay
rate (b) for the B4 microcosm set from the rate measurement during day 28
37 and from the model simulation
Microcosm
Rate measurement
kmaxXo/Ks
(day')
(day')
6.6
3.2
5.0
0.28
0.20
0.15
0.21 ± 0.064
B4-1
B4-2
B4-3
Average
Model simulation
b
5.0± 1.62
kmaxXo/Ks
(day1)
b
(day')
9.7
6.3
9.9
8.6 ±2.01
0.15
0.15
0.15
0.15
All parameters used in the model simulation for the B4 microcosm set are
presented in Table 4.25. In the B4 microcosm set the cells were washed to remove
the mineral salts media. Similar kinetic parameters including butane maximum
specific rate (km
But),
1,1,1 -TCA maximum specific rate (kmax,TCA) and
transformation capacity (Ta) were obtained as in the BR3 and BR4 microcosm sets.
This indicates that these parameters, including the transformation ability of
bioaugmented microcosms, are not influenced by the addition of nutrients.
Table 4.25 Parameters for model development in the B4 microcosm set
Parameter
mg/L
Y,mg!mg
X0,
b,day'
KS,BUt, mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
Microcosm set B4
0.5 (assumed number)
0.7
0.15
0.11
2.2 ± 0
kmax,TCA, mg/mg-day
0.37
0.13 ± 0.024
T,mg/mg
0.33±0.103
126
4.3.3.4 The simulation of butane utilization and 1,1J-TCA
transformation in the BRC microcosm set
Butane utilization and 1,1,1 -TCA transformation data for indigenous
microorganisms in the BRC microcosm set (Table 4.9) were also modeled to
determine kinetic parameters. All kinetic parameters for butane utilization and
1,1,1-TCA transformation in the indigenous microcosms were unknown. The value
of butane and 1,1,1 -TCA half-saturation constant (Ks,But and KS,TCA) could not be
determined from the kinetic experiments since butane utilization and 1,1,1 -TCA
transformation were slow, and 1,1,1 -TCA transformation stopped after the
microcosms were exposed to the second addition of 1,1,1-TCA (800 p.g/L). The
kinetic parameters of bioaugmented butane-utilizers in the BR3 microcosm set
including butane and 1,1,1 -TCA half-saturation constant (Ks,But and KS,TCA), butane
and 1,1,1 -TCA maximum specific rate
(kmax,But
and kmax,TCA), cellular yield (Y), and
cell decay rate (b) were therefore used to simulate butane utilization and 1,1,1 -TCA
transformation in the BRC microcosm set (Table 4.19).
Initially the simulation of butane utilization and 1,1,1 -TCA transformation
occurring in the indigenous microcosms was conducted by not considering the
effect of transformation product toxicity. Figure 4.60 presents the simulation of
butane utilization and 1,1,1 -TCA transformation for microcosm BRC- 1. The
microcosm data were divided into 7 sections (A, B, C, D, E and F). Each section
represented the data from each addition of butane and 1,1,1 -TCA, and was modeled
separately, as shown in Figure 4.61. The initial biomass in the indigenous
microcosm was unknown. So the values of biomass were varied to achieve a
reasonable fit between the model and the microcosm data in section A. The
estimated initial biomass for all microcosms is presented in Table 4.26.
127
5
Xo 0004 mg/L
X=22.36mgIL
X0.O3mg/L
I
2
X42.05mg/L
J
a
BRC-1
X=37.95mgJL
1
20406080
00
100
Time, day
0.16
BRC-1
X=9.28mg/L
Model
E
0
0.08
'
004
0
0
20
60
40
80
100
Time, day
transformation
Figure 4.60 Simulations of butane utilization and 1,1,1-TCA
capacity
for microcosm BRC-1 without the term for transformation
128
Section A: The first addition of butane on day 0
5
U)
u,3
.13
ml
0
0
2
4
6
8
10
Time, day
Section B: The second addition of butane on day 9
4
o
BRC-1
Model
E
U)
U)
2
C
.131
0
9
9.2
9.4
9.6
9.8
10
10.2
10.4
Time, day
Section C: The third addition of butane on day 10
r
4
U. I
o
C'
0.08
U)
U)
BRC-1
Model
E
0.06
0)
C
'13
m
0.02
0
10
10.2
10.4
10.6
10.8
11
11.2
Time, day
Figure 4.61 Simulations of butane utilization and 1,1,1-TCA transformation in
each section for microcosm BRC-1 without the term for transformation
capacity
129
Section D: The fourth addition of butane on day 10
U.-,
a)
E
0.3
U,
U)
0.2
a)
0.1
0
10
10.5
11.5
11
12
13
12.5
13.5
Time, day
Section E: The fifth addition of butane on day 20
5
0)
4
15
7
19
TI3252729
Time, day
Section F: The first addition of 1,1,1-TCA on day 28
0.02
BRC-1
Model
0.015
U)
0.01
4
0.005
4
0125
27
29
31
Time, day
Figure 4.61, Continued
33
35
130
Section 0: The second addition of 1,1,1-TCA on day 32
0.15
0)
.
$
E
Model
0.1
...
..
U
'.;
BRC-1
0.05
0
15
35
55
75
95
115
Time, day
Figure 4.61, Continued
The model fit the data well during the first 10 days of incubation, as shown
in section A and B, but started deviating from the data on day 10, as shown in
section C and D (Figure 4.61). This indicates that the kinetic parameters of
bioaugmented microcosm do not fit the non-augmented data well. As shown in
section D (Figure 4.61), the initial rates of butane utilization at moderate
concentrations of butane were slower than the model prediction. One possibility is
that the indigenous microorganisms have a lower butane maximum specific rate
(kmax,But) than the bioaugmented microorganisms. The rate of butane utilization
followed first-order kinetics when the concentration was below 100 tg/L, as shown
in section D (Figure 4.61). Thus the butane half-saturation constant
(Ks,But)
of the
indigenous microorganisms should be higher than that of bioaugmented
microorganisms. The deviation of model from the microcosm data increased with
time, especially after day 20 or section E (data not shown). Thus the biomass was
adjusted in section E to make the model fit the data. Table 4.27 presents the initial
biomass in section E that was obtained from model simulations and adjusted
biomass. The estimated biomass resulting from model simulations was much
higher than that obtained from the adjusted values. Based on the cell decay rate
131
equation (equation D.7), the indigenous microorganisms had four to ten times the
cell decay rate (b) of the bioaugmented microorganisms. As shown in section E
(Figure 4.61), the initial rate of butane utilization at moderate concentrations of
butane, after the lag phase, was slower than the model prediction. This
corroborates the results from section D indicating that the indigenous
microorganisms have a lower butane maximum specific rate (k,Bt) than the
bioaugmented microorganisms. However, the slower rate of butane utilization
might be due to nutrient limitations that were not included in the model
simulations. The results from the simulation of other indigenous microcosms were
similar as shown in appendix Figures G.42 to G.45.
Table 4.26 Initial biomass (X0) for the BRC microcosm set.
Microcosm
X0
(mg/L)
0.004
0.006
BRC-1
BRC-2
BRC-3
BRC-4
BRC-5
Average
0.001
0.004
0.005
0.004 ± 0.002
Table 4.27 Initial biomass (X0) in section E for the BRC microcosm set
obtained from the adjustment and the model simulation.
xo
Microcosm
BRC-1
BRC-2
BRC-3
BRC-4
BRC-5
Starting day
28
26
25
32
25
(mgIL)
Adjusted
Modeled
0.03
0.2
10.27
10.79
1
14
0.001
0.7
10.92
10.82
132
The simulations of 1,1,1 -TCA transfonnation were performed by using the
same kinetic parameters as the bioaugmented microorganisms in the BR3
microcosm set. The initial biomass (X = 19.10 mg/L) was obtained from the
previous simulations using the revised biomass. The model overestimated the
transformation rate of 1,1,1 -TCA, as shown in section F and G (Figure 4.61).
Similar to butane utilization, the initial rate of 1,1,1 -TCA transformation at high
concentration was slower than the model prediction, as shown in section G. This
shows that the 1,1,1 -TCA maximum specific rate
(kmax,TCA)
of the bio augmented
microorganisms is higher than that of the indigenous microorganisms. The
fluctuation of data points and also the slow transformation rate in section G
provided a difficulty in estimating the value of 1,1,1 -TCA half-saturation constant
(KS,TCA).
The slow disappearance of 1,1,1-TCA after day 50 was possibly due to
microcosm leakage rather than the microbial transformation. As shown in
appendix Figure G.45, there was no 1,1,1-TCA transformation in microcosm BRC5, but slow disappearance of 1,1,1-TCA was observed after day 50. Thus the
results from the simulations were performed using the microcosm data before day
50.
Another simulation of 1,1,1 -TCA transformation was performed to evaluate
the effect of transformation product toxicity. Figure 4.62 presents the simulation of
1,1,1 -TCA transformation for microcosm BRC- 1 over a range of transformation
capacities. The values of all kinetic parameters except the transformation capacity
(Ta) were maintained constant (Table 4.19). The initial biomass (X = 19.10 mgIL)
was also the same as the previous simulation of 1,1,1 -TCA transformation which
used the revised biomass. Unlike the bioaugmented microorganisms, the
simulations of 1,1,1 -TCA transformation at both high and low concentrations for
indigenous microorganisms were sensitive to the transformation capacity. As
shown in section F, the model with a low transformation capacity (T = 0.01 mg
1,1,1 -TCA/mg cells) fit the microcosm data at low concentration well although it
slightly under predicted the initial rate of 1,1,1 -TCA transformation. The results
from section G show that the inclusion of transformation capacity did not help to
133
Section F: The first addition of 1,1,1-TCA on day 28
0.02 -r----------BRC-1
0.01
0.015
- Tc = 0.1
Tc = 0.2
0.01
- No Tc
r
0.005
NoTcTerm ..J
nI
u-r---------
.-..-----
26
28
30
32
34
Time, day
Biomass (X) at starting of model simulation
(mg/L)
= 0.01
19.10
T = 0.1
T = 0.2
No T term
19.10
19.10
19.10
Section G: The second addition of 1,1,1-TCA on day 32
0.16
0,
E
0.12
0.08
0.04
0
I
0
20
40
60
80
100
Time, day
T = 0.01
0.24
Biomass (X) at starting of model simulation
(mgIL)
T = 0.1
T 0.2
8.23
8.76
No T term
9.28
Figure 4.62 Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of transformation capacities
134
achieve a better fit to the microcosm data. The slow transformation occurring over
a long period of time could not be explained. The results from the simulations for
the other microcosms are similar as shown in appendix Figures G.46 to G.48. The
simulation for microcosm BRC-5 was not performed since no transformation of
1,1,1-TCA occurred. Table 4.28 presents the values of transformation capacity (Ta)
for the indigenous microorganisms. The values were obtained from the simulation
at low concentration (section F). It is likely that the indigenous microorganisms
had a lower transformation capacity than the bioaugmented microorganisms.
Table 4.28 Transformation capacity (Ta) values achieved in the BRC
microcosm set
Microcosm
BRC-1
BRC-2
BRC-3
BRC-4
Average
T
(mg 1,1,1-TCA/mg cells)
0.01
0.01
0.02
0.01
0.01 ± 0.005
Although the model with low transformation capacity fit the microcosm
data at low concentrations well, it could not fit the microcosm data at the higher
concentrations at later time. One of hypotheses is that the indigenous
microorganisms had a different decay rate from the bioaugmented microorganisms.
Figure 4.63 presents the simulation of 1,1,1 -TCA for microcosm BRC- 1 over a
range of decay rates (b). In this simulation, the indigenous microorganisms were
assumed to have high transformation capacity, as presented in Table 4.29. These
values were estimated from the previous simulation of 1,1,1 -TCA transformation at
high concentrations in section G, Figure 4.62. The values of initial biomass (X =
19.10 mg/L) and the other kinetic parameters including the maximum specific rate
of butane utilization
(km,But)
and 1,1,1 -TCA transformation (kmax,TCA), cell decay
rate (b), butane and 1,1,1 -TCA half-saturation constants (Ks,Bt and KS,TCA), and
135
cellular yield (Y) were maintained constant from the previous model simulation
(Table 4.19). The simulation was sensitive to decay rates at both low and high
concentrations. As shown in Figure 4.63, the high decay rate model (b = 1.3
day1)
better fit the microcosm data at low concentrations (section F), but it did not fit the
microcosm data at high concentrations (section G). The reason for the slow 1,1,1
TCA transformation in section G was not known. The variation of decay rates did
not achieve a good model fit to the microcosm data. This indicates that other
kinetic parameters for the indigenous microorganisms are most likely different
from those for the bioaugmented microorganisms. The results from the simulations
for other microcosms are similar, as shown in appendix Figures G.49 to G.5 1.
Table 4.30 presents the values of decay rate for the indigenous microorganisms.
These values were estimated from the simulation at low concentrations (section F).
The decay rates ranged from 1.2 to 1.5 day1. In this case, the high decay rate
represented the enzyme decay rate resulting from the 1,1,1 -TCA transformation
rather than the cell decay rate. Unlike the bioaugmented microcosms, long-term
1,1,1-TCA transformation did not occur in indigenous microcosms.
Table 4.29 Transformation capacity (Ta) values used in the simulation of 1,1,1TCA transformation for the BRC microcosm set
Microcosm
T
(mg 1,1,1 -TCA/mg cells)
BRC-1
BRC-2
BRC-3
BRC-4
0.1
0.4
0.3
0.6
136
Section F: The first addition of 1,1,1-TCA on day 28
0.016
.
BRC-1
b=0.15
b=0.2
E0.012
(0
b = 1.3
----------- b = 0.3
0.008
b= 1.3
- 0.004
b=0.15
25
27
29
31
33
35
Time, day
Biomass (X) at starting of model simulation
(mg/L)
b=O.15
b=0.2
b=0.3
b=1.3
19.10
19.10
19.10
19.10
Section G: The second addition of 1,1,1 -TCA on day 32
D'
0.16
_____b=0.15
0.12
2::
0.08
b= 1.3
'- 0.04
0-i--0
I
20
40
60
80
100
Time, day
Biomass (X) at starting of model simulation
(mg/L)
b=0.15
b=0.2
b=0.3
b=1.3
8.23
6.45
3.95
0.026
Figure 4.63 Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of decay rates.
137
Table 4.30 Decay rate (b) values achieved in the BRC microcosm set
Microcosm
b
(day1)
BRC-1
BRC-2
BRC-3
BRC-4
Average
1.3
1.3
1.2
1.5
1.33 ± 0.13
The simulation of 1,1,1-TCA for microcosm BRC-1 over a range of 1,1,1TCA maximum specific rates (kmax,TCA) is presented in Figure 4.64. Similar to the
previous simulation, the indigenous microorganisms were assumed to have high
transformation capacity, as presented in Table 4.29. The values of initial biomass
(X = 19.10 mg/L) and the other kinetic parameters including the maximum specific
rate of butane utilization (kmax,But), cell decay rate (b), butane and 1,1,1 -TCA halfsaturation constants (K,But and KS,TCA), and cellular yield (Y) were also maintained
constant from the previous model simulation (Table 4.19). The simulation was
sensitive to the kmax,TCA values at both low and high concentrations. However,
changing only the kmaX,TCA value did not achieve a good model fit to the microcosm
data at both low and high concentrations. The value of 1,1,1-TCA half-saturation
constant (KSTCA) is possibly another factor controlling the shape of the model fit at
the low concentrations (section F). However, the microcosm data at low
concentrations (section F) presented between the models having kmax,TCA values
ranging from 0.02 to 0.12 mg/mg-day. The slow transformation of 1,1,1 -TCA at
high concentrations in section G was not greatly explained by the kmax,TCA. The
simulations for other microcosms are similar, as presented in appendix Figures
G.52 to G.54. Table 4.31 presents a range of 1,1,1-TCA maximum specific rates
(kmaxTCA) estimated from the simulation at low concentrations (section F). It is
impossible to indicate the exact kmax,TCA values for the indigenous microorganisms
since a good model fit was not obtained. The indigenous microorganisms were
likely to have lower krnax,TCA values than the bioaugmented microorganisms.
138
Section F: The first addition of 1,1,1-TCA on day 28
0.016
BRC-1
kmax = 0.02
0)
-----kmax = 0.02
E 0.012
\ \\\
U)
U)
0.008
kmax = 0.05
kmax = 0.09
4\
\\\
kmax = 0.12
'"N,
'-: 0.004
kmax= 0.12
I
25
J \ N.
27
29
31
35
33
Time, day
Biomass (X) at starting of model simulation
(mg/L)
kmax.TCA = 0.02
kmax,TCA = 0.05
kmaxTCA = 0.09
kmax,TCA = 0.12
19.10
19.10
19.10
19.10
Section G: The second addition of 1,1,1-TCA on day 32
0.2
BRC-1
0)
= 0.02
0.16
kmax = 0.05
0.12
kmax = 0.09
kmax= 0.12
0.08
..
0.04
0+
I
I
0
20
40
60
80
100
Time, day
Biomass (X) at starting of model simulation
(mg/L)
kmax,TCA = 0.02
kmax,TCA = 0.05
kmax,TCA = 0.09
kmax,TCA = 0.12
8.24
8.17
8.23
8.25
Figure 4.64 Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of 1,1,1-TCA maximum specific rates
139
Table 4.31 1,1,1-TCA maximum specific rates (kmax,TCA) values achieved in the
BRC microcosm set.
Microcosm
kmax,TCA
(mg/mg-day)
0.02-0.12
BRC-1
BRC-2
BRC-3
BRC-4
Average
0.02
0.02
0.02
0.06 ±
0.09
0.05
0.12
0.046
Figure 4.65 presents the simulation of 1,1,1-TCA for microcosm BRC-1
over a range of 1,1,1 -TCA half-saturation constants (KS,TCA). The values of initial
biomass and all kinetic parameters except KS,TCA were maintained constant from the
previous simulations. Good model fits were not achieved by changing only the
KS,TCA values. The simulation was sensitive to the
KS,TCA
values at low
concentrations (section F) rather than at high concentrations (section G) since the
simulation of initial transformation at high concentrations followed zero-order
kinetics. Furthermore, the high K values could not explain the slow transformation
of 1,1,1 -TCA in section G. The results from the simulations for other microcosms
are similar as shown in appendix Figures G.55 to G.57. The models with KS,TCA
higher than 0.37 mg/L were likely to provide better fit to the microcosm data at low
concentrations (section F). Estimates of KSTCA values for the indigenous
microcosms are presented in Table 4.32. The indigenous microorganisms seem to
have higher KS,TCA values than the bioaugmented microorganisms.
140
Section F: The first addition of 1,1,1-TCA on day 28
0.016
I
C)
Ks0.37
Ks=2
E 0.012
Ks=1
(0
0)
Ks = 1.5
0.008
0.004
.
Ks0.37
I
25
I
29
27
33
31
35
Time, day
Biomass (X) at starting of model simulation
(mgIL)
KS,TCA = 0.37
KS,TCA = 1
KS,TCA = 1.5
KS.TCA = 2
19.10
19.10
19.10
19.10
Section G: The second addition of 1,1,1-TCA on day 32
0.2
BRC-1
C)
0.16
Ks = 0.37
.
0.12
Ks1
*
Ks1.5
I..
-Ks=2
0.08
0.04
I
I
0
20
40
60
80
100
Time, day
Biomass (X) at starting of model simulation
(mg/L)
KS,TCA = 0.37
KSTCA = 1
8.23
8.16
KSTCA
8.20
1.5
KS,TCA =2
8.27
Figure 4.65 Simulations of 1,1,1-TCA transformation for microcosm BRC-1
over a range of 1,1,1-TCA half-saturation constants
141
Table 4.32 1,1,1-TCA half-saturation constant (KS,TCA) values achieved in the
BRC microcosm set.
Microcosm
BRC-1
I3RC-2
BRC-3
BRC-4
Average
KS,TCA
(mg/L)
0.37 1.5
0.37 1.5
0.37-2
0.37-2
1.06 ± 0.761
Table 4.33 presents the kinetic parameters used for the model development
for indigenous microcosms. The model simulations for the indigenous microcosms
were conducted by using all kinetic parameters for the BR3 bioaugmented
microcosms. The initial biomass in the indigenous microcosms was the average
value obtained from the variation in the simulations (Table 4.27). The kinetic
parameters in parentheses, including cell decay rate (b), 1,1,1 -TCA half-saturation
constant (KS,TCA) and 1,1,1 -TCA maximum specific rate (kmax,TCA), were estimated
from the variation of single kinetic parameter at a time in the simulations using
high transformtation capacity.
Table 4.33 Parameters for model development in the BRC microcosm set.
Parameter
X0,
mg/L
Y,mg/mg
b, day'
mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
KS,BUt,
kmaxTcA, mg/mg-day
T, mg /mg
Note
Microcosm set B4
0.004 ± o.002a
0.7
0.15 (1.33 ±
0.11
2.2
0.37 (1.06 ±
0.09 (0.06 ±
0.01 ± 0.005
a = Average value from Table 4.27
b = Obtained from a variation of single kinetic parameter at a time in model
simulation using high transformation capacity
Table 4.34 Parameters used in the simulation of butane utilization and 1,1,1-TCA transformation
Parameter
Xo, mg/L
Y, mg/mg
b,day'
K,nt,mgIL
kmax,But,
mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
Vg, L
HCC,BUt,
mg-L/mg-L
HCC,TCA, mg-L/mg-L
Augmented Microcosm
without cell washing
GW/Soil
GW/Soil
BR3 set
BR4 set
0.47
0.45
0.7
0.15 ± 0.006
0.11±0.014
1.76 ± 0.023
0.37 ± 0.05
0.09 ± 0.027
0.11 ± 0.064
0.1
0.057
38.05
0.55
Medium
BR4-M
0.45
0.7
0.15
0.11
1.79 ± 0.15
0.37
0.16 ± 0.038
0.3 ± 0
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.1
0.057
38.05
0.55
Augmented Microcosm
with cell washing
GW/Soil
B4 set
0.5a
Indigenous Microcosm
OW/Soil
BRC set
0.004 ± 0002b
0.7
0.15 (1.33 ± 0.13)c
0.11
1.76
0.37 (1.06 ± 0.761)c
0.09 (0.06 ± 0.046)c
0.1
0.7
0.15
0.11
2.2 ± 0
0.37
0.13 ± 0.0236
0.33 ± 0.103
0.075
0.057
38.05
0.55
0.082
38.05
0.55
0.057
38.05
0.55
0.01 ± 0.005
0.1
Note a = Assumed number
b = Obtained from the variation in model simulation (Table 4.27)
c = Estimated from a variation of single kinetic parameter at a time in model simulation using high transformation capacity
NJ
143
Table 4.34 presents the values of parameters for the simulation of butane
utilization and 1,1,1 -TCA transformation for the BR3, BR4 and B4 bioaugmented
microcosm sets, the BR4-M bioaugmented microcosm, and the BRC indigenous
microcosm set. The butane and 1,1,1 -TCA half-saturation constants
(Ks,But
and
KS,TCA) and cell decay rate (b) values of the bioaugmented microcosms were
obtained from the model simulations in the BR3 microcosm set. The butane and
1,1,1 -TCA maximum specific rates
(kmax,But
and kmaX,TCA) and transformation
capacity (Ta) values achieved in all bioaugmented microcosms were similar. These
microcosms were inoculated with the same culture that was grown at different
times. This indicates that the bioaugmented microcosms have a reproducible
performance. Additionally, the kinetic parameters obtained from the simulations
were not influenced by the addition of nutrients. The consistency of the
transformation ability of bioaugmented microorganisms will be required to insure
success in field bioaugmentation applications.
Since the kinetic parameters for the BRC indigenous microcosm set could
not be determined, the parameters for the BR3 bioaugmented microcosm set were
used in the model simulation. The model fit the butane utilization well in early
time, but overestimated the butane utilization and 1,1,1 -TCA transformation after
10 days of incubation. The results from the simulations showed that the indigenous
microorganisms likely had higher values of butane half-saturation constant
and cell decay rate (b), and lower values of butane and 1,1,1 -TCA maximum
specific rate (kmax But and kmIXTCA) than the bioaugmented microorganisms.
However, the slower butane utilization could have been caused by the other factors,
possibly nutrient limitation. The results also indicated that the indigenous
microorganisms had lower transformation capacity than the bioaugmented
microorganisms.
The values of decay rate (b), 1,1,1 -TCA maximum specific rate (kmax,TCA),
and 1,1,1 -TCA half-saturation constant (KS,TCA) in parentheses were estimated from
the variation of a single kinetic parameter at a time. In these simulations, the
indigenous microorganisms were assumed to have high transformation capacity. It
144
was difficult to determine how the values differed for the indigenous
microorganisms compared to the bioaugmented microorganisms. The indigenous
microorganisms potentially had higher decay rates, higher 1,1,1 -TCA halfsaturation constants and lower 1,1,1 -TCA maximum specific rates than the
bioaugmented microorganisms. The estimated value of high decay rate possibly
represented the enzyme decay rate resulting from the 1,1,1 -TCA transformation
rather than the cell decay rate.
Different butane-utilizing cultures are likely present in the indigenous and
bioaugmented microcosms. A slower rate of 1,1,1-TCA transformation was
probably due to the indigenous microorganisms being less effective towards 1,1,1
TCA transformation. Unlike the bioaugmented microcosms, TCA transformation
in the indigenous microcosms was not active over long periods of time. However,
the model construct that was used could not accurately model the results obtained
in the indigenous microcosms. The model did a much better job simulating the
results obtained in the bioaugmented microcosms. This was partly due to better
estimations of vital model parameters.
145
CHAPTER 5
SUMMARY AND ENGINEERING SIGNIFICANCE
5.1 SUMMARY
Microcosm studies were conducted to develop a culture to be bioaugmented
into the subsurface for the cometabolic treatment of 1,1,1 -TCA, TCE and 1,1 -DCE.
The culture was tested in groundwater and aquifer solid microcosms that mimic
conditions where a field test was to be performed. The culture was originally
acquired from existing microcosms (Jitnuyanont 1998) used for the 1,1,1 -TCA
transformation tests.
The results presented in these studies demonstrated that all propane- and
butane-utilizers in the existing microcosms could cometabolicaily transform 1,1,1 -
TCA. The indigenous propane and butane-utilizers transformed TCA more slowly
and had lower transformation yields than bioaugmented microorganisms (Table
4.2). The bioaugmented propane- and butane-utilizers showed long-term
transformation ability for 1,1,1 -TCA in the absence of butane utilization, indicating
that the enzyme system responsible for transformation was present and active long
after the substrate was utilized. After prolonged treatment, transformation
efficiency in bioaugmented propane-utilizing microcosms decreased. The final
transformation yields of bioaugmented propane-utilizers were lower than the
average transformation yields. Unlike bioaugmented propane-utilizers,
bioaugmented butane-utilizers maintained transformation yield values over the
study period. The bio augmented butane-utilizers also showed longer 1,1,1 -TCA
transformation periods in the absence of butane utilization than the bioaugmented
propane-utilizers. To ensure that there was sufficient active biomass, the final
transformation yield was used to determine which microcosm was suitable for the
bioaugmentation of additional microcosms. The maximum final transformation
146
yields in bioaugmented propane- and butane-utilizers were 0.0 19 and 0.03 6 mg
TCA!mg substrate. Thus bioaugmented butane-utilizers in microcosm B6 were
chosen to be the parent microcosm for bioaugmentation studies.
Similar to the butane-utilizers in parent microcosm B6, the butane-utilizers
in all bioaugmented microcosms were able to transform 1,1,1 -TCA over long
periods in the absence of butane utilization. The indigenous butane-utilizers
showed less ability to transform 1,1,1 -TCA than those in the existing microcosms
(Jitnuyanont, 1998). Thus the bioaugmented microcosms had more reproducible
performance than the non-augmented microcosms.
The slurry-inoculated and media-culture inoculated microcosms (B2 and B4
sets) had similar values of average transformation yield, 0.079 ± 0.005 and 0.06 1 ±
0.012 mg TCAJmg butane, respectively. Jitnuyanont (1998) reported a similar
transformation yield for butane-utilizers, which was determined during the first 190
days of incubation (0.07 mg TCA/mg butane). Although all bioaugmented butaneutilizers behaved similarly during the first 10 days of incubation, slurry-inoculated
microcosms (B3 set), which contained no aquifer solids but only mineral salts
growth media, showed much lower transformation ability. The transformation
yield for the B3 microcosm set was 0.024 ± 0.001 mg TCA/mg butane, which was
two to three times less than the other microcosm sets.
The transformation rates of 1,1,1 -TCA in the slurry-inoculated and media-
culture inoculated microcosms (B2 and B4 sets) were also measured in the absence
of butane. The values of the pseudo first-order rate parameter (kmXü/Ks) and cell
decay rate (b) for these microcosms were determined through the rate regression
method. The transformation rates were assumed to follow first-order kinetics. The
average cell decay rates in both slurry-inoculated and media-culture inoculated
microcosms (B2 and B4 sets) were similar (Table 4.5). The pseudo-first-order rate
parameters (kmX0!Ks) were within a factor of two of each other (Table 4.5). This
could have resulted from the different amounts of microbial cells in two inoculums,
and indicates kinetic factors did not change significantly.
147
The similarity of transformation yields and cell decay rates between the
slurry-inoculated and media-culture inoculated microcosms (B2 and B4 sets)
suggest that growing the culture on growth media before bioaugmentation might
not change the activity of the microbial community. However, the mixed cultures
probably changed when growing in mineral salts growth media for longer than 20
days, as indicated by the differences in transformation yields.
Competitive inhibition of 1,1,1 -TCA transformation by butane was
observed. The results from rate measurements demonstrated that the decrease in
transformation rate at high concentration, ranging from 1,500 to 2,600 ig/L, tends
to correlate with the mass of transformed 1,1,1 -TCA rather than with time, while at
low concentration, ranging from 300 to 500 .tg/L, the decrease in rate tends to
correlate with either time or the amount of 1,1,1 -TCA transformed. It may be that
repeated transformation of low amounts of 1,1,1 -TCA did not drain energy
reserves, but served to induce enzyme activity. Additionally, limited and slow
transformation of 1,1,1 -TCA appears to be correlated with a slow rate of butane
uptake. 1,1,1 -TCA transformation product toxicity, a drain in energy reserves, and
cell decay might all be contributing to this loss in activity.
In addition to 1,1,1 -TCA, the bioaugmented butane-utilizers were able to
transform other chlorinated solvents such as 1,1 -DCE and TCE. Initial aqueous
concentrations of 1,1,1-TCA, TCE, and 1,1-DCE ranged from 390 to 590 tgIL, 540
to 1400 j.igIL, and 100 to 730 tg/L, respectively. The transformation of 1,l-DCE
was very fast, while the transformation rate of 1,1,1 -TCA and TCE were slower.
The inhibition of 1,1,1 -TCA and TCE transformation by 1,1 -DCE was also
observed. The transformation efficiency decreased with mass of chlorinated
solvent mixture transformed. Transformation product toxicity from chlorinated
solvents, especially from 1,1 -DCE, and possibly TCE at high concentrations, was
suspected to be the maj or cause of the limited transformation of chlorinated
solvents and the cessation in butane utilization.
148
Based on the good performance of 1,1,1 -TCA transformation,
bioaugmented butane-utilizers in microcosm B2- 1 was chosen to be the culture
seed to obtain a uniform culture for use in bioaugmentaion studies both in the
laboratory and in field tests. The culture was grown in mineral salt media,
harvested, placed in 1 ml vials with 7%DMSO (Dimethyl Sulfoxide), and stored in
196 °C liquid nitrogen for future studies. When the cultures were harvested, the
measured 0D600 was approximate 0.36. The calculated yield was around 0.7 mg
cell / mg butane, and the density of the frozen culture was 2500 mg TSS IL.
Microcosm studies using the frozen butane-utilizing culture were conducted
to compare 1,1,1 -TCA transformation ability between indigenous and
bioaugmented butane-utilizers. No 1,1,1 -TCA transformation was observed in the
indigenous microcosms. With long-term exposure to 1,1,1-TCA, the rate of butane
utilization in the indigenous microcosms slowed. The microcosms bioaugmented
with the frozen butane-utilizing culture showed the same performance as the
microcosms bioaugmented with slurry. Long-term transformation of 1,1,1 -TCA in
the absence of butane utilization was achieved at rapid rates. The values of cell
decay rate in the frozen-culture inoculated microcosms were determined through
the rate regression method (Table 4.8). These values were similar to those in the
slurry-inoculated and media-culture inoculated microcosms. This indicates that the
mixed cultures are likely similar after being k'pt in the frozen condition and then
grown in media before bioaugmentation. The transformation of a mixture of
chlorinated solvents, 1,1,1 -TCA, 1,1 -DCE and TCE, also shows similar trends.
The transformation rate of 1,1 -DCE was the fastest, while the transformation rate
of 1,1,1-TCA and TCE were slower. The competitive inhibition of 1,1,1-TCA and
TCE transformation by butane and 1,1 -DCE was observed. After transforming the
mixture of these chlorinated solvents, the rates of butane utilization and chlorinated
solvent transformation slowed down.
Results from the kinetic experiments demonstrated that the butane and
1,1,1 -TCA half-saturation constants
(K,But
and KS,TCA) in the frozen-culture
bioaugmented microcosms (BR3 and BR4 sets) were similar (Table 4.14). These
149
microcosms were bioaugmented with frozen butane-utilizing cultures after one
growth step in mineral media, but from batches of cells grown at different times.
This illustrates the very reproducibility of the bioaugmentation process. The values
of butane and 1,1,1 -TCA half-saturation constant (K.But and KS,TCA) for the
indigenous microcosms could not be determined from kinetic experiments since the
butane utilization and 1,1,1 -TCA transformation was slow, and 1,1,1 -TCA
transformation stopped after the microcosms were exposed to the second addition
of TCA (800 tg/L).
The results of microcosm studies in the BR3, BR4 and B4 bioaugmented
microcosm sets, and the BRC non-augmented microcosm set were selected to
conduct a model simulation of butane utilization and 1,1,1 -TCA transformation.
Values of kinetic parameters were determined including the maximum specific rate
of butane utilization (kmax,But) and 1,1,1 -TCA transformation
(kmax,TCA),
cell decay
rate (b), and transformation capacity (Ta). The bioaugmented microcosms were
inoculated with the media-culture from batches of cells grown at different times.
The bioaugmented cultures in the BR3 and BR4 microcosm sets were not washed
with groundwater to rinse away the mineral salts media prior to inoculation, while
the cultures bioaugmented in the B4 microcosm set were washed.
The results from the simulation of butane utilization and 1,1,1 -TCA
transformation for the BR3 bioaugmented microcosm set showed that the model fit
data well at both and high TCA concentrations (Figure 4.47). The butane and
1,1,1 -TCA half-saturation constants
(Ks,But
and KS.TCA) obtained from the kinetic
experiment provided good matches between the simulation and the microcosm
results. The model started to deviate from the microcosm data at later time. Little
improvement was obtained by including a transformation capacity term. The
results from the data at low concentrations were not sensitive to the transformation
capacity term, since the mass of TCA transformed was much less than the
transformation capacity term (Figure 4.52). The lack of sensitivity to
transformation capacity was also due to the high amount of biornass available. Fits
to the transformation capacity model were obtained from microcosm data at high
150
concentrations. The values of kinetic parameters including maximum specific rate
of butane utilization (kmax,But) and 1,1,1 -TCA transformation
(kmax,TCA),
cell decay
rate (b), and transformation capacity (Ta) were similar among the microorganisms
in the BR3 bioaugmented microcosm set (Tables 4.16 to 4.18). Again, this
indicates the reproducibility of bioaugmentation process.
The microcosm data of butane utilization and 1,1,1 -TCA transformation in
the BR4 bioaugmented microcosm set and the BR4-M bioaugmented microcosm
were modeled by using the same butane and 1,1,1 -TCA half-saturation constant
(KS,BUt
and KS,TCA), cellular yield (Y), and cell decay rate (b) values as the BR3
microcosm set (Figures 4.53 to 4.54), Reasonable fits between the model
simulation and data were obtained. The results from the 1,1,1 -TCA transformation
at early time were not sensitive to the transformation capacity term (Figures 4.55a
and 4.55b). This was possibly due to the high transformation capacity of 1,1,1TCA. The simulation of 1,1,1 -TCA transformation at later time still had low
sensitivity at low concentrations, but became more sensitive at higher
concentrations (Figures 4.55e and 4.55f). The high biomass also made the
simulation less sensitive to the transformation capacity values. The bioaugmented
microorganisms had high transformation capacity (0.3 mg 1,1,1-TCA/mg cells).
The results from the model simulation at the tested concentration showed little
difference between having no transformation term (T
= cc)
and a T value of 0.3
mg/mg. Although the effect of the transformation product toxicity was included in
the simulation, it did not improve the model fit to the data. The slower butane
uptake rate at later time may be caused by nutrient limitations. Effects of enzyme
induction or a change of microbial community that were not included in the model
simulations are possible factors resulting in the higher transformation rates. All
bio augmented microcosms, either constructed with groundwater and aquifer
material (BR4 set) or containing only growth media (BR4-M), had similar kinetic
parameter values (Table 4.20). This indicates the reproducibility of
bioaugmentation process, and indicates the same butane-utilizing microbial activity
is present in both media and groundwater microcosms.
151
The results from the model simulation for the B4 bioaugmented microcosm
set demonstrated that the model fit the data well after day 20 (Figures 4.57 to 4.58).
The rate of butane utilization during the 10 days of incubation was slower than the
model prediction. This implies that the values of km,But between the two periods
are possible different. Additionally, the slower butane utilization rate was probably
due to the inhibition of 1,1,1 -TCA, which was not considered in the model. The
model also overestimated butane utilization and 1,1,1 -TCA transformation at later
time. The inclusion of a transformation capacity term did not help to achieve a
better fit to the data (Figure 4.59). The model with no or high transformation
capacity term (T =
or T
0.4 mg/mg) better fit the data at early time, while it
under predicted the transformation of 1,1,1 -TCA at later time. The slower
transformation of 1,1,1 -TCA at later time could not be completely explained by
incorporating the transformation capacity term. Slow deactivation of enzyme
might be another factor, resulting in a decrease of kmax,TCA as a function of the mass
of 1,1,1 -TCA transformed. Other factors, such as the change of microbial
community, cannot be ruled out. The cell decay rate obtained from the rate
measurement of 1,1,1 -TCA transformation was higher than that from the
simulation of butane utilization (Table 4.24). The higher b value probably
represents both cell and enzyme decay rate. The values of pseudo-first-order rate
parameter (kmXo/Ks) obtained from the model simulation were higher than that
from the rate regression method (Table 4.24). This was possibly due to the higher
value of initial biomass (Xo) in the model simulation since the value of cell decay
rate (b) from the model simulation was lower than that obtained from the rate
regression measurement. The results obtained from the model simulations and the
rate measurements indicate the practicality of the simplified analysis in the rate
regression measurement. The rate regression method provided fairly reasonable
values of the pseudo-first-order rate parameter
(kmaxXo/Ks),
within factor of 2. A
good agreement between the values of pseudo-first-order rate parameter
(kXoIK) and cell decay rate (b) was also obtained.
152
The values of butane maximum specific utilization rate
(kmax,But),
TCA
maximum specific transformation rate (kmax,TCA) and transformation capacity
(Ta)
in the B4 bioaugmented microcosm set were similar and also similar to those of the
BR3 and BR4 bioaugmented microcosm sets (Table 4.34). This indicates that the
bioaugmented microorganisms have very reproducible performance. Additionally,
the kinetic parameters obtained from the simulations were not influenced by the
addition of nutrients over a one-month period of operation.
The simulations of butane utilization and TCA transformation for the
indigenous microcosms were conducted by using the same kinetic parameters as
the BR3 bioaugmented microcosm set (Figure 4.60). The model fit the butane
utilization well in early time, but it overestimated the butane utilization and 1,1,1-
TCA transformation after 10 days of incubation. The results from these
simulations showed that the indigenous microorganisms potentially had higher
values of butane half-saturation constant (KS,BUt) and cell decay rate (b), and lower
values of butane and TCA maximum specific rate
(km,But and kmax,TCA) than the
bioaugmented microorganisms. However, the slower rate of butane utilization may
have been caused by the other factors such as nutrient limitations. The indigenous
microorganisms had ten to thirty times lower transformation capacity (Ta) values
than the bioaugmented microorganisms (Table 4.28). However, the inclusion of
transformation capacity could not explain the slow transformation of 1,1,1 -TCA at
later time (Figure 4.62). Additionally, changing the value, of only one kinetic
parameter at a time could not achieve a good fit between model and microcosm
data. Results from the sensitivity analysis of the simulation using high
transformation capacity show that the indigenous microorganisms seem to have
either higher values of decay rate (b) and TCA half-saturation constant (KS,TCA), or
lower values of 1,1,1 -TCA maximum specific rate
(kmax,TCA)
than the
bioaugmented microorganisms (Table 4.34). A best combination of these values
was not determined. The very high value of decay rate possibly represented the
rapid enzyme decay rate resulting from the TCA transformation rather than the cell
decay rate.
153
Different butane-utilizing cultures are likely present in the indigenous and
bioaugmented microcosms. A slower rate of TCA transformation was probably
due to the indigenous microorganisms being less effective towards TCA
transformation. Unlike the bioaugmented microcosms, TCA transformation in the
indigenous microcosms was not active over long periods of time without butane
present.
5.2 ENGINEERING SIGNIFICANCE
The transformation of chlorinated solvents including TCE, t-DCE, c-DCE
and VC through aerobic cometabolism by indigenous methane-, phenol-, and
toluene-utilizing cultures has been found in the Moffett field (Mountain View, CA)
(Semprini et al., 1990; Semprini et al., 1991; Hopkins et al., 1993; Hopkins et aL,
1995). However, the transformation of l,1,l-TCA, the major contaminant in th
native groundwater, was not observed. Jitnuyanont (1998) found that propane- and
butane-oxidizing bacteria were more effective in 1,1,1 -TCA transformation, and
that bioaugmentation was a good strategy to reduce the long lag time required to
stimulate the indigenous microorganisms. The transformation of 1,1,1 -TCA and
other CAHs, including TCE, and 1,1 -DCE, by both indigenous and bioaugmented
propane-and butane-utilizers was further studied in order to develop the appropriate
cultures for bioaugmentation studies both in the laboratory and the field tests.
In this study, both propane- and butane-utilizers in the existing microcosms
(Jitnuyanont, 1998) could cometabolically transform 1,1,1 -TCA. However, the
transformation efficiency in bioaugmented propane-utilizers decreased after
prolong treatment, while bioaugmented butane-utilizers maintained transformation
yield values over the study period. As a result, the mixed butane-utilizing culture
was developed for bioaugmentation studies.
The bioaugmented microcosms had reproducible performance, maintaining
the transformation ability. The indigenous butane-utilizers transformed 1,1,1-TCA
154
less effectively, and their transformation abilities were inconsistent. Thus
bioaugmentation will likely be required to insure success in the field
demonstration. The transformation efficiency of bioaugmented butane-utilizers
decreased with continued transformation of l,1,1-TCA. After the transformation of
a mixture of 1,1,1 -TCA, TCE and 1,1 -DCE, a limitation of butane utilization and
chlorinated transformation was also observed. This probably resulted from
transformation product toxicity, especially from 1,1 -DCE. Reinoculating the
cultures could be another strategy to maintain the transformation performance.
Bioaugmentation is a good strategy for enhancing bioremediation when
indigenous microorganisms are unable to degrade the existing contaminants. The
inability of indigenous microorganisms to degrade existing contaminants is
probably due to the lack of the appropriate enzyme(s), low population of
microorganisms, and contaminant toxicity. Augmentation with proven
contaminant-degrading microorganisms can lead to a higher degree of confidence
in remediation success, and can also eliminate long lag periods required to
stimulate indigenous microorganisms.
To produce reliable bioaugmentation cultures, the developed cultures were
stored with 7%DMSO (Dimethyl Sulfoxide) in 196 °C liquid nitrogen. Prior to
use, the frozen cultures were thawed, washed with growth media to rinse away
DMSO and then grown in mineral salts growth media. The growth media used for
culture development is presented in appendix A.3.
The kinetic parameters for butane utilization and 1,l,l-TCA transformation
were also evaluated by modeling the results of microcosm studies. The kinetics of
cometabolism is an important factor for application of bioremediation. Predictive
models for the cometabolic process will be useful for engineering design to plan
and monitor site remediation, to conduct risk and exposure assessments and also to
evaluate the project cost and duration. (Alvarez-Cohen and McCarty, 1991a;
Semprini et al., 1998). The kinetic parameters obtained from these microcosm
studies can now be used in modeling the field demonstration.
155
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propane-utilizers for transforming of 1,1,1 -trichioroethane in Moffett Field
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161
APPENDICES
162
APPENDIX A
MICROCOSM PREPARATION AND OPERATION
A.! AQUIFER MATERIAL
The core material was taken from the Moffett Field site in California. It
was kept in cold storage before unpacking. The aquifer material consisted of
particle sizes ranging from silts to large cobbles. All cores were unpacked and
blended together, under a laminar flow hood, prior to transferring them into the
microcosms. They were unpacked under a laminar flow hood and wet sieved
through a no. 8 sieve (2.38 mm opening) to remove the large particles. The core
materials used in microcosm fabrication are listed in Table A. 1
A.2 GROUNDWATER
Groundwater was collected from the Moffett Field site in California and
kept in cold storage prior to use. The background aqueous concentrations of 1,1,1TCA in the Moffett groundwater ranged from 2.51 to 3.73 g/L. The nitrate
concentration was in the range of 40 mg/L as nitrate.
Table A.! List of core material used in microcosm fabrication.
a) Microcosm studies with groundwater and soil from the original microcosms
(Jitnuyanont 1998)
Location
SU39-14
Depth
I
18
163
Table A.1, Continued.
b) Microcosm studies with the frozen culture
Location
Depth
_____________________________
(ft)
16-16.5
14.5-15
16-16.5
15-15.5
15-15.5
15.5-16
14.5-15
16-16.5
15.5-16
16-16.5
14.5-15
16-16.5
18.5-19
SU39-PP1
SU39-PP2
SU39-PP3
SU39-PP4
SU39-PP5
SU39-PP6
SU39-FP1
SU39-FP2
SU39-FP3
SU39-17
SU39-18
SU39-19
SU39-20
A.3 MINERAL SALT GROWTH MEDIUM
The mineral salt growth media was prepared in demineralized water and
autoclaved prior to use. The composition was as follows:
1. Phosphate Buffer
-
K2HPO4*3H20
2030.9
mg/L
-
NaH2PO4*H20
739.0
mg/L
2. MgSO4
60.2
mg/L
3. CaC12
11.1
mg/L
4.NaNO3
153.0
mg/L
164
5. Trace Element Solution
FeSO4*7H2O
6283.0
j.ig/L
- MnCl2*4H20
300.8
p.g/L
ZnSO4*7H2O
146.6
tgIL
61.8
tg/L
108.9
ig/L
-H3B03
- Na2MoO4*2J12O
- NiC12*6H20
23.8
- CuCl2*2H20
17.0
jiglL
- C0C12*6H20
23.8
pg/L
165
APPENDIX B
CAH ANAYSIS
To quantify the concentration of 1,1,1 -TCA, TCE and 1,1 -DCE, a 100 p.L
headspace sample was injected into a Hewlett Packard (Wilmington, DE) 5890 gas
chromatograph equipped with a 63Ni electron capture detector (ECD). Separation
was obtained by an HP-624 30 m x 0.25 mm x 1.4 mm film thickness stainless steel
packed column (Hewlett Packard, Wilmington, DE) operated isothermally at 80 °C.
A flow rate of 60 mL/min of argonlmethane (95%:5%) mixture at a head pressure
of 80 psi was used as the carrier gas. A Hewlett Packard 3393 integrator was
coimected to the gas chromatograph, providing the peak area for gaseous
concentration analysis by comparing with a standard curve prepared with external
standards.
B.1 PREPARATION OF CAH STOCK SOTLUTION
A 10 mL volumetric flask was filled with methanol nearly to the meniscus
and placed on a balance. One drop of 1,1,1 -TCA, TCE or 1,1 -DCE solvents from a
10 !.IL microsyringe was then added in the flask without touching the sides of the
flask. After the added weight was recorded, the flask was capped, shaken
vigorously, and stored in refrigerator for later use.
B.2 STANDARD CURVE PREPARATION
A 125 ml amber serum bottle was filled with 15 mL of demineralized water
and then a given amount of the stock solution of water saturated with 1,1,1 -TCA,
TCE or 1,l-DCE was added. The bottle was shaken for 20 to 30 minutes to allow
166
equilibration. Various volumes of headspace sample were taken with a 100 L gas
tight syringe (Hamilton 170 iN), diluted to 100 tL, and injected into gas
chromatography to achieve different concentrations. Standard curves were
generated by plotting the peak area versus gas concentration. The chromatograms
of various concentrations of CAHs are shown in Figure B. 1.
*
LOAD NETH SE1TCE
*
* RUN 4 4726
START
0
*
AlT ?A '
* RUN 4 4725
START
DEC II, 2888
DEC 11, 2880
14:28:86
14:20:32
41
I .077
1. 843
3.471
2.94?
4.4e1
0.082 mg/L C of 1,1-DCE
0.027 mg/L C oil ,l -DCE
3.8U
0.090 mg/L C of [,1,1-TCA
0,030 mg/L C of 1,1 ,1TCA
5.991
0.108 rng/L Ci of TCE
7.IBQ
.663
0.036 nIWL Ci of ICE
S TDP
STOP
RUM#
RUN*
4725
DEC
i1
2880
14:28:32
ARFPI
4726
18382
10071
3.880
5.472
6.663
8648
91873
31250
AREA TVPE
P13
P8
PB
PB
PB
TOTAl. AREA= 152224
WIDTH
.056
.068
.851
.854
.863
AREA
6.82821
6.61591
5.68110
60.35382
28.52895
14:28:86
#REA
RT
RI
1.843
2.947
DEC 11, 2808
1.841
3.471
4.401
5.991
7.180
AREA TYPE
8944
PB
9497
PR
23838
PB
262376
P8
86098
PB
TOTAL AREA= 389953
Mt3L FACTORI . 0088E#88
MUL FACTOR=I 8088E+08
Figure B.1 Chromatograms for 1,1-DCE, 1,1,1-TCA and TCE.
WIDTH
.052
.958
.058
.855
.064
AREA
2.29361
2.43542
5.98789
67.28403
22.07987
167
*
*
RUN * 4727
DEC 11, 2800
START
14:36:15
4 * 4728
*
14:43:47
DEC 11, 2088
1F
IF
:9.989
1.832
F"835
2.845
2.849
0.137 mgfL C of 1,1 -DCE
- 3.773
3,775,
i.354
0.150 mg/L C of I,1,1-TCA
0.l92mgILC!ofl,1-DCE
0.210 nig/L C1 of 1,1,1-TCA
5.369
5.369
0.181 mg/LC10fTCE
0.253 nig/L
C1
of TCE
6.545
6.545
STOP
STOP
RUJ*
4727
DEC 11
2889
14:36:15
RUH*
DEC 11, 2088
4728
14:43:47
AREA
PT
1832
2.845
3.773
4.354
!.360
6.545
AREA TYPE
863?
PB
8958
38282
7236
398397
134125
RB
PB
PB
PB
PB
TOTAL AREA= 587555
MUL FACTOR=1 . 8880E+88
Figure B.!, Continued.
WIDTH
.050
.058
.045
.057
.855
.063
AREA'I
1.46999
1.52462
5.14828
1.23154
67.88592
22.82765
4REA
PT
1.835
2.849
3.775
5.368
6.545
TOTAL AREA
AREA TYPF
8836
PB
7742
35716
485344
160020
RB
PB
PB
PB
696858
MilL FACTORI . 8000EfOO
WIDTH
.851
.055
.843
.855
.063
AREA
1.15318
1.11099
5.12529
69.64749
22.96387
168
*
*
JJH * 4729
DEC i1
2088
14:51:18
START
iF
1.028
1.823
2.888
3.813 0.247 mg/L C of I ,I-DCE
0.270 mg/L C of i,l,I-TCA
5.399
0.325 mg/L C of ICE
6.583
STOP
RUNt
4729
RI
1.823
2.888
3.813
5.399
6.589
DEC 11, 2800
AREA T'fPE
8535
7982
43821
682575
196390
PB
P8
PR
PB
PR
TflTAI AREA= 859383
PIUL FACTOR=1 . 8088E1-88
Figure B.1, Continued.
1DTH
.052
.856
.843
.856
.863
14:51:18
AREA
.99325
.92889
5.09968
70.12368
22.85457
169
APPENDIX C
PROPANE AND BUTANE ANAYSIS
To quantify the concentration of butane and propane, a 100 L headspace
sample was injected into a Hewlett Packard 5890 gas chromatograph equipped with
a flame ionization detector (FID), coupled with a GS-Q 30 m x 0.53 mm stainless
steel packed column (J&W Scientific, Folsom, CA). A flowrate of 25 mL/min of
nitrogen gas at a head pressure of 60 psi was used as the carrier gas. A Hewlett
Packard 3393 integrator was connected to the gas chromatograph, providing the
peak area for gaseous concentration analysis by comparing with a standard curve
prepared with external standards.
C.1 STANDARD CURVE PREPARATION
A gas container was filled up with demineralized water and then closed
with a rubber cap without creating any headspace. Various amounts of butane or
propane and air were added into the gas container to achieve different
concentrations. A headspace sample was taken with a 100 tL gas tight syringe
(Hamilton 170 iN) and injected into the gas chromatograph. Standard curves were
generated by plotting the peak area versus gas concentration. The chromatograms
of various concentrations of butane are shown in Figure C.!.
170
*
*
LOAD ?4ETH PROPAHEN
*
REiN * 4721
START
DEC 11. 2088
12;51:87
8.4213
.27 mg!L 0
] 2.385
.253
1
2.54 rng/L Ci
1 5.483
6.67.5
R!JN*
7.681
3.81 mg/LC
DEC 11, 2000
4721
NETHOD HAME
18.892
(18.345
I 18.812
5.08 rng/L C
1
12.065
13.651
14,121
6.35 mg!LC
115.326
M:PROPAHEI4.MET
AREA'.
AREA TYPE
RI
P8
1865
.428
657
PB
.988
PP
55634
1.816
P8
2.305 21218128
08
883
3.708
908
88
4.155
5.860
5.483
7.131
7.152
7.601
8.584
8.892
18.345
10812
11.707
12.865
13.651
105826
40424032
472
PP
PB
1197
1483
148727
PR
PR
PP
59722384
892
98
1789
190956
88811392
Figure C.1: Chromatograms for butane.
OP
P8
88
PP
P8
872 I 88
14121
1874
88
15. 801
233488
PP
PB
15.326 180852888
STOP
1251: 0?
TOTAl. AREA3. 0377E+88
14UL FACTOR1 . 8008E+08
WIDTH
.817
.042
.242
.194
.889
.038
.231
.209
.887
.815
.035
.226
.238
.816
.034
.217
.259
.813
.829
.206
.269
AREA
.88861
.80022
.81831
6.98483
.00029
.88838
.0345?
13.38726
.08016
.88839
.88946
.84896
19. 66089
08829
.88856
.86286
26.60243
.88829
.88962
.87686
33.19966
171
APPENDIX D
THE TRANSFORMATION RATE OF l,1,l-TCA
D.1 DERIVATION OF MONOD KINETICS
The transformation rate of 1,l,1-TCA was described by Monod kinetics.
The experiments were designed to avoid competitive inhibition. The effects of
product toxicity and reducing power limitation were also assumed to be negligible.
The transformation rate is provided by equation D. 1, relating the transformation
rate to the aqueous phase concentration.
aMkmXCiv
at
(Di)
Kç+Cj
where,
M
=
Total mass of 1,1,1-TCA at time t, mg
C1
=
1,1,1 -TCA concentration in aqueous phase, mg/L
Half-saturation constant for 1,1,1 -TCA, mg/L
kmax
X
t
=
Maximum specific rate of 1,1,1 -TCA, mg/mg-day
=
Active microbial concentration, mg/L
Time, day
Since the aqueous concentration of 1,1,1 -TCA was relatively low (assuming
C1 <<
Ks), the transformation rate (equation D. 1) was simplified to followa pseudo
first order model.
ÔM
kinaxXCi
at
Kç
(D.2)
172
The mass balance on 1,1,1 -TCA in the microcosms can be wriften as
M=CiV1+CgVg
(D.3)
where,
Cg
=
1,1,1-TCA concentration in gas phase, mg/L
V1
=
Liquid volume, L
Vg
=
Gas volume, L
The equilibrium two-phase (air and liquid) partitioning was assumed based
on Henry's Law. The dimensionless Henry partition coefficient (H) was used to
describe the distribution of 1,1,1-TCA between phases. Equation D.3 was
rearranged and expressed in terms of the aqueous concentration.
M
V1
(D.4)
+HVg
Substituting equation D.4 into D.2,
aMkmaxXl
M
K.;
kmaxXMl
K.;
Lefting
X(
p
K,.;
_
VI
Vi+HccVg
v,
Vi+HccVgJ'
0t
Integrating equation D.5,
Vi+HccVg)
(D.5)
173
U
S= P Jot
M0
in
Al
M = Pt
(D.6)
M0
where,
M0
Initial total mass of 1,1,1 -TCA, mg
Equation D.6 represents the linear equation y = mx + b, allowing the y-axis
to be ln M/M0, and the x-axis to be time. The regression line of experimental data
plotted in the graph should follow this linear equation, providing the value of
pseudo first-order rate coefficient (P) from the slope of the regression line.
The cell decay rate equation was also modified from Monod kinetics,
expressed as
X = Xebt
(D.7)
where,
Xo
b
Initial total cell mass, mg/L
=
Cell decay rate, day'
Substituting equation D.7 into the P term,
kmaxX0
Kç
VP
(D.8)
V+HccVg)
Equation D.8 can symbolize y = me. The value of pseudo first-order rate
coefficient (P) will be plotted against time. The values of
obtained from the exponential regression line.
kniaxXo/Ks
and b will be
174
D.2 EXAMPLE OF CALCULATION
Figure D.1 presents the experimental data of 1,1,1 -TCA obtained from the
transformation rate measurement for microcosm B2-1. The experimental data are
plotted on a semi-logarithmic scale to test for first-order transformation, as shown
in Figure D.2. The slope of the regression line in each graph presents the pseudo
first-order rate coefficient (P), the combination term of kinetic parameters and
biomass as illustrated in equation D.5.
Is;
0.06
a,
E
0.04
002
30
31
32
33
34
Time, day
Figure D.1 1,1,1-TCA transformation data for microcosm B2-1.
35
175
a) The 1St addition of 1,1,1 -TCA
b) The 2nd addition of 1,1,1 -TCA
0.0
3T5.7:;x
-2
0.40
0.60
y-4.6829x
R2=0.9971
=
-3
-4
Time, day
c) The
3rd
addition of 1,1,1 -TCA
Time, day
d) The
4th
addition of 1,1,1 -TCA
Time, day
Time, day
e) The 5th addition of 1,1,1-TCA
Time, day
Figure D.2 1,1,1-TCA transformation rate data plotted on a semi-logarithmic
scale.
176
The values of the pseudo first-order rate coefficient (P) from each graph in
Figure D.2 are then plotted against time, as shown in Table D.1 and Figure D.3.
The exponential regression line in the graph is obtained from Microsoft Excel
2000. The slope and the power of the exponential regression line will provide the
values of the pseudo first-order rate parameter (kmXo/Ks) and cell decay rate (b),
respectively, as following:
kmXOI
K,
Cell decay rate (b)
= Power of the exponential regression line
= 0.22
Pseudo first-order rate parameter
(D.8)
V+HccVg)
day1
= Slope of the exponential regression line
(kmaxXo/Ks)
T +HVg
5.4817
0.75
0.75 + (0.55)(0.82)
=
(
=
8.77 day'
Table D.1 The values of the pseudo first-order rate coefficient (P) and time.
Starting Day
Time
(day)
30.49
31.17
31.80
32.48
33.51
0
0.68
1.31
1.99
3.02
P
(day1)
5.77
4.68
3.98
3.34
3.03
177
10
8
-.
y =
B2-1
5.4817e64x
R2 = 0.956
6
0
4
2
TF
4
Time, day
Figure 11.3 The decrease in pseudo-first-order rate coefficient (P) as a
function.
178
APPENDIX E
THE KINEITC EXPERIMENT OF BUTANE AND 1,1,1-TCA
E.1 DERIVATION OF MONOD KINEITCS
The rates of butane utilization and 1,1,1 -TCA transformation were
described by Monod kinetics. The experiments were designed to avoid competitive
inhibition. The effects of product toxicity and reducing power limitation were also
assumed to be negligible. The kinetic rate was described by equation E. 1 relating
the transformation rate to each concentration in aqueous phase.
3MkmXCI
.v
Kç+C1
ft
(E.1)
where,
M
=
Total mass of butane or 1,1,1 -TCA at time t, mg
C1
=
Butane or 1,1,1 -TCA concentration in aqueous phase, mg/L
=
Half-saturation constant for butane or 1,1,1 -TCA, mg/L
kmax
=
Maximum specific rate of butane or 1,1,1 -TCA, mg/mg-day
X
=
Active microbial concentration, mg/L
t
=
Time, day
The mass balance on butane or 1,1,1-TCA in the microcosms can be written
F1
M=C1V1 +CgVg
where,
Cg
=
Butane or 1,1,1 -TCA concentration in gas phase, mg/L
V1
=
Liquid volume, L
Vg
Gas volume, L
(E.2)
179
The equilibrium two-phase (air and liquid) partitioning was assumed based
on Henry's Law. The dimensionless Henry partition coefficient (H) was used to
describe the distribution of 1,1,1-TCA between phases. Equation E.2 was
rearranged and expressed in terms of the aqueous concentration.
C=
M
(E.3)
E.1.1 Pseudo first-order model
For the relatively low aqueous concentration (assuming C1 <<Ks), the
transformation rate (equation E. 1) was simplified to follow a pseudo first order
model.
=
5t
kmaxXCi
(E.4)
.
K
Substituting equation E.3 into E.4,
aMkmaxXl
Kç
M
Ij/+HccVg)
kmaxXMl
_
J/
Kç
Letting p
V1
kmaxX I
Kç
Vi+HccVgj'
aM
at
Integrating equation E.5,
(E.5)
180
=
M0
Al
M
ln=Pt
M0
(E.6)
where,
M0
=
Initial total mass of butane or 1,1,1 -TCA, mg
Equation E. 6 represents the linear equation y mx + b, allowing the y-axis
to be In M/M0, and the x-axis to be time. The regression line of the experimental
data plotted in the graph should follow this linear equation, providing a P value
from the slope of the regression line.
E.1.2 Zero-order model
For the relatively high aqueous concentration (assuming C1>>
Ks),
the
transformation rate (equation E. 1) was simplified to follow a zero-order model.
'=kmaxXVi
(E.7)
Integrating equation E.7,
M
JM =
kmax AT Jat
0
M M0
kmXVt
M =M
kmaxXVit
(E.8)
Similar to the pseudo first-order model, equation E.8 can symbolize y =
b+mx. The experimental data in high aqueous concentration will be plotted against
time. The value of kmaxXVi will be obtained from the slope of the regression line.
181
Since the value of P,
V1, Vg,
and kmaxXVi (from the zero-order model)
were known, K can be solved. The following is the example, showing how these
values are determined.
E.2 EXAMPLE OF CALCULATION
Figure E. 1 presents the experimental data of butane obtained from the
kinetic experiment in microcosm BR3-2. The data at high concentration tend to
follow zero-order kinetics and the data at low concentration seem to follow first-
order kinetics. The data at high concentration are plotted against time, as shown in
Figure E.2. The slope of the linear regression presents the value of kmaxXVi as
illustrated in equation E.8.
kmaxXVi
=
Slope of the linear regression line
=
0.18
4
BR3-2
Zero Order Rate
E
2
C,
I
/
13.00
13.50
14.00
First Order Rate
14.50
15.00
Time, day
Figure E.1 Experimental data for the kinetic experiment conducted in
microcosm BR3-2.
182
Figure E.3 presents the data at low concentration plotted on a logarithmic
scale. The slope of the linear regression line presents the pseudo first-order rate
coefficient (P), the combination term of kinetic parameters and biomass as
illustrated in equation E. 5.
Pseudo first-order rate coefficient (P)
=
Slope of the linear regression line
0.73
Dividing kmaxXVi by P
kXXV1
kma
P
VI
kmaxXl
K
Vi+HccVg
Ks(Vi+HccVg)
kmaxXV
So
S
(Vi+HccVg)
0.18
0.7299
0.1 + (38.05)(0.57)
=
0.011 mgIL
183
4
I:
0
0
5
15
10
20
Time, hr
Figure E.2 Experimental data at high concentration plotted against time to
test for zero-order kinetics.
0
22
23
24
25
26
Z7
-2
BR3-2
0
y = -O.7299x + 12.332
0
R2 = 0.9895
p
Time, hr
Figure E.3 Experimental data at low concentration plotted on a logarithmic
scale to test for first-order kinetics.
184
APPENDIX F
MODEL DEVELOPMENT
F.! BUTANE UTILIZATION AND 1,1,1-TCA
TRANSFORMATION
The rates of butane utilization and 1,1,1 -TCA transformations were
described by Monod kinetics that relates the transformation rate to each
concentration in aqueous phase. To simplify the kinetics expressions, the
experiments were designed to avoid the effects of competitive inhibition. The
effect of reducing power limitation was also assumed to be negligible. The rates of
butane utilization and l,1,1-TCA transformations were described by equations F.1
and F.2, respectively.
kfflBU, XCIB
dMBU,
di
+
dIVITCA
kmaXTCAXCZTCA
di
KSTCA +C,TCA
(F.l)
CIBUt
.V
(F.2)
where,
MBUt
=
Total mass of butane, mg
MTCA
=
Total mass of 1,1,1 -TCA, mg
C,,Bt
=
Butane concentration in liquid phase, mg/L
C1,TCA
=
1,1,1-TCA concentration in liquid phase, mg/L
kmax,But
=
Maximum specific rate of butane, mg/mg-day
kmax,TCA
=
Maximum specific rate of butane or 1,1,1 -TCA, mg/mg-day
=
Half-saturation constant for butane, mg/L
KS,TCA
=
Half-saturation constant for butane or 1,1,1 -TCA, mg/L
X
=
Active microbial concentration, mg/L
185
t
Time, day
The mass balance on butane and 1,1,1 -TCA in the microcosms can be
written as equations F.3 and F.4, respectively.
MBUI = CIBUVl + CgBuIVg
(F.3)
+ C g,TCA Vg
(F.4)
=
C1TCA V1
where,
Cg,But
Butane concentration in gas phase, mg/L
Cg,TCA
1,1,1-TCA concentration in gas phase, mg/L
Vg
Gas volume, L
VI
Liquid volume, L
Since the experiments were conducted for long periods of time, equilibrium
two-phase (air and liquid) partitioning was assumed based on Henry's Law. The
dimensionless Henry partition coefficient (Fl) was used to describe the
distribution of butane and 1,1,1-TCA between phases. Equations F.3 and F.4 were
rearranged and expressed in terms of the aqueous concentration of butane and
1,1,1 -TCA, respectively.
MBUI
CIBUt =
V1
+
(F.5)
Vg
MTCA
+
(F.6)
Vg
186
Substituting equations F.5 and F.6 and F.6 into F.1 and F.2, respectively,
Mi
kmButX
dMBU
di'
+
+ H
[
MBU
[VP+HGBU,
Vg
J
M
kmTCA X
dMTCA
dt
[
MTCA
KSTCA +
(F.7)
But Vg J.
Iv
Vj + HCCTcAVg)
I
(F.8)
+ HCCTCAVj
F.2 MICROBIAL GROWTH
Similar to the rate of butane utilization and 1,1,1 -TCA transformation,
Monod kinetics was also used to express the rate of microbial growth as a function
of growth consumption and cell decay:
dXpjydMBut bX
(F.9)
V1dt
di'
where,
=
Net specific cellular growth rate, day'
Y
=
Cellular yield of butane, mg cell! mg butane
b
=
Cell decay rate, day'
Substituting equation F.7 into F.9,
kmBulX
=Y
dt
KSBU +
1_Mt
+ Hcc But Vg J
[
MBU,
+ HCCButVg
J_bX
(F.lO)
[I:
Since the transformation product toxicity is another factor that effects cell
decay, it was also incorporated into the model. The effect of transformation
product toxicity on cell activity is quantified in terms of the transformation capacity
(Ta),
representing it as a constant defined by the amount of 1,1,1 -TCA compound
degraded divided by the amount of cell inactivated. The transformation capacity
term (Ta) was incorporated into equation F.9 to include the effect of transformation
product toxicity on cell activity.
dX
= ,
di'
dMBI4(
V1dt
' dM1
(F.l1)
bX
Vdt
T
where,
T
= Transformation capacity for 1,1,1 -TCA, mg TCA / mg cells
Substituting equations F.7 and F.8 into F. 11,
dt
kmaxButX
=Y
MBUt
K58, + I
[
MBUI
J/, + HccBuVg
Ve+HccButVgJ
kInaxTCAX
-
Ks,TCA
+I
MTCA -'
bX
+ H CC,TCA Vg)
M7
+ HCCTCA Vg
(F.12)
J
To develop the model, the values of all parameters were introduced into the
above equations. The differential equations F.7, F.8, and F.1O or F.12 were solved
in STELLA®. Numerical integration in time was performed in STELLA® using a
fourth-order Runge-Kutta method. Map, diagram and equations for model
simulation in STELLA® are shown in Figures F.1, F.2 and F.3, respectively.
188
KSTCA
Ks Butane
K
1]
;
i
0.000
Kmax Butane
________
0.14
0.070
KmaXTCA
________
I
I
I,
0.00 5.000
To Conceptual
Modei
Tc
________
jr
,
:0:
00 2.300
Q.d'oo
1 .000
9:30 PM 211 312001 Table 1 (T
Butane
Days
aOIH I
0i
9
8
X
0.0000
0.0000
0 0702 .853640e
0.0702 .61 3342e
18 d
0.0000
0.0702 .380490E
18.3
1 8.4
0.0000
0,0000
0.0702 .1 54761
0.0702 ,935935e
18.4
0.0000
0.0702 .123802e
18.4
0.0702 .5181 SOc
1 8.4
0.0000
0.0000
1 8.4
0.0000
0.0702 .1 25546e
1 8.4
0.0000
0.0702 .9381 99e
Fin
0.0000
0,0702 .756503E
01
...
,C
1 8.3
18.3
i:TCe
:
TCA
PU
Figure F.! Map for model simulation in STELLA®.
0.0702 .31 8803e
189
TCA pulse4
liii
TCA pulsel
TCAputse2
TCA pulses
TCA pulse3
TCA pulse6
TCA ZcTCA
n
Navigate to
L
model
parameter
Butane
Butane pulsel
Butane pulse2
/'Butie Luss
Kmax Butane
Butane pulse3
Butane pulse4
Figure F.2 Diagram for model simulation in STELLA®.
Hcc Butane
'-'
Ks Butane
190
Butane(t) = Butane(t dt) + (Butane_pulse 1 + Butane_pulse2 + Butane_pulse3 +
Butane_pulse4 - Butane_Loss) * dt
INIT Butane = 0
Butane_pulsel
pulse (0,4.25, 0)
Butane_pulse2 = pulse (0, 13.38,0)
Butane_pulse3
pulse (0,5.11,0)
Butane_pulse4
pulse(0,6.27,0)
Butane_Loss = (Kmax_Butane * X * VL * (Butane / (VL + Vg * Hcc_Butane))) /
(Ks_Butane + (Butane I (VL + Vg * Hcc_Butane)))
TCA(t) = TCA(t dt) + (TCA_pulsel + TCA_pulse2 + TCA_pulse3 + TCA_pulse4
+ TCA_pulse5 + TCA_pulse6 - TCA_Loss) * dt
INIT TCA
0.089
TCA_pulsel = pulse (0,15.87,0)
TCA_pulse2 = pulse (0,8.8,0)
TCA_pulse3 = pulse (0,11.79,0)
TCA_pulse4 = pulse (0,5.34,0)
TCA_pulse5 = pulse (0,6.98,0)
TCA_pulse6 = pulse (0,9.0,0)
TCA_Loss = (Kmax_TCA * X * VL * (TCA 1 (VL + Vg * Hcc_TCA))) 1
(Ks_TCA + (TCA I (VL + Vg * Hcc_TCA)))
Figure F.3 Equations for model simulation in STELLA®.
191
X(t) = X(t - dt) + (X_pro - X_Loss) * dt
JNITX= 19.68
X_pro = Y * Butane_Loss I VL
X_Loss = (b * X) + (TCA_Loss / (VL * Te))
b=0.15
11cc_Butane
38.05
Hcc_TCA = 0.548
Kmax Butane = 1.79
KmaxTCA 0.5
Ks_Butane = 0.11
KsTCA = 0.37
Tc= 0.01
Vg0.057
VL=0.1
Y=O.7
Figure F.3, Continued.
192
APPENDIX G
LABORATORY DATA
G.1 CULTURE DEVELOPMENT FOR BIOAUGMENTATION
STUDIES
-a--- P1
E
(5
0
I-.
0..
50
100
200
150
Time, day
0.1
--- P1
E
uc 0.06
(0
0.04
I-
': 0 02
[II
0
50
100
150
200
Time, day
Figure G.1 Propane utilization and 1,1,1-TCA transformation achieved in
indigenous microcosm P1.
193
7
E5
U,
U,
Cu
C)
C
Cu
0.
22
a-
1
[I]
0
50
100
150
200
250
Time, day
0.1
0)
0.08
-.--- P2
E
0.06
0.04
0.02
[II
0
50
150
Time, day
100
200
250
Figure G.2 Propane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm P2.
194
[;1
5
D)
E
4
U)
3
C)
Cu
2
0
1
50
100
150
200
250
150
200
250
Time, day
0.1
0.06
0.04
0.02
I
50
100
Time, day
Figure G.3 Propane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm P7.
195
E]
7
E
(0
4
0)
m
2
I
[I]
0
50
100
150
200
250
Time, day
0.1
-.- B2
0.08
E
0.06
CO
0.04
::
0.02
[1
0
50
100
150
200
250
Time, day
Figure G.4 Butane utilization and 1,1,1-TCA transformation achieved in
bioaugmented microcosm B2.
196
G.2 MICROCOSM STUDIES WITH GROUNDWATER AND
SOIL FROM MICROCOSM B6
0.05
0.04
0.03
0.02
0.01
0
50
100
Time, day
200
150
Figure G.5 Control bottles of the Bi, B2 and B4 microcosm sets.
Table G.1 The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B2-1
a) During day 30
35
Starting Day
Time
(day)
30.49
31.17
31.80
32.48
33.51
0
P
(day')
1.99
3.02
5.77
4.68
3.98
3.34
3.03
Time
(day)
(day')
0.68
1.31
During day 3 8-45
Starting Day
38.30
39.95
44.01
0
1.65
5.71
P
10.41
2.62
3.12
197
Table G.2 The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B2-2
a) During day 30 35
Starting Day
30.49
31.17
31.80
32.48
33.12
Time
(day)
0
0.68
P
(day')
2.64
6.14
6.17
5.19
4.07
3.50
Time
(day)
(day')
0
1.65
5.71
10.75
1.48
2.93
1.31
1.99
b) During day 3 8-45
Starting Day
38.30
39.95
44.01
P
Table G.3 The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B2-3
a) During day 30 - 35
Starting Day
30.49
31.17
31.80
32.48
33.51
Time
(day)
0
0.68
1.31
1.99
3.02
P
(day1)
5.33
4.34
3.40
296
2.63
198
Table G.3, Continued
b) During day 38-45
Starting Day
38.52
39.95
44.01
Time
(day)
(day1)
0
1.42
5.49
6.85
1.87
1.70
P
Table GA The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B4-1
a) During day 30 - 35
Starting Day
Time
(day)
(day')
0
0.68
1.67
2.70
4.54
3.14
2.42
2.08
Starting Day
Time
(day)
P
38.52
39.95
44.01
0
1.42
30.81
31.48
32.48
33.51
P
b) During day 3 8-45
5.49
(day')
6.56
1.83
1.57
199
Table G.5 The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B4-2
a) During day 30 - 35
Starting Day
31.17
32.48
Time
(day)
(day')
0
1.31
2.11
1.62
Time
(day)
0
1.42
P
P
b) During day 38-45
Starting Day
39.74
42.29
(day')
1.3
0.12
Table G.6 The values of the pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement for microcosm
B4-3
a) During day 30 35
Starting Day
Time
(day)
(day')
30.81
31.48
32.48
33.51
0
3.25
0.68
2.67
1.67
2.41
2.70
2.10
Time
(day)
(day1)
P
b) During day 3 8-45
Starting Day
38.52
39.95
44.01
0
1.42
5.49
P
4.57
0.77
0.61
4
-.-- B2-2
E
0
'I)
110
130
150
170
190
170
190
Time, day
0.07
1,1,1 -TCA
0.05
_ii_TCE
0.04
1,1-DCE
0.03
0.02
0
I 0.01
ii
110
130
150
Time, day
Figure G.6 Butane utilization and chlorinated compound transformation
achieved in slurry-inoculated microcosm B2-2
201
5
4
E
0
C
ml
80
100
120
Time, day
140
160
0.12
0. 1
0.08
E
u' 0.06
U)
! 0.04
P2
0.02
80
100
120
140
160
Time, day
Figure G.7 Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM1-2
202
5
4
E
U)
U)
2
100
120
140
160
Time, day
0.14
0.12
0.1
0.08
U)
0.06
2
0.04
0
'
0.02
80
100
120
140
160
Time, day
Figure G.8 Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BM3-2
203
G.3 MICROCOSM STUDIES WITH FROZEN BUTANEUTILIZING CULTURE
Table G.7 The values of pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement in microcosm
BR2-2
Starting Day
Time
(day)
28.28
29.26
30.29
31.35
32.70
0
0.98
2.01
3.08
4.43
P
(day1)
2.7
2.02
1.79
1.56
1,15
Table G.8 The values of pseudo-first-order rate coefficient (P) and time
obtained from the 1,1,1-TCA transformation rate measurement in microcosm
BR2-3
Starting Day
Time
(day)
28
28.54
29.26
30.10
30.97
32.26
0
0.54
1.26
2.10
2.97
4.26
P
(day")
3.46
3.14
2.37
2.37
1.95
1.74
204
BR2-3
a)
t1
1
0450
60
70
80
90
Time, day
0.14
0.12
-.-1,1,1-TCA
0.1
-.--- TCE
0.08
-h-- 1,1-DCE
0.06
0
I- 0.04
0.02
0
50
60
70
80
90
Time, day
Figure G.9 Butane utilization and chlorinated compound transformation
achieved in the media-culture inoculated microcosm BR2-3
205
0
4
C)
0
0
E
0
0
0
0
0
..8R3-1
0
0
*
0
0
0
0
4-
0
0
0
0
0
0
p0
0
0
5
10
20
15
Time, day
Figure G.1O Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-1
5
0
0
000
C)
E
0
0
0
0
0BR3-3
G)
0
5
10
15
20
Time, day
Figure G.11 Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-3
206
4
0)
E
2
a,
5
20
15
10
Time, day
Figure G.12 Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-4
5
0
E
0
0
0
0
0
0)
0)
oBR35
0i
a)
ml
I- -0
p
5
10
15
20
Time, day
Figure G.13 Overall results from the butane kinetic experiments conducted in
the bioaugmented microcosm, BR3-5
207
Table G.9 The results from the butane kinetic experiments conducted in the
BR3 microcosm set
a) The fourth addition of butane
Microcosm
First-order kinetics
Slope
kmax*XIKs
(day')
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
0.64
0.81
0.90
0.87
0.84
14.57
18.45
20.43
19.81
19.06
Zero-order kinetics
kmax*X
Slope
(mg/L-day)
0.17
1.74
1.92
0.19
0.22
2.23
0.22
2.17
2.28
0.23
(mg/L)
0.119
0.104
0.109
0.11
0.12
b) The fifth addition of butane
Microcosm
First-order kinetics
Slope
km*X/Ks
(day1)
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
0.64
0.81
0.90
0.87
0.84
14.57
18.45
20.43
19.81
19.06
Zero-order kinetics
km*X
Slope
(mgIL-day)
0.18
1.11
1.8
0.21
0.23
0.23
2.14
2.29
2.31
0.11
(mg/L)
0.077
0.109
0.102
0.113
0.132
208
0.1
A
ABR31
0.08
A
'I)
A
(0
A
A
0.04
A
- 0.02
A
A
A
0
14.50
15.50
17.50
16.50
A A
19.50
18.50
Time, day
Figure G.14 Overall results from the 1,1,1-TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-1
0.1
A
A BR3-3
0.08
A
A
(0
A
(4)
((4
A
A
0.04
A
A
A
.' 0.02
£
A
£
AA
A
0
_________
14.50
A
I
15.50
16.50
17.50
18.50
-H
19.50
Time, day
Figure G.15 Overall results from the 1,1,1-TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-3
209
0.1
A
A BR3-4
0.08
A
A
A
(I)
0.06
£
0.04
A
A
.-
AA
0.02
A
14.50
.-.
15.50
16.50
A
17.50
18.50
19.50
Time, day
Figure G.16 Overall results from the 1,1,1-TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-4
0.1
A
ABR3-5
A
0) 0.08
E
A
0.06
A
A
0.04
A
A
A
- 0.02
£
A
A
AA
14.50
A
15.50
16.50
17.50
18.50
19.50
Time, day
Figure G.17 Overall results from the 1,1,1-TCA kinetic experiments conducted
in the bioaugmented microcosm, BR3-5
210
Table G.1O The results from the 1,1,1-TCA kinetic experiments conducted in
the BR3 microcosm set
Microcosm
First-order kinetics
Slope
kmax*X!Ks
(day1)
BR3-1
BR3-2
BR3-3
BR3-4
BR3-5
0.06
0.139
0.15
0.17
0.17
0.08
0.18
0.19
0.23
0.23
Zero-order kinetics
km*X
Slope
(mg/L-day)
0.0033
0.033
0.0081
0.0063
0.0076
0.0078
0.081
0.063
0.076
0.078
(mg/L)
0.400
0.443
0.324
0.335
0.345
211
4
0
a)3
0
E
0
0
0
(1)
(0
0
oBR4-M
0)
0
C
0
0J
[1
0
5
10
20
15
Time, day
2.5
a)
E
ABR4-M
2
4
(0
(0
A
ii
A
0
A
a
A
A
A
'-0.5
I
0
5
10
15
Time, day
Figure G.18 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-M
20
212
4
E
0
0
[ii
0
5
10
15
20
15
20
Time, day
3
2.5
E
u
H
A
A
2
H
£
1.5
C-)
£
0
5
10
Time, day
Figure G.19 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-1
213
4
0
*
H
E
0
*
U)
(0
0
0
0
o BR4-2
0
C
(U
0
4-.
0
to
0
0
5
10
15
20
15
20
Time, day
3
2.5
E
ci
2
(0
(U
<1.5
C.)
I-:.
0.5
0
LI1 I
0
5
10
Time, day
Figure G.20 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-2
214
4
E
0
0
02
a)
C
0
5
10
15
20
Time, day
2.5
E
2
U)
U,
C.)
I-
1
O.5
0
5
10
15
Time, day
Figure G.21 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-3
20
215
4
0
0
*
0
E
0
0
a,
(0
*
0
o BR4-4
0
w
*
0
0
0
5
10
15
20
15
20
Time, day
0.4
0,
0.3
U,
(0
a,
<0.2
C)
-:
0.1
5
10
Time, day
Figure G.22 Overall results from the butane and 1,1,1-TCA kinetic
experiments conducted in bioaugmented microcosm BR4-4
216
Table G.11 The results from the butane kinetic experiments conducted in the
BR4 microcosm set
a) The third addition of butane
Microcosm
First-order kinetics
Slope
kmax*X!Ks
(day')
BR4-M
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
2.08
47.08
1
22.6
24.42
21.50
23.67
23.64
1.08
0.95
1.04
1.04
Zero-order kinetics
kmax*X
Slope
(mgIL-day)
0.28
2.85
0.26
0.28
2.14
2.65
2.78
0.3
3
0.27
2.68
0.21
K
(mg/L)
0.06 1
0.095
0.108
0.129
0.127
0.113
b) The fifth addition of butane
First-order kinetics
Slope
km*X/Ks
BR4-M
1.68
38.06
Zero-order kinetics
km*X
Slope
(mg/L-day)
0.27
2.67
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
1.01
0.94
0.96
0.92
22.80
21.30
21.77
20.96
24.91
0.24
0.2
0.25
0.25
0.25
Microcosm
(day')
1.1
2.40
1.99
2.47
2.45
2.49
(mgIL)
0.071
0.105
0.093
0.116
0.117
0.100
217
Table G.12 The results from the 1,1,1-TCA kinetic experiments conducted in
the BR4 microcosm set
a) The first and second additions of 1,1,1 -TCA
First-order kinetics
km*X!Ks
Slope
BR4-M
0.36
0.48
Zero-order kinetics
km*X
Slope
(mgIL-day)
0.0146
0.146
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
0.25
0.51
0.39
0.46
0.53
0.33
0.67
0.51
0.61
0.7
0.0172
0.0321
0.0132
0.0281
0.0298
Microcosm
(day')
0.172
0.321
0.132
0.281
0.298
(mg/L)
0.306
0.519
0.480
0.259
0.464
0.427
b) The third and fourth additions of 1,1,1 -TCA
Microcosm
First-order kinetics
km*X/K
Slope
(day1)
BR4-M
0.53
0.7
BR4-1
BR4-2
BR4-3
BR4-4
BR4-5
0.35
0.53
0.39
0.54
0.58
0.46
0.70
0.52
0.71
0.76
Zero-order kinetics
km*X
Slope
(mg/L-day)
0.144
0.0144
K
(mg/L)
0.206
0.0167
0.0185
0.0096
0.0205
0.0209
0.365
0.263
0.186
0.289
0.276
0.167
0.185
0.096
0.205
0.209
Table G.13 Parameters for model simulations in Figures G.23
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KSTCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
Vg, L
= 0.01 mg TCA 1mg cells
The lS
The 2nd
addition of
TCA
addition of
TCA
15.33
15.87
30.51 a
0.7
0.15
0.11
1.79
15.87
18.46
19,68a
0.37
0.05
0.01
0.1
0.7
0.15
0.11
1.79
0.37
0.05
0.01
0.1
0.057
0.057
HCC,BUt, mg-L/mg-L
38.05
38.05
HCC,TCA, mg-L/mg-L
0.55
0.55
Note a = Obtained from the previous simulation
T 0.1 mg TCA 1mg cells
The lS
The 2'
addition of
addition of
TCA
TCA
15.33
15.87
30.51 a
0.7
0.15
0.11
1.79
0.37
0.05
0.1
0.1
0.057
38.05
0.55
15.87
18.46
27.20 a
0.7
0.15
0.11
1.79
0.37
0.05
0.1
T 0.2 mg TCA 1mg cells
The 1st
The 2nd
addition of
addition of
TCA
TCA
15.33
15.87
3051
0.7
0.15
0.11
1.79
0,37
0.05
0.2
a
15.87
18.46
27.67 a
0.7
0.15
0.11
1.79
0.37
0.05
0.2
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
Table G.14 Parameters for model simulations in Figure G.24
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day4
T = 0.01 mg TCA 1mg cells
The lS
The 2nd
T 0.2 mg TCA 1mg cells
The 21
The 1st
addition of
TCA
addition of
TCA
addition of
TCA
addition of
TCA
addition of
TCA
addition of
TCA
15.01
15.47
16.86
18.40a
0.7
0.15
0.11
1.74
0.37
0.12
0.01
15.01
15.47
15.47
16.86
15.01
15.47
15.47
16.86
29.06a
0.7
0.15
0.11
1.74
0.37
0.12
0.2
15.47
3355a
0.7
0.15
KS,BU,mg/L
kmax,But, mg/mg-day
0.11
1.74
KS,TCA, mg/L
kmax,TCA, mg/mg-day
0.37
0.12
T, mg/mg
VL,L
0.01
0.1
0.1
0.057
0.057
38.05
HCC,BUt, mg-L/mg-L
38.05
0.55
HCCTCA, mg-L/mg-L
0.55
Note a = Obtained from the previous simulation
Vg, L
T 0.1 mg TCA 1mg cells
The 1st
The 2nd
3355a
25.58a
3355a
0.7
0.15
0.7
0.15
0.11
1.74
0.7
0.15
0.11
1.74
0.37
0.12
0.37
0.12
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.11
1.74
0.37
0.12
0.2
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
220
0.1
Tc = 0.01 mg/mg
0.08
U)
U)
.!
0.06
BR3-1
-Tc=0.O1
0.04
--Tc=O.1
NoTc term
Tc0.2
0.02
NoTc
0
15
16
17
18
20
19
Time, day
Section
Biomass (X) at starting of model simulation
(mg/L)
E
F
T=0.01
'f=0.l
T=0.2
NoTterm
30.51
19.68
30.51
30.51
27.67
30.51
28.14
27.20
Figure G.23 Simulations of 1,1,1-TCA transformation for microcosm BR3-1
over a range transformation capacities
0.12
0.1
Te
E
0.01 mg/mg
0.08
BR3-2
< 0.06
0.01
'k
0.04
--Tc= 0.05
NoTc term
0.02
0
__
14.5
15.5
16.5
17.5
18.5
Time, day
Section
E
F
Biomass (X) at starting of model simulation, mg/L
0.01
33.55
18.40
T = 0.05
T = 0.07
No T term
33.55
25.58
33.55
29.06
33.55
31.31
Figure G.24 Simulations of 1,1,1-TCA transformation for microcosm BR3-2
over a range transformation capacities
Table G.15 Parameters for model simulations in Figure G.25
0.01 mg TCA 1mg cells
The
The 2
addition of
addition of
TCA
TCA
15
15.46
15.46
16.84
33.56a
21.85a
0.7
0.7
0.15
0.15
T
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
kmax,But, mg/mg-day
KS,TCA,mg/L
kmax,TCA,
mg/mg-day
T,mg/mg
VL,L
15
0.11
1.76
0.11
1.76
0.37
0.37
15.46
33.56a
0.7
0.15
0.11
1.76
0.37
0.11
0.01
0.1
0.11
0.01
0.1
0.11
0.1
0.1
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
11cc,But, mg-L/mg-L
T 0.1 mg TCA /mg cells
The
The 2nd
addition of
addition of
TCA
TCA
0.057
38.05
0.55
15.46
16.84
30.24 a
0.7
0.15
0.11
1.76
0.37
0.11
0.1
0.1
0.057
38.05
0.55
T = 0.2 mg TCA 1mg cells
The 1st
The 2nd
addition of
TCA
addition of
TCA
15
15.46
16.84
31.21a
15.46
33.56a
0.7
0.15
0.11
1.76
0.37
0.11
0.2
0.7
0.15
0.11
1.76
0.37
0.11
0.2
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
Table G.16 Parameters for model simulations in Figure G.26
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b,day'
KS,BUt,mg/L
kmax,But, mg/mg-day
KSTCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
T = 0.01 mg TCA 1mg cells
The 1St
The 2nd
addition of
TCA
addition of
TCA
addition of
TCA
addition of
TCA
15
15.46
16.85
23.71a
0.7
0.14
0.11
15
15.46
37.17a
0.7
0.14
0.11
15.46
37.17a
0.7
0.14
0.11
15.46
16.85
1.73
1.73
0.37
0.09
0.01
0.1
T = 0.2 mg TCA /mg cells
The 21
The 1st
addition of
TCA
15
addition of
TCA
15.46
16.85
34.11a
0,7
0.14
0.7
0.14
0.11
15.46
37.17a
0.7
0.14
0.11
1.73
1.73
1.73
0.11
1.73
0.37
0.09
0.37
0.09
0.37
0.09
0.01
0.1
0.1
0.1
0.1
0.1
0.37
0.09
0.2
0.37
0.09
0.2
0.057
38.05
0.55
0.057
38.05
0.55
0.057
0.057
38.05
HCC,BUt, mg-L/mg-L
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
T = 0.1 mg TCA /mg cells
The 1St
The 2nd
3353a
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
223
0.1
m=ooi mg/mg
a 0.08
0.06
BR3-3
-..Tc001
0.04
0.04
No Tc term J \
Ic = 0.06
0.02
No Tc
0
-
14.5
15.5
16.5
17.5
18.5
Time, day
Section
Biomass (X) at starting of model simulation, mgIL
T = 0.01
T = 0.04
= 0.06
No I term
E
35.56
21.85
F
35.56
30.24
35.56
35.56
33.19
31.21
Figure G.25 Simulations of 1,1,1-TCA transformation for microcosm BR3-3
over a range transformation capacities
0.1
Tc = 0.01 mglmg
0.08
.
0.06
BR3-4
0.01
0.04
-Tc=0.2
NoTcterm
0.02
0
14.5
15.5
16.5
17.5
18.5
Time, day
Section
E
F
Biomass (X) at starting of model simulation, mg/L
T = 0.01
T = 0.1
Tc = 0.2
No Tc term
37.17
23.71
37.17
33.53
37.17
34.11
37.17
34.69
Figure G.26 Simulations of 1,1,1-TCA transformation for microcosm BR3-4
over a range transformation capacities
Table G.17 Parameters for model simulations in Figure G.27
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
The 1st
addition
The 2nd
addition
The 3rd
addition
The
addition
HCC,BUt, mg-L/mg-L
HCC,TCA, mg-L/mg-L
The 5th
addition
The 3rd
addition
The 4t)
addition
of
of
of
of
of
of
of
of
of
butane
4.55
butane
TCA
6.89
butane
11.67
butane
TCA
TCA
5.71
18.29a
6.67
36.21a
7.1
13.23
13.23
13.68
13.68
13.92
13.92
15.26
27.15a
29.4a
28.36a
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
TCA
6.67
6.89
35.14a
0.7
0.15
0.7
0.15
0.11
0.7
0.15
0.11
1.58
0.37
0
4.55
0.45
0.7
0.15
0.11
1.58
0.37
5.71
34a
0.37
0.7
0.15
0.11
1.58
0.37
0.1
0.1
0.11
1.58
16.6a
0.7
0.15
0.37
0.37
0.7
0.15
0.11
1.58
0.37
0.1
0.1
0.1
0.1
0.11
1.58
_b
0.1
0.1
0.1
0.057
0.057
0.057
38.05
38.05
38.05
0.55
0.55
0.55
Note a = Obtained from the previous simulation
b = Not used in the simulation
Vg, L
The 4th
addition
butane
T, mg/mg
VL,L
The 2nd
addition
1.58
_b
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
225
4
Xo0.45mg/L
0)
X=16.6mg/L
E
U)
BR4-1
Model
I
r
X36.21mg/L
X=27.l5mg/L
00
5
10
20
15
Time, day
0.4
X=34mg/L
0)
E 0.3
4
0.2
X=28.36mg/L
BR4-1
Model
I.;.
-: 0.1
X=35.14m9/L
0 -L0
5
10
\
,,4\X=29.4m/L
15
20
Time, day
Figure G.27 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-1 without the term for transformation capacity
Table G.18 Parameters for model simulations in Figure G.28
addition
The 2nd
addition
The 3rd
addition
of
of
of
butane
butane
4.05
4.8
butane
4.8
5.24
18.64a
36.4a
0.7
0.15
0.11
1.74
0.37
0.7
0.15
0.11
1.74
0.37
The 1st
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
0
b, day'
4.05
0.45
0.7
0.15
kmax,But, mg/mg-day
0.11
1.74
KS,TCA, mg/L
0.37
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
_b
0.1
_b
0.1
0.1
0.057
0.057
0.057
38.05
HCC,BUI, mg-L/mg-L
38.05
38.05
HCC,TCA, mg-L/mg-L
0.55
0.55
0.55
Note a Obtained from the previous simulation
b = Not used in the simulation
Vg, L
addition
The 2nd
addition
The 4th
addition
of
of
of
of
of
of
TCA
5.24
5.47
37.89a
0.7
0.15
TCA
5.47
5.68
butane
TCA
TCA
36.6a
13.47
13.68
28.69a
0.11
1.74
0.11
1.74
0.37
0.19
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
butane
13.02
13.47
26.93a
0.7
0.15
13.68
14.58
27.8a
0.7
0.15
0.11
1.74
0.37
0.19
_b
_b
_b
The 1st
0.7
0.15
12.17
13.02
13.4a
The 5th
addition
The 3rd
addition
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
_b
_b
0.11
1.74
The 4th
addition
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
227
4
Xo = 0.45 mg/L
X=13.4mg/L
E
o
BR4-2
Model
X36.4mgIL
OH0
X=26.93mgJL
I
Tim1,0c1ay
20
15
0.4
X36.6mg/L
BR4-2
Model
0)
E
0
U)
X27.Bmg/L
X=37.89mg/L
I
Timday
X28.69mg/L
15
20
Figure G.28 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-2 without the term for transformation capacity.
Table G.19 Parameters for model simulations in Figure G.29
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
KS,BU,mg/L
kniax But, mg/mg-day
KSTCA,
mg/L
addition
The 2nd
addition
The 3rd
addition
of
of
of
of
butane
butane
0
3.8
TCA
5.39
5.64
37.8a
0.7
0.15
The 1st
3.8
0.45
0.7
0.15
0.11
1.74
0.37
4.96
butane
4.96
5.39
16.22a
3607a
0.7
0.15
0.11
1.74
0.37
0.7
0.15
0.11
1.74
0.37
0.1
0.1
kmaX,TCA, mg/mg-day
T,_mg/mg
VL,L
0.1
0.057
0.057
0.057
38.05
38.05
38.05
0.55
0.55
0.55
Note a = Obtained from the previous simulation
b = Not used in the simulation
Vg, L
HCC,BUt, mg-L/mg-L
HCC,TCA, mg-L/mg-L
The 4th
addition
The 5th
addition
The 3rd
addition
The 4
addition
of
of
of
of
of
TCA
5.64
butane
butane
TCA
TCA
13.01
3641a
11.67
13.01
14.74a
13.68
15.08
29.31a
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
13.47
28.67a
0.7
0.15
0.11
1.74
0.37
0.14
13.47
13.68
30.25a
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0,11
1.74
0.37
0.14
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.057
38.05
0.55
0.057
38.05
0.057
38.05
0.55
The 1st
addition
0.11
1.74
The 2nd
addition
5.83
0.55
0.55
t'J
229
4
Xo=0.45mg/L
X14.74mg/L
E
o
(0
(0
(0
BR4-3
Model
C)
(C
4-
X36.07mg/L
0
X=28.67mg/L
A
o-
5
0
Tim9day
15
20
0.4
X=36.4lmgIL
BR4-3
Model
0,
U'
U)
(0
X=29.3lmg/L
0
FX=37.8mg/L
X= 30.25 mgIL
0
0
5
iim9ciay
15
20
Figure G.29 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-3 without the term for transformation capacity
Table G.20 Parameters for model simulations in Figure G.30
Parameters
The 1St
addition
The 21
addition
The 3rd
addition
The
addition
The 2nd
addition
The 4th
addition
The 5th
addition
The 3rd
addition
The 4th
addition
of
of
of
of
of
of
of
of
of
butane
butane
3.54
4.34
butane
4.34
4.74
TCA
4.74
4.96
TCA
4.96
5.17
butane
butane
12.52
12.97
29.99a
TCA
12.97
13.17
31.86a
0.7
0.15
TCA
13.17
14.07
30.92a
0.7
Startingday
0
Ending day
Xo,mgIL
Y, mg/mg
3.54
0.45
0.7
0.15
b,day'
KSBU,mg/L
mg/mg-day
KSTCA, mg/L
kmax,But,
kmçjç, mg/mg-day
T, mg/mg
VL,L
18.15a
379a
3945a
38.17a
11.42
12.52
14.48a
0.7
0.15
0.11
0.7
0.15
0.11
0.7
0.15
0.11
0.7
0.15
0.11
0.7
0.15
0.11
0.7
0.15
0.11
1.93
1.93
1.93
1.93
1.93
1.93
0.11
1.93
0.37
0.37
0.37
0.37
0.16
0.37
0.16
0.37
0.16
0.37
0.16
0.37
0.16
_b
U
_b
_b
_b
_b
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.11
1.93
0.1
0.1
0.057
0.057
0.057
38.05
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
b Not used in the simulation
Vg, L
0.15
0.11
1.93
0.37
0.16
_b
t'.)
231
4
Xo = 0.45 mgJL
6R4-5
Model
0,
E
X
14.48 mg/L
(a)
(I)
0
0)
C
X=37.gmg/L
X 29.99 mg/L
0
0
0
5
Tin, day
20
15
0.4
-T
X 38.17 mgIL
E 0.3
Ui.
BR4-5
Model
0)
1
X= 30.92 mg/L
0.2
0.1 -1
X= 39.45 mg/L
a!
5
10
X=31.86mg/L
15
i
20
Time, day
Figure G.30 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-5 without the term for transformation capacity
Table G.M Parameters for model simulations in Figure G.31
0.1 mg TCA/mg cells
a) T
The 1st
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
KS,BU,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TcA,mg/mgday
T,mg/mg
VL,L
The
2nd
The
5th
The
3rd
The 4th
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
4.5
4.73
43.92a
4.73
4.97
11.67
12.53
12.53
12.75
12.75
12.98
41.2a
12.98
14.17
28.66a
31.41a
29.48a
0.7
0.15
0.11
2.2
0.37
0.11
0.7
0.15
0.11
2.2
0.37
0.11
11.63a
0.7
0.15
0.7
0.15
0.11
2.2
0.37
0.11
0.7
0.15
0.11
2.2
0.37
0.11
0.7
0.15
0.11
2.2
0.37
0.11
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
The 4th
0.11
2.2
0.37
0.11
0.1
0.1
0.057
38.05
0.55
w
Table G.21, Continued
0.2 mg TCA/mg cells
b) T
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TcA,mg/mgday
T, mg/mg
VL,L
2nd
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
4.5
4.73
43.92a
4.73
4.97
12.53
12.75
12.75
12.98
12.98
14.17
41.81'
11.67
12.53
13.2a
30.21a
32.92a
31.36a
0.7
0.15
0.11
2.2
0.37
0.11
0.2
0.7
0.15
0.11
2.2
0.37
0.11
0.2
0.7
0.15
0.11
2.2
0.37
0.11
0.2
0.7
0.15
0.11
2.2
0.37
0.2
0.7
0.15
0.11
2.2
0.37
0.11
0.2
0.7
0.15
0.11
2.2
0.37
0.11
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
The
0.057
0.057
38.05
38.05
0.55
0.55
HCCTCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
HCC,BUt, mg-L/mg-L
The 4th
The 3rd
addition of
TCA
The 1st
5th
The
0.11
The 4th
addition of
TCA
Table G.21, Continued
0.3 mg TCA/mg cells
c) T
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
kmax
But, mg/mg-day
KS,TCA,
mg/L
kmax,TcA,mg/mgday
T, mg/mg
VL,L
The 1st
The 2nd
The 4th
The 5th
The 3rd
The 4th
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
4.5
4.73
43.92a
0.7
0.15
0.11
2.2
0.37
0.11
4.73
4.97
11.67
12.53
13.77a
12.53
12.75
12.75
12.98
30.71a
3341a
12.98
14.17
31.98a
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.3
0.1
42.02a
0.7
0.15
0.11
2.2
0.37
0.11
0.3
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
NJ
235
a) The first addition of 1,1,1 -TCA on day 4
0.02
0.015
jBR4M
Tc=0.1 mg/mg
(I,
U)
iziii::
0.01
1
-: 0.005
INoTclerm
1
i1
4.45
4.5
4.55
4.6
4.65
4.7
4.75
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.1
T=O.2
T=O.3
NoTterm
43.92
43.92
43.92
43.92
b) The second addition of 1,1,l-TCA on day 4
0.4
O.3
U)
0,
BR4-M
0.2
= 0.1
'-
---Tc=0.2
-Ic = 0.3
0.1
No Tc
4.7
4.75
4.8
4.85
4.9
4.95
5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=O.3
NoTterm
41.20
41.81
42.02
42.43
Figure G.31 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-M over a range transformation capacities
236
c) The fourth addition of butane on day 11
1gImg
2.:
E
-
2
Tc=O.2
1.5
No Tc Term
0.5
11.6
11.8
12
12.2
12.4
12.6
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=0.3
NoTterm
11.63
13.20
13.77
14.98
d) The fifth addition of butane on day 12
0.6
0)
E
0.4
U,
U,
C)
I
H
12.5
12.55
12.6
12.65
12.7
12.75
12.8
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.1
Tz=0.2
T=0.3
NoTterm
28.66
30.21
30.71
31.74
Figure G.31, Continued
237
e) The third addition of 1,1,1-TCA on day 12
0.016
BR4-M
-Ic = 0.1
O.012
Tc = 0.2
U)
Tc = 0.1 mg/mg
0,008
-Tc = 0.3
-NoTc
NoTcTerm
'. 0.004
.
12.7
12.75
12.8
13
12.95
12.9
12.85
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=O.1
T=O.2
T=O.3
NoTterm
31.41
32.92
33.41
34.42
f) The fourth addition of 1,1,1 -TCA on day 12
0.2
0.15
U)
U)
0.1
C.)
I.-
1
0.05
12.8
13
13.2
13.4
13.6
13.8
14
14.2
14.4
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.1
T=O.2
T=O.3
NoTterm
29.48
31.36
31.98
33.25
Figure G.31, Continued
Table G.22 Parameters for model simulations in Figure G.32
a) T = 0.1 mg TCA/mg cells
Parameters
Startingday
Endingday
Xo,mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TcA,mg/mgday
T,mg/mg
VL,L
The 1st
The 2nd
The 4th
addition of
TCA
6.67
addition of
TCA
6.89
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
6.89
7.1
13.23
13.68
13.68
13.92
24.8a
27.2a
13.92
15.26
25.47a
5th
The
3rd
The 4th
354a
32.97a
11.67
13.23
13.42a
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
HCC,BUt, mg-L/mg-L
The
QO
Table G.22, Continued
b) T = 0.2 mg TCA/mg cells
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
The 1
addition of
TCA
6.67
6.89
The 2nd
addition of
TCA
6.89
The 4th
The
5th
The
3rd
The 4th
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
13.23
13.68
13.68
13.92
25.90a
28.23a
13.92
15.26
26.45a
354a
33.48a
11.67
13.23
14.92a
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
0.7
0.15
0.11
1.58
1.58
0.37
0.37
0.1
0.1
0.1
0.2
0.2
0.2
VL,L
0.1
0.1
0.1
0.1
0.2
0.1
0.1
T, mg/mg
0.1
0.2
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA,mg/L
kmax,TCA, mg/mg-day
7.1
0.057
0.057
38.05
38.05
0.55
0.55
Note a = Obtained from the previous simulation
Vg, L
HCC,BUt, mg-L/mg-L
HCC,TCA, mg-L/mg-L
0.2
0.1
Table G.22, Continued
c) T = 0.3 mg TCA/mg cells
The 1st
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b, day'
KS,BU,mgI'L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
The
2nd
HCC,BUt, mg-L/mg-L
HCC,TCA, mg-L/mg-L
The
5th
The
3rd
The 4th
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
6.67
6.89
6.89
11.67
13.23
13.23
13.68
13.68
13.92
15.46a
0.7
0.15
26.30a
28.61a
13.92
15.26
27.33a
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
1.58
0.37
0.7
0.15
0.11
0.1
0.1
0.3
0.1
0.1
0.3
0.1
0.1
0.3
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
354a
0.7
0.15
0.11
1.58
0.37
0.1
0.3
0.1
7.1
33.65a
0.7
0.15
0.11
1.58
0.37
0.1
0.3
0.1
0.057
0.057
38.05
38.05
0.55
0.55
Note a = Obtained from the previous simulation
Vg, L
The 4th
0.11
1.58
0.37
0.1
0.3
1.58
0.37
241
a) The first addition of 1,1,1 -TCA on day 6
0.02
BR4-1
0.015
Tc = 0.1
61)
U,
0.01
'- 0.005
6.65
6.7
6.75
6.85
6.8
6.9
6.95
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.1
TO.2
T=O.3
NoTterm
35.4
35.4
35.4
35.4
b) The second addition of 1,1,1 -TCA on day 6
0.4
E 0.3
U,
BR4-1
O.2
-Tc = 0.1
= 0.2
0.1
-Tc = 0.3
- No Tc
0
6.85
6.9
6.95
7
7,05
7.1
7.15
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=O.l
T=O.2
T=O.3
NoTterm
32.97
33.48
33.65
34
Figure G.32 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-1 over a range transformation capacities
242
c) The fourth addition of butane on day 11
BR4-1
0.1
Tc
Tc = 0.2
0.lmglmg
-Ic = 0.3
1.5
No Tc
0
0
o.:HNocteJ
0
0
11.5
12
13
12.5
13.5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T0.1
13.42
T=0.2
T=O.3
NoTterm
14.92
15.46
16.60
d) The fifth addition of butane on day 13
0.8
0.6
E
:O2
0
13.2
13.3
13.4
13.5
13.6
13.7
13.8
Time, day
T0.1
Biomass (X) at starting of model simulation
(mg/L)
24.80
Figure G.32, Continued
T=0.2
T=0.3
NoTterm
25.90
26.30
27.15
243
e) The third addition of 1,1,1 -TCA on day 13
0.016
1
BR4-1
.
-Tc=0.1
0.012
m:eOTC
0.008
'. 0 004
0
13.65
13.7
13.75
13.9
13.85
13.8
13.95
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=0.3
NoTterm
27.2
28.23
28.61
29.40
f) The fourth addition of 1,1,1 -TCA on day 13
0.25
1
0.2
0.1
E
0.2
0.15
0.3
0.1
- 0.05
0
13.5
14
15
14.5
15.5
Time, day
T0.l
Biomass (X) at starting of model simulation
(mg/L)
25.47
Figure G.32, Continued
T=O.2
T=0.3
NoTterm
26.84
27.33
28.36
Table G.23 Parameters for model simulations in Figure G.33
a) T = 0.1 mg TCAImg cells
The 4th
The 5th
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
12.17
13.02
13.47
13.68
9.06a
13.02
13.47
19.44a
21.67a
13.68
14.58
19.98a
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
0.7
0.15
0.11
1.74
0.37
0.19
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
The 1st
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T,mg/mg
VL,L
addition of
TCA
5.24
5.47
37.89a
0.7
0.15
0.11
1.74
The 2'
addition of
TCA
5.47
5.68
3475a
0.057
0.057
38.05
38.05
HCC,BIJt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
I
The 3
The 4th
Table G.23, Continued
b) T = 0.2 mg TCAImg cells
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
The
1St
addition of
TCA
5.24
5.47
37.89a
0.7
0.15
0.11
1.74
0.37
0.19
0.2
0.1
I
The 4th
The 5th
addition of
butane
addition of
butane
12.17
13.02
13.02
13.47
13.68
11.ola
23.09a
25.loa
13.68
14.58
23.78a
0.37
0.19
0.2
0.7
0.15
0.11
1.74
0.37
0.19
0.2
0.7
0.15
0.11
1.74
0.37
0.19
0.2
0.7
0.15
0.11
1.74
0.37
0.19
0.2
0.7
0.15
0.11
1.74
0.37
0.19
0.2
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
The 2'
addition of
TCA
5.47
5.68
35.67a
0.7
0.15
0.11
1.74
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
The 3rd
addition of
TCA
13.47
The 4th
addition of
TCA
Table G.23, Continued
0.3 mg TCA/mg cells
c) T
The 1st
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b, day'
KB,mg/L
kmax,But, mg/mg-day
KSTCA, mg/L
kmaxTcA, mg/mg-day
T, mg/mg
VL,L
The
2nd
The
5th
The
3rd
The 4th
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
5.24
5.47
37.89a
0.7
0.15
0.11
1.74
0.37
0.19
5.47
5.68
12.17
13.02
13.02
13.47
13.47
13.68
35.89a
0.7
24.39a
0.7
26.31a
0.7
0.15
11.76a
0.7
0.15
13.68
14.58
25.12a
0.11
1.74
0.11
1.74
0.37
0.19
0.37
0.19
0.15
0.11
1.74
0.37
0.19
0.3
0.1
0.3
0.1
0.3
0.1
0.15
0.11
1.74
0.37
0.19
0.3
0.7
0.15
0.11
1.74
0.37
0.19
0.3
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.3
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L!mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
The 4th
247
a) The first addition of 1,1,1 -TCA on day 5
0.025
BR4-2
C) 0.02
- Ic = 0.1
Tc = 0.2
0:15
\ço.imgimg
= 0.3
- No Tc
0.005
No Tc Term
0
5.2
5.25
5.35
5.3
5.45
5.4
5.5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=0.3
NoTterm
37.89
37.89
37.89
37.89
b) The second addition of 1,1,1 -TCA on day 5
0.4
BR4-2
0.1
Tc=0.2
'
0.1
-Tc = 0.3
-No Tc
0 -b--
5.45
5.5
5.55
5.6
5.65
5.7
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=O.1
T=0.2
T=O.3
NoTterm
34.75
35.67
35.89
36.60
Figure G.33 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-2 over a range transformation capacities
248
c) The fourth addition of butane on day 12
2.5
c=0.1mg/mg
0)
-Tc-0.1
-Tc=0.3
Tc
NoTe Ter1
12
12.2
12.4
12.6
13
12.8
13.2
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.l
TO.2
T=0.3
NoTterm
9.06
11.01
11.76
1140
d) The fifth addition of butane on day 13
o
BR4-2
0
0)
E
-Tc0.2
0.4
(I)
-Tc0.3
-NoTe
0
0.2
12.9
13
13.1
13.2
13.3
13.4
13.5
Time, day
TO.1
Biomass (X) at starting of model simulation
(mg/L)
19.44
Figure G.33, Continued
T=O.2
T=O.3
NoTterm
23.09
24.39
26.93
249
e) The third addition of 1,1,1 -TCA on day 13
0.016
U,
U,
0.&
C.)
I-
.
0.0
0
13.45
13.5
13.55
13.7
13.65
13.6
Time, day
Biomass (X) at starting of model simulation
(mgIL)
= 0.1
21.67
T = 0.2
T = 0.3
No T term
25.10
26.31
28.69
f) The fourth addition of 1,1,1 -TCA on day 13
BR4-2
= 0.1
0.15
-Tc=0.2
U,
= 0.3
0.1
-No Ic
0
'7
0.05
0
13.6
13.8
14
14.2
14.4
14.6
14.8
Time, day
T=0.1
Biomass (X) at starting of model simulation
(mg/L)
19.98
Figure G.33, Continued
T=0.2
T=0.3
NoTterm
23.78
25.12
27.8
Table G.24 Parameters for model simulations in Figure G.34
a) T = 0.1 mg TCA/mg cells
The 4th
The 5th
The 3rd
The 4th
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
3494a
13.01
13.47
11.28a
13.47
13.68
26.loa
13.68
15.08
27.85a
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
5.64
5.83
25.96a
0.7
0.15
0.11
1.74
0.37
0.14
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
The 1St
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b, day'
mg/mg-day
KS,TCA, mg/L
kmaxTcA, mg/mg-day
kmax,But,
T,mg/mg
VL,L
addition of
TCA
5.64
5.83
37.8a
The 2'
addition of
TCA
11.67
13.01
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
mg-L/mg-L
0.55
0.55
HCC,TCA,
Note a = Obtained from the previous simulation
Vg, L
t'J
Table G.24, Continued
b) T = 0.2 mg TCA/mg cells
Parameters
Startingday
Ending day
X0, mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
The 1st
The 2''
The 4th
addition of
TCA
addition of
TCA
addition of
butane
addition of
butane
addition of
TCA
5.64
5.83
37.8 a
0.7
0.15
0.11
1.74
0.37
0.14
0.2
11.67
13.01
35.67a
13.01
13.47
12.89a
13.47
13.68
27.28a
13.68
15.08
28.95a
0.7
0.15
0.11
1.74
0.37
0.14
0.2
0.7
0.15
0.11
1.74
0.37
0.14
0.2
0.7
0.15
0.7
0.15
0.11
1.74
0.37
0.14
0.2
0.1
0.1
0.057
Vg, L
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
The
5th
0.11
1.74
0.37
0.14
0.2
The
3rd
The 4
addition of
TCA
5.64
5.83
27.52a
0.7
0.15
0.11
1.74
0.37
0.14
0.2
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
L1
Table G.24, Continued
0.3 mg TCA/mg cells
c) T
Parameters
Startingday
Ending day
Xo,mg/L
Y, mg/mg
b,day'
KS,BUt,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TcA,mg/mgday
T, mg/mg
VL,L
The
1St
addition of
TCA
5.64
5.83
37.8a
0.7
0.15
addition of
butane
13.01
13.01
35.92a
13.47
13.48a
0.7
0.15
0.7
0.15
0.11
1.74
0.37
0.14
0.3
0.11
1.74
0.11
1.74
0.37
0.14
0.37
0.14
0.3
0.1
0.3
0.1
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
Hcc,But, mg-L/mg-L
The 4th
The 2'
addition of
TCA
11.67
5th
The 3rd
The 4th
addition of
butane
addition of
TCA
addition of
TCA
13.47
13.68
27.72a
13.68
15.08
29,36a
0.7
0.15
0.11
1.74
0.37
0.14
0.7
0.15
0.11
1.74
0.37
0.14
0.3
5.64
5.83
28.09a
0.7
0.15
0.11
1.74
0.37
0.14
0.3
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
The
0.1
0.3
0.1
0.057
38.05
0.55
0.057
38.05
0.55
253
a) The first addition of 1,1,1 -TCA on day 5
0.02
a,
E
U,
0.0
0.00
0
5.35
5.4
5.45
5.5
5.55
5.65
5.6
5.7
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T0.2
T=0.3
NoTterm
37.80
37.80
37.80
37.80
b) The second addition of 1,1,1 -TCA on day 5
0.4
a)
E 0.3
U)
T02
0.1
5.65
5.7
5.75
5.8
5.85
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=O.1
T=0.2
T=0.3
NoTterm
34.94
35.67
35.92
36.41
Figure G.34 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-3 over a range transformation capacities
254
c) The fourth addition of butane on day 11
BR4-3
a
2.:
= 0.1
= 0.1 mg/mg
= 0.3
j1.
No Tc
0
NoTcTerm _/
0.5
0
0
0
0
11.5
12
13
12.5
13.5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.l
11.28
T=O.2
T=O.3
NoTterm
12.89
13.48
14.74
d) The fifth addition of butane on day 13
0.6
E
0.4
C)
C
Li
12.9
13
13.1
13.2
13.3
13.4
13.5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.1
T=O.2
T=O.3
NoTterm
26.10
27.28
27.72
28.67
Figure G.34, Continued
255
e) The third addition of 1,1,1 -TCA on day 13
0.016
BR4-3
0.012
Tc0.
gImgImg
-Ic = 0.1
- Tc=0.2
U'
Tc=0.3
0.008
C.)
_I
NoTcTerm
I-;.
0.004
0
13.45
13.5
13.55
13.6
13.7
13.65
Time, day
T
Biomass (X) at starting of model simulation
(mg/L)
T 0.3
0.2
T
28.95
29.36
0.1
27.85
No T term
I
I
30.25
f) The fourth addition of 1,1,1 -TCA on day 13
0.2
BR4-3
Tc = 0.1
0.15
\NoTc
-Tc0.2
u5
U)
Tc
0.1
C.)
I-;.
0.3
0.05
I
0
13.5
14
15
14.5
15.5
Time, day
T0.1
Biomass (X) at starting of model simulation
(mg/L)
25.96
Figure G.34, Continued
T=0.2
27.52
T=0.3
I
28.09
NoTterm
I
29.31
Table G.25 Parameters for model simulations in Figure G.35
a) T = 0.1 mg TCAImg cells
Parameters
Startingday
Ending day
The 1st
The 2d
The 4th
addition of
TCA
4.74
4.96
addition of
TCA
4.96
addition of
butane
X0, mg!L
39.83a
Y, mg/mg
0.7
0.15
0.11
1.95
0.37
0.16
5.17
36.89a
0.7
0.15
0.11
1.95
0.37
0.16
0.1
0.1
0.1
0.1
b, day'
K5,B,mg/L
mg/mg-day
KS,TCA, mg/L
kmax,But,
kmax,TCA, mg/mg-day
T,mg/mg
VL,L
0.057
0.057
mg-L/mg-L
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
The 3rd
The 4th
addition of
butane
addition of
TCA
addition of
TCA
11.67
12.52
9.61a
12.52
13.01
22.57a
13.01
13.21
24.69a
13.21
14.07
23.34a
0.7
0.15
0.11
1.95
0.37
0.16
0.7
0.15
0.11
0.37
0.16
0.7
0.15
0.11
1,95
0.37
0.16
0.7
0.15
0.11
1.95
0.37
0.16
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
5th
The
1.95
L'J
Table G.25, Continued
0.2 mg TCA!mg cells
b) T
The 1st
Parameters
Starting day
Ending day
Xo,mg/L
addition of
TCA
4.74
4.96
The
2nd
addition of
TCA
4.96
The 4th
The
5th
The
3rd
The 4th
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
12.52
13.01
26.13a
13.01
13.21
13.21
14.07
26.84a
0.7
0.15
0.11
0.7
0.15
0.11
1.95
1.95
0.11
1.95
T, mg/mg
0.7
0.15
0.11
1.95
0.37
0.16
0.2
11.67
12.52
11.64a
0.7
0.15
0.11
0.37
0.16
0.2
0.37
0.16
0.2
0.37
0.16
0.2
27.99a
0.7
0.15
0.11
1.95
0.37
0.16
0.2
VL,L
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
Y,mg/mg
b, day'
K5,B,mg/L
kmax,But, mg/mg-day
KS,TCA, mgIL
kmax,TcA,mg/mgdaY
39.83a
5.17
37.71a
0.057
0.057
38.05
38.05
0.55
0.55
IICC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
11cc,But, mg-L/mg-L
0.7
0.15
1.95
0.37
0.16
0.2
Table G.25, Continued
0.3 mg TCA/mg cells
c) T
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
KS,B11,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmaxTcA,mg/mgday
T, mg/mg
VL,L
The 1st
The 2nd
The 4th
addition of
TCA
4.74
4.96
addition of
TCA
4.96
5.17
addition of
butane
addition of
butane
addition of
TCA
addition of
TCA
11.67
12.52
12.41a
12.52
13.01
27.23a
13.01
13.21
13.21
14.07
0.7
0.15
0.11
1.95
0.37
0.16
0.7
0.15
0.11
29.Ola
0.7
0.15
0.11
1.95
1.95
0.37
0.16
0.37
0.16
0.3
0.1
0.3
0.1
0.3
0.1
27.93a
0.7
0.15
0.11
1.95
0.37
0.16
0.3
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
39.83a
0.7
0.15
0.11
1.95
0.37
0.16
0.3
0.1
37.71k'
0.7
0.15
0.11
1.95
0.37
0.16
0.3
0.1
0.057
0.057
38.05
38.05
HCCBUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
The
5th
The
3rd
The 4th
0.1
0.057
38.05
0.55
00
259
a) The first addition of 1,1,1-TCA on day 4
0.02
BR4-4
-Tc = 0.1
0.015
Tc = 0.2
-Tc = 0.3
(
0.1 mg/mg
Tc
o.oi
No Tc
C.)
I-.
"%..__.
-0.005
NoTcTerm _J
0
41
4.75
4.8
4.85
4.9
4.95
5
Time, day
Biomass (X) at starting of model simulation
(mg/L)
0.1
39.83
T = 0.2
T = 0.3
No T term
39.83
39.83
39.83
b) The second addition of 1,1,1 -TCA on day 4
0.4
-Tc=0.1
-Tc=0.2
r
0.1
-Ic = 0.3
-NoTc
0
4.9
4.95
5
5.05
5.1
5.15
5.2
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=O.2
T=0.3
NoTterm
36.89
37.71
37.99
38.54
Figure G.35 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BR4-4 over a range transformation capacities
260
c) The fourth addition of butane on day 11
'.1
2.5
a)
U)
U)
1.5
C
1
.
0.5
0
11.6
11.8
12
12.2
12.6
12.4
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.l
T=0.2
T=0.3
No T term
9.61
11.64
12.41
14.09
d) The fifth addition of butane on day 12
0.6
a)
E
O.4
0.2
0
12.4
12.5
12.6
12.7
12.8
12.9
13
13.1
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.l
T=O.2
T=0.3
NoTterm
22.57
26.13
27.23
29.14
Figure G.35, Continued
261
e) The third addition of 1,1,1 -TCA on day 13
0.012
BR4-4
Tc = 0.1
C)
E
Tc = 0.2
u'O.008
U)
Tc = 0.3
No Tc
0
0.004
0
13
13.05
13.2
13.15
13j.
13.25
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.1
T=0.2
T=0.3
NoTterm
24.69
27.99
29.01
30.79
f) The fourth addition of 1,1,1 -TCA on day 13
0.25
13
13.2
13.4
13.8
13.6
14
14.2
Time, day
Biomass (X) at starting of model simulation
(mgIL)
23.34
Figure G.35, Continued
T=0.2
T=0.3
NoTterm
26.84
27.93
29.88
262
Table G.26 Parameters for model simulations in Figures G.36 and G.37
Parameters
Figure G.36
Figure G.37
0
33
25.02
41.02
23.87a
0.7
0.15
Starting day
Ending day
X0, mg/L
0.5
0.7
0.15
0.11
2.2
Y,mg/mg
b,day'
mg/L
kmax But, mg/mg-day
KS But,
0.11
2.2
0.37
0.37
KS,TCA, mg/L
kmax,TCA, mg/mg-day
0.15
T,_mg/mg
VL, L
11cc,But,
0.075
0.082
38.05
0.55
0.075
0.082
38.05
0.55
Vg,L
mg-L/mg-L
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
b = Not used in the simulation
Table G.27 Parameters for model simulations in Figure G.38
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b,day'
mg/L
kmax,But, mg/mg-day
KS,BUt,
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T,mg/mg
VL, L
Vg, L
T = 0.2
mg TCA/mg cells
T0
0.4
mg TCA/mg cells
25.02
41.02
23.87a
0.7
0.15
25.02
41.02
23.87a
0.7
0.15
25.02
41.02
23.87a
0.7
0.15
0.11
2.2
0.37
0.15
0.1
0.075
0.11
2.2
0.37
0.15
0.2
0.075
0.11
2.2
T 0.1
mg TCA/mg cells
0.082
38.05
HCC,TCA, mg-L/mg-L
0.55
Note a = Obtained from the orevious simulation
HCC,BUt,
mg-L/mg-L
0.082
38.05
0.55
0.37
0.15
0.4
0.075
0.082
38.05
0.55
263
5
X=5.86mgIL
X18.91mgfL
4
/
0)
E
U)
U)
0
0
4-
/
0
2
0
o
B4-1
Model
00
0
20
10
40
30
Time, day
50
Figure G.36 Simulations of butane utilization for microcosm B4-lwithout the
term for transformation capacity
0.4
0.3
0)
U)
T
0.1
0
24
29
34
Time, day
39
44
Figure G.37 Simulations of 1,1,1-TCA transformation for microcosm B4-1
without the term for transformation capacity
264
a) 1,1,1 -TCA transformation from day 25 to 30
0.2
0)
E
0,15
0.1
C.)
I-;.
0.05
0
24
25
28
27
Time, day
26
29
Biomass (X) at starting of model simulation
(mg/L)
T = 0.2
= 0.4
23.87
23.87
T 0.1
23.87
30
No T term
23.87
b) Butane utilization from day 31 to 33
4
E
o
(n
B4-1
-Tc = 0.1 mg/mg
-Tc = 0.2 mg/mg
Tc
0.4 mg/mg
-No Tc term
I
030
32
34
36
Time, day
38
40
Biomass (X) at starting of model simulation
(mg/L)
T=0.1
T=0.2
T=0.4
NoTterm
0.11
2.59
6.30
10.02
Figure G.38 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm B4-1 over a range transformation capacities
265
c) l,1,1-TCA transformation from day 33 to 41
0.5
0.4
U)
0,
-
0.1
32
34
36
38
Time, day
40
42
Biomass (X) at starting of model simulation
T=O.l
0.19
Figure G.38, Continued
T
0.2
14.8
T = 0.4
No T term
34.37
37.07
Table G.28 Parameters for model simulations in Figures G.39 and G.40
Parameters
Figure G.39
Figure GAO
Starting day
Ending day
Xo,mg/L
0
34
0.5
Y,mglmg
0.7
0.15
0.11
2.2
0.37
25.38
91.03
23.35a
0.7
0.15
b,day'
KS,BU,mg/L
kmax,But, mg/mg-day
KS,TCA,
mg/L
0.11
2.2
0.37
0.10
kmax,TCA, mg/mg-day
T,_mg/mg
L
Vg, L
0.075
0.082
38.05
0.55
0.075
0.082
HCC,BUt, mg-L/mg-L
38.05
FIcC,TCA, mg-L/mg-L
0.55
Note a Obtained from the previous simulation
b Not used in the simulation
VL,
Table G.29 Parameters for model simulations in Figure G.41
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b,day'
KS,BUt, mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T,mg/mg
L
Vg, L
0.1
mg TCA/mg cells
0.2
mg TCA/mg cells
T 0.4
mg TCA/mg cells
25.38
91.03
23.35 a
0.7
0.15
25.38
91.03
23.35 a
0.7
0.15
0.11
2.2
0.11
2.2
0.37
0.10
0.2
0.075
0.082
38.05
0.55
0.37
0.10
0.4
0.075
0.082
38.05
0.55
25.38
91.03
23.35 a
0.7
0.15
0.11
2.2
0.37
0.10
0.1
0.075
0.082
HCC,BUt, mg-L/mg-L
38.05
HCC,TCA, mg-L/mg-L
0.55
Note a = Obtained from the Drevious simulation
VL,
267
5
x21.4mg/L,,r
/
4
x=5mg/L
x10.3mg/L
/
0)
E
c2
o
B4-2
Mod&
0
60
40
Time, day
20
80
Figure G.39 Simulations of butane utilization for microcosm B4-2without the
term for transformation capacity
0.3
B4-2
X = 23.35 mg/L
0)
Mode'
E
X = 32.15 mg/L
ui' 0.2
4,
C)
'.
.
.
0
20
40
80
Tim
100
day
Figure G.40 Simulations of 1,1,1-TCA transformation for microcosm B4-2
without the term for transformation capacity
a) 1,1,1 -TCA transformation from day 25 to 32
0.15
a)
E
0.1
u
U)
0
0.05
0
25
27.
26
30
29
28
Time, day
Biomass (X) at starting of model simulation
(mg/L)
TO.1
23.35
T=O.2
T=O.4
NoTterm
23.35
23.35
23.35
b) Butane utilization from day 31 to 34
4
B4-2
o
a3
E
Tc = 0.1 mg/mg
Tc = 0.2 mg/mg
U)
U)
-
No Ic term
C)
a
Tc = 0.4 mg/mg
1
0---
30
31
32
33
Time, day
34
35
36
Biomass (X) at starting of model simulation
(mgIL)
T=0.1
T=0.2
T=0.4
NoTterm
1.66
5.77
8.08
10.33
Figure G.41 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm B4-2 over a range transformation capacities
c) 1,1,1 -TCA transformation from day 33 to 43
0.3
B4-2
Ic = 0.1 mg/mg
a)
E
Tc = 0.2 mg/mg
uc 0.2
Tc = 0.4 mg/mg
(j)
NoTc term
33
43
53
63
73
83
93
103
Time, day
Biomass (X) at starting of model simulation
T = 0.1
29.86
Figure G.41, Continued
= 0.2
30.21
T = 0.4
No T term
31.08
33.98
Table G.30 Parameters for model simulations in Figure G.42
Parameter
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
KS,BUt, mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
/
linax,TCA, mg/mg-uay
The 2nd
The 3id
The 4l
The 5th
addition of
butane
addition of
butane
addition of
butane
addition of
butane
9.12
10.98
21.59a
0.7
0.15
10.98
11.2
38.45a
11.2
addition of
TCA
26.49
addition of
TCA
0
20.06
37.78a
addition of
butane
20.06
26.49
0.2'
31.54
95
8.81a
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.37
0.7
0.15
0.11
1.76
0.37
The
9.12
0.006c
0.7
0.15
0.11
1.76
0.37
1
T, mg/mg
VL,L
1St
0.11
1.76
-
-
-
-
_b
_b
_b
_b
_b
0.1
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
a = Obtained from the previous simulation
b Not used in the simulation
c = Obtained from the variation
Vg, L
The
1St
18.79k'
0.11
1.76
0.37
The 2''
31.54
0.7
0.15
0.11
1.76
0.37
_b
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
271
4.(
X=0.2mg/L
3
0)
2
X38.45mg/L
(U
o
I
X = 37.78 mg/L
BRC-2
- Model
0
0
20
40
60
Time, day
80
100
60
Time, day
80
100
0.12
0.1
E
u' 0.08
U)
0.06
0.04
0.02
20
40
Figure G.42 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BRC-2 without the term for transformation capacity
Table G.31 Parameters for model simulations in Figure G.43
The 1st
Parameter
Starting day
Ending day
Xo,mg/L
Y, mg/mg
kmax,But, mg/mg-day
KS,TCA, mgIL
1
/
2nd
addition of
butane
addition of
butane
0
10.98
12.94
22.07a
10.98
0.001c
b, day'
The
0.7
0.15
0.11
1.76
0.37
The 3t1
addition of
butane
12.94
The 4th
addition of
butane
13.25
37.31a
20.06
36.21a
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.7
0.15
0.11
1.76
0.11
1.76
0.37
0.37
13.25
The 5th
addition of
butane
20.06
25.06
The 1st
addition of
TCA
25.06
l'
32.85
19.56a
0.7
0.15
0.7
0.15
0.11
1.76
0.11
1.76
0.37
0.37
The
2nd
addition of
TCA
32.85
95.3
6.08a
0.7
0.15
0.11
1.76
0.37
i
rnax,TCA, mg/mg-uay
T
Ic, mg/mg
-
-
-
-
/
VL,L
0.1
0.1
0.057
0.057
38.05
38.05
0.55
0.55
Note a Obtained from the previous simulation
b = Not used in the simulation
c Obtained from the variation
Vg, L
HCC,BUt, mg-L/mg-L
HCC,TCA, mg-L/mg-L
-
-
-
-
-
-
-
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
273
5
Xo = 0.001 mg/L
X=22.O7mg/L
X=lmg/L
I
0)
E
(0
X=37.31 mg/L
2
BRC-3
I
Model
X=36.21 mg/L
L.
0
0
40
20
60
80
100
Time, day
0.16
X=6.O8mg/L
E 0.12
BRC-3
Model
0.08
1
.
X=19.56mg/L
0.0:
Time, day
Figure G.43 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BRC-3 without the term for transformation capacity
Table G.32 Parameters for model simulations in Figure G.44
Parameter
The 1St
The 2nd
The 3id
addition of
butane
addition of
butane
The 4th
addition of
butane
10.2
10.98
20.06
Starting day
0
addition of
butane
9.12
Endingday
9.12
0.004c
10.2
23.91a
4433a
3994a
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
_b
mg/L
Y, mg/mg
X0,
b, day'
kmaxBut, mg/mg-day
KS,TCA, mg/L
Km,TCA, mg/mg-uay
1
T, mg/mg
VL,L
addition of
butane
20.06
31.54
The 1St
The 2nd
addition of
TCA
addition of
TCA
31.54
38.05
38.05
19.26a
95.1
7.25a
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
_b
_b
_b
/
-
_b
_b
0.1
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
b = Not used in the simulation
c = Obtained from the variation
Vg, L
10.98
5th
The
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
275
Xo=004mg/L
I-
X=23.9lmgfL
X=0.00lmg/L
3
2
X=44.33 mg/L
BRC
1
ModeI
X =39.94 m gIL
0
0
2v
40
80
100
80
100
Time, day
0.2
c, 0.16
E
(I)
U)
0.08
'c-.
0.04
0
0
20
40
60
Time, day
Figure G.44 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BRC-4 without the term for transformation capacity
Table G.33 Parameters for model simulations in Figure G.45
Parameter
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
K,mg/L
kmax,8ut, mg/mg-day
KS,TCA, mg/L
i
1'-max,TCA, mg/mg-uay
1
T, mg/mg
VL,L
The
1st
The 2nd
31(1
The 4th
addition of
butane
addition of
butane
addition of
butane
addition of
butane
0
9.13
12.95
14.98
27.19a
14.98
0.005c
9.13
12.95
21.08a
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
0.7
0.15
0.11
1.76
0.37
20.07
20.58a
0.7
0.15
0.11
The
5th
addition of
butane
20.07
25.06
0.7c
0.7
0.15
The
1St
addition of
TCA
25.06
95.1
19.17a
1.76
0.11
1.76
0.7
0.15
0.11
1.76
0.37
0.37
0.37
/
-
-
-
_b
_b
_b
0.1
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
b Not used in the simulation
c Obtained from the variation
Vg, L
The
-
-
_b
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
277
4/[
Xo0.005mg/L
X21.O8mg/L
X=0.7mglL
E
c,c
U)
0
2
_______
_________
_____
X2 7.19 mg/L
BRC-5
Model
______ X=20.58mgIL
°
1
0
20
40
60
80
100
80
100
Time, day
0.025
0.02
0.015
C,
I-
'-'0.005
0
0
20
40
60
Time, day
Figure G.45 Simulations of butane utilization and 1,1,1-TCA transformation
for microcosm BRC-5 without the term for transformation capacity
Table G.34 Parameters for model simulations in Figures G.46
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
K,8,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
T = 0.01 mg TCA 1mg cells
The 1st
The 2nd
addition of
TCA
26.49
31.54
18.79a
addition of
TCA
0.7
0.15
0.11
1.76
0.37
0.09
0.01
0.7
0.15
0.11
1.76
0.37
0.09
0.01
0.1
0.1
31.54
95
o.16a
0.057
0.057
38.05
38.05
0.55
0.55
Note a = Obtained from the previous simulation
Vg, L
11cc,But, mg-L/mg-L
HCCTCA, mg-L/mg-L
T = 0.3 mg TCA 1mg cells
The 1st
The 2nd
addition of
TCA
26.49
addition of
TCA
31.54
18.79a
0.7
0.15
95
0.11
1.76
0.37
0.09
0.3
0.1
0.057
38.05
0.55
31.54
8.46a
0.7
0.15
0.11
1.76
0.37
0.09
0.3
0.1
0.057
38.05
0.55
T = 0.4 mg TCA 1mg cells
The
The 2
addition of
TCA
26.49
31.54
addition of
TCA
31.54
95
l8.79'
8.55k'
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
279
Section F: The first addition of 1,1,1-TCA on day 26
0.02
BRC-2
--Tc = 0.01
C' 0.016
I
Tc=0.4
0.012
NoTc
'-
0.008
= 0.01
0.004
NoTcTerm
0
24
26
32
30
28
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=O.Ol
T=O.3
T=O.4
NoTterm
18.79
18.79
18.79
18.79
Section G: The second addition of 1,1,1-TCA on day 31
(
.I)
U. IL
0.1
C'
E
u5
0.08
0.06
T
0.04
0.02
0
0
20
40
60
80
100
Time, day
T = 0.01
0.16
Biomass (X) at starting of model simulation
(mg/L)
T = 0.4
T 0.3
8.46
8.55
No T term
8.81
Figure G.46 Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of transformation capacities
Table G.35 Parameters for model simulations in Figures G.47
0.01 mg TCA 7mg cells
The 15t
The 2nd
addition of
addition of
TCA
TCA
25.06
32.85
32.85
95.3
19.56a
1.43*10a
0.7
0.7
0.15
0.15
T
Parameters
Starting day
Ending day
Xo,mgIL
Y, mg/mg
b, day'
KS,BU,mg/L
kmax,But, mg/mg-day
0.11
1.76
KS,TCA, mg/L
kmax,TCA, mg/mg-day
0.37
0.09
T, mg/mg
0.01
0.1
VL,L
0.11
1.76
0.37
11cc,But,
mg-L/mg-L
St
addition of
TCA
25.06
32.85
19.56a
0.7
0.15
0.11
1.76
0.09
0.37
0.09
0.01
0.1
0.3
0.1
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
'Vg, L
T 0.3 mg TCA 7mg cells
The 1
The
0.057
38.05
0.55
= 0.4 mg TCA 1mg cells
The 2nd
addition of
TCA
32.85
524a
The
addition of
TCA
25.06
32.85
19.56a
0.7
0.15
0.11
1.76
0.37
0.09
0.3
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
addition of
TCA
32.85
95.3
95.3
8.46a
0.7
0.15
0.11
1.76
0.37
0.09
0.4
oc
281
Section F: The first addition of 1,1,1-TCA on day 25
0.04
BRC-3
= 0.01
0.03
Tc= 0.01
Tc = 0.3
to
NoTc
0.02
4
'-01
4
NoTclerm
.
4
0
20
22
24
28
26
30
32
34
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T
0.01
19.56
T = 0.2
T = 0.3
No T term
19.56
19.56
19.56
Section G: The second addition of 1,1,1-TCA on day 32
0.16
0,14
0.12
1
0.1
BRC-3
0.08
0.060.04
Tc0.01
Tc=0.2
Tc=O.3
0.0: NoTc
0
20
L'_
\,,
40
60
80
100
120
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.01
1.43*10b0
T=0.2
T=0.3
NoTterm
5.24
5.52
6.08
Figure G.47 Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of transformation capacities
Table G.36 Parameters for model simulations in Figures G.48
Parameters
Startingday
Ending day
Xo, mg/L
Y, mg/mg
b, day'
K,BU,mgIL
kinax,But, mg/mg-day
KS,TCA,
mgIL
mg/mg-day
kIX,TCA,
T, mg/mg
VL,L
T = 0.01 mg TCA 1mg cells
The lS
The 2'
addition of
TCA
addition of
TCA
31.54
38.05
19.26 a
0.7
0.15
38.05
0.11
1.76
0.37
0.09
0.01
0.1
95.1
0.04a
0.7
0.15
0.11
1.76
0.37
0.09
0.01
0.1
0.057
0.057
mg-L/mg-L
38.05
38.05
HCC,BUt,
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
T = 0.3 mg TCA /mg cells
The 1st
The 2''
T 0.4 mg TCA /mg cells
The 1st
The 2'
addition of
TCA
31.54
addition of
TCA
addition of
TCA
addition of
TCA
38.05
38.05
38.05
19.26 a
0.7
0.15
0.11
1.76
0.37
0.09
95.1
31.54
38.05
19.26 a
0.7
0.15
0.3
0.1
0.3
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.7
0.15
0.11
1.76
0.37
0.09
95.1
0.37
0.09
0.4
7.14 a
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.11
1.76
283
Section F: The first addition of 1,1,1-TCA on day 31
.
0.016
I
E
BRC-4
Tc=0.6
Tc=0.8
0.012
NoTe
0.008
Tc=0.01
1
L
' 0.004
Nolelerm
._/\\\
4
28
30
34
32
38
36
40
Time, day
Biomass (X) at starting of model simulation
(mg/L)
T=0.01
T=0.6
T=0.8
NoTterm
19.26
19.26
19.26
19.26
Section G: The second addition of 1,1,1-TCA on day 38
0.2
El
0)
0.16 -I
u.
0.12
0
'
.
BRC-4
0.01
Tc=0.6
Tc=0.8
u.uo
NoTe
004
4
.
.
0
0
20
40
60
80
100
Time, day
Biomass (X) at starting of model simulation
(mgIL)
T=0.01
Tz=0.6
T=0.8
NoTterm
0.04
7.11
7.14
7.25
Figure G.48: Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of transformation capacities
Table G.37 Parameters for model simulations in Figures G.49
b 0.15 day'
Parameters
Starting day
Ending day
1St
The
addition
of TCA
26.49
31.54
The 2
addition
of TCA
31.54
b= 0.2 day'
The 2'
The 1t
addition
of TCA
26.49
31.54
addition
of TCA
31.54
X0, mgIL
18.79a
95
8.55a
18.79k'
95
6.64a
Y, mg/mg
T, mg/mg
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.7
0.2
0,11
1.76
0.37
0.09
0.4
0.7
0.2
0.11
1.76
0.37
0.09
0.4
VL,L
0.1
0.1
0.1
0.057
38.05
0.55
b, day'
KSBU,mg/L
kma,çBut, mg/mg-day
KS,TCA,
mg/L
kmax,TCA, mg/mg-day
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
11CC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
b= 0.3 day'
The 15t
The 2'
addition
addition
of TCA
of TCA
26.49
31.54
31.54
95
18.79a
4.00a
0.7
0.7
0.3
0.3
b= 1.3 day'
The lS
The 2nd
addition
addition
of TCA
of TCA
26.49
31.54
31.54
95
18.79a
0.024a
0.7
0.7
1.3
1.3
0.11
1.76
0.11
1.76
0.11
1.76
0.11
1.76
0.37
0.09
0.4
0.37
0.09
0.4
0.37
0.09
0.4
0.37
0.09
0.4
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
285
Section F: The first addition of 1,1,1-TCA on day 26
0.02
BRC-2
00 016
E
b = 0.15
b = 0.2
b=1.3
0.012
-----b = 0.3
b = 1.3
0.008
15JS..
0.004
25
26
27
29
Time, day
30
28
31
32
Biomass (X) at starting of model simulation
(mgIL)
b=O.15
b=O.2
b=0.3
b=1.3
18.79
18.79
18.79
18.79
Section G: The second addition of 1,1,1 -TCA on day 31
0.2
.
0.16
BRC-2
b=0.15
b=0.2
Li
0.12
0.08
0
20
40
60
Time, day
80
100
Biomass (X) at starting of model simulation
(mgIL)
b=0.15
b=O.2
b=0.3
b=1.3
8.55
6.64
4.00
0.024
Figure G.49: Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of decay rates
Table G.38 Parameters for model simulations in Figures G.50
b= 0.15 day'
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
b, day'
K,B,mg/L
kmax,But, mg/mg-day
KS,TCA,
mg/L
kmax,TCA, mg/mg-day
T, mg/mg
VL,L
The ls
addition
of TCA
25.06
32.85
19.56a
0.7
0.15
0.11
1.76
0.37
0.09
0.3
0.1
2nd
The
addition
of TCA
32.85
95.3
5.52a
0.7
0.15
0.11
1.76
0.37
0.09
0.3
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
b= 0.2 day
The 1St
The 2'
addition
addition
of TCA
of TCA
25.06
32.85
32.85
95.3
373a
19.56a
0.7
0.2
b= 0.3 day'
The 2'
The 1St
addition
addition
of TCA
of TCA
25.06
32.85
32.85
95.3
b= 1.2 day'
The 1st
addition
of TCA
25.06
32.85
The 2'
addition
of TCA
32.85
19.56a
1.69a
19.56a
95.3
0.Oola
0.7
0.7
0.7
0.7
0.3
0.11
1.76
1.2
0.11
1.76
1.2
0.11
1.76
0.7
0.2
0.11
1.76
0.37
0.09
0.11
1.76
0.3
0.11
1.76
0.37
0.09
0.37
0,09
0.37
0.09
0.37
0.09
0.37
0.09
0.3
0.1
0.3
0.1
0.3
0.1
0.3
0.1
0.3
0.1
0.3
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
287
Section F: The first addition of 1,1,1-TCA on day 25
0.04
.
,
C)
BRC-3
b=0.15
Eo.03
b0.2
b=1.2
V.
0.02
\
..
2
0.01
b =015
I
22
24
26
28
Time, day
32
30
34
Biomass (X) at starting of model simulation
(mg/L)
b=0.15
b=O.2
b=0.3
b=1.2
19.56
19.56
19.56
19.56
Section G: The second addition of 1,1,1-TCA on day 32
BRC-3
C)
b = 0.15
E 0.16
b=0.2
U)
U)
-b= 0.3
0.12
b= 1.2
C-)
'--
.'
0.08
aI.i
0
0
20
40
60
Time, day
80
100
Biomass (X) at starting of model simulation
(mgIL)
b=0.15
b=0.2
b=0.3
b=l.2
5.52
3.73
1.69
0.001
Figure G.50: Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of decay rates
Table G.39 Parameters for model simulations in Figures G.51
b= 0.15 day'
Parameters
2nd
95.1
711a
19.26a
T, mg/mg
0.7
0.15
0.11
1.76
0.37
0.09
0.6
0.7
0.15
0.11
1.76
0.37
0.09
0.6
0.7
0.2
0.11
1.76
0.37
0.09
0.6
VL,L
0.1
0.1
Y, mg/mg
b, day'
KS,BU,mg/L
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
19.26a
2nd
The
addition
of TCA
38.05
Endingday
Xo,mg/L
The
addition
of TCA
38.05
b= 0.2 day'
The
addition
of TCA
31.54
38.05
Starting day
The
addition
of TCA
31.54
38.05
lS
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
HCC,BUt, mg-L/mg-L
lS
b= 0.3 day'
The 2m1
addition
of TCA
38.05
95.1
The
addition
of TCA
31.54
38.05
513a
19.26a
2.67a
0.7
0.2
0.7
0.7
0.3
0.11
1.76
0.37
0.09
0.6
0.1
0.057
38.05
0.55
lS
0.11
1.76
0.3
0.11
1.76
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
95.1
b 1.5 day'
The 1St
The 2'"
addition
addition
of TCA
of TCA
31.54
38.05
38.05
95.1
19.26a
o.00r'
0.7
0.7
1.5
1.5
0.11
1.76
0.11
1.76
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
289
Section F: The first addition of 1,1,1-TCA on day 31
0.02
BRC-4
.
C)
b=0.15
E 0.015
b=0.2
U)
b=1.5
\\,r
0.01
b= 1.5
-0.005
b=0.15
I
28
30
32
36
34
38
40
Time, day
Biomass (X) at starting of model simulation
(mg/L)
b=0.15
b=0.2
b=0.3
bz=1.5
19.26
19.26
19.26
19.26
Section G: The second addition of 1,1,1-TCA on day 38
b=0.15
C)
E 0.16
U)
0.12
l-;
:'i
0.08
T004
.
0
20
.
60
40
Time, day
Biomass (X) at starting of model simulation
(mgIL)
80
100
b=0.15
b0.2
b0.3
b1.5
7.11
5.13
2.67
0.001
Figure G.51: Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of decay rates
Table G.40 Parameters for model simulations in Figures G.52
0.02 mg/mg-day
The
The
kmax =
Parameters
Starting day
Ending day
Xo, mg/L
Y, mg/mg
1St
addition
of TCA
26.49
31.54
addition
of TCA
31.54
18.79a
95
8.54a
T, mg/mg
0.7
0.15
0.11
1.76
0.37
0.09
0.4
VL,L
0.1
b,day1
KS,BU,mg/L
kmax,But, mg/mg-day
KS,TCA,
mg/L
kmax,TCA, mg/mg-day
1st
addition
of TCA
26.49
31.54
addition
of TCA
31.54
18.79a
95
8.53a
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.7
0.2
0.11
1.76
0.37
0.09
0.4
0.7
0.2
0.11
1.76
0.37
0.09
0.4
0.1
0.1
0.057
38.05
0.55
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
FIcc,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
0.05 mg/mg-day
The 2t
The
km
0.09 mg/mg-day
The
The 2nd
km
1St
addition
of TCA
26.49
31.54
addition
of TCA
31.54
18.79a
95
8.55a
0.7
0.12 mg/mg-day
The 1st
The 2?
km
addition
of TCA
26.49
31.54
addition
of TCA
31.54
18.79a
95
8.55a
0.7
0.7
0.7
0.3
0.11
1.76
0.3
0.11
1.76
1.5
1.5
0.11
1.76
0.11
1.76
0.37
0.09
0.4
0.37
0.09
0.4
0.37
0.09
0.4
0.37
0.09
0.4
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
291
Section F: The first addition of 1,1,1-TCA on day 26
0.02
'O 016
BRC-2
kmax = 0 02
._._kmax=0.02I
kmax= 0.05
o.o12
'N
0.008
kmax = 0.09
kmax = 0.12
0.004
= 0.12
1
32
Time, day
Biomass (X) at starting of model simulation
(mgIL)
kinaX,TCA = 0.02
kmax,TCA = 0.05
kmax,TCA = 0.09
18.79
18.79
18.79
kmax,TCA
0.12
18.79
Section G: The second addition of 1,1,1 -TCA on day 31
0.2
BRC-2
D
U)
0.16
= 0.02
kmax=0.05
O2
kmax = 0.09
kmax=0.12
0.08
- 0.04
0
0
20
40
60
80
100
Time, day
kmax,TCA = 0.02
8.54
Biomass (X) at starting of model simulation
(mg/L)
0.09
0.05
kmaxTCA
kmax,TCA
8.53
8.55
kmax,TCA
0.12
8.55
Figure G.52 Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of 1,1,1-TCA maximum specific rates
Table G.41 Parameters for model simulations in Figures G.53
kmax
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
b, day'
kmax,But, mg/mg-day
KS,TCA, mg/L
kmaxTcA, mg/mg-day
T, mg/mg
VL,L
0.02 mg/mg-day
The
addition
of TCA
31.54
38.05
lS
The 2''
addition
of TCA
38.05
19.26a
95.1
7.11a
0.7
0.15
0.11
1.76
0.37
0.09
0.6
0.1
1st
addition
of TCA
31.54
38.05
addition
of TCA
38.05
95.1
0.09 mg/mg-day
The
The 2'
km
1st
addition
of TCA
31.54
38.05
addition
of TCA
38.05
0.12 mg/mg-day
km
1st
The
addition
of TCA
31.54
38.05
The 2'
addition
of TCA
38.05
95.1
19.26a
95.1
2.67a
0.7
0.7
0.7
o.o0V
0.7
0.3
0.11
1.76
0.3
0.11
1.76
1.5
0.11
1.76
0.11
1.76
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0,057
38.05
0.55
19.26a
5j3a
0.7
0.15
0.11
1.76
0.37
0.09
0.6
0.7
0.2
0.11
1.76
0.37
0.09
0.6
0.7
0.2
0.11
1.76
0.37
0.09
0.6
0.1
0.1
0.057
38.05
0.55
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
0.05 mg/mg-day
The 2'
The
kmax
19.26a
1.5
293
Section F: The first addition of 1,1,1-TCA on day 25
0.04
BRC-3
r
E 0.03
0.02
kmax
kmax = 0.02
.
U)
0.02
0.01
kmax= 0.12
kmax = 0.05
kmax = 0.09
kmax= 0.12
J
.
-,----,------
0
21
23
25
29
27
33
31
35
Time, day
Biomass (X) at starting of model simulation
(mg/L)
kmax,TCA = 0.02
kmax,TCA
19.56
0.05
kmax,TCA = 0.09
kmax,TCA = 0.12
19.56
19.56
19.56
Section G: The second addition of 1,1,1-TCA on day 32
BRC-3
0.2
= 0.02
E 0.16
kmax = 0.05
U)
U)
kmax= 0.09
0.12
0.08
1
0.04
0
0
40
20
60
80
100
120
Time, day
Biomass (X) at starting of model simulation
(mg/L)
kmax,TCA = 0.02
kmax,TCA = 0.05
kmax,TCA = 0.09
kmax,TCA = 0.12
5.42
5.47
5.52
5.53
Figure G.53 Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of 1,1,1-TCA maximum specific rates
Table G.42 Parameters for model simulations in Figures G.54
0.02 mg/mg-day
The 1st
The
addition
addition
of TCA
of TCA
31.54
38.05
38.05
95.1
kmax =
Parameters
Startingday
Endingday
Xo,mg/L
Y, mg/mg
19.26a
711a
T, mg/mg
0.7
0.15
0.11
1.76
0.37
0.09
0.6
0.7
0.15
0.11
1.76
0.37
0.09
0.6
VL,L
0.1
0.1
b, day'
kmax,But, mg/mg-day
mg/L
kmax,TCA, mg/mg-day
KS,TCA,
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
mg-L/mg-L
0.55
HCC,TCA,
Note a = Obtained from the previous simulation
Vg, L
= 0.05 mg/mg-day
The 1st
The 2fl"
addition
addition
of TCA
of TCA
31.54
38.05
95.1
38.05
5.13a
19.26a
0.7
0.7
0.2
0.2
km
=0.09 mg/mg-day
The 1st
The 2
addition
addition
of TCA
of TCA
31.54
38.05
38.05
95.1
km
km = 0.12 mg/mg-day
2id
lS
The
addition
of TCA
31.54
38.05
The
addition
of TCA
38.05
95.1
19.26a
2.67a
19.26a
o.00r'
0.7
0.7
0.7
0.7
0.3
0.11
1.76
0.3
0.11
1.76
1,5
0.11
1.76
1.5
0.11
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.11
1.76
0.37
0.09
0.6
0.11
1.76
0.37
1.76
295
Section F: The first addition of 1,1,1-TCA on day 31
002
BRC-4
E0.015
kmax = 0.02
kmax = 0.02
kmax = 0.05
kmax=0.09
kmax=0.12
N
0.01
U
0.005
kmax =0.12
J
0
28
30
32
34
36
38
40
Time, day
= 0.02
19.26
kmax,TCA
Biomass (X) at starting of model simulation
(mg/L)
kmax,TCA 0.05
kmax,TCA = 0.09
19.26
19.26
kmax,TCA
0.12
19.26
Section G: The second addition of 1,1,1 -TCA on day 38
0.2
E 0.16
0.12
BRC-4
----kmax =
0.02
kmax= 0.05
kmax = 0.09
kmax=0.12
::
Time, day
kmax,TCA
= 0.02
7.09
Biomass (X) at starting of model simulation
(mg/L)
kmax,TCA = 0.05
kmax,TCA = 0.09
7.11
7.10
= 0.12
7.11
kmax,TCA
Figure G.54 Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of 1,1,1-TCA maximum specific rates
Table G.43 Parameters for model simulations in Figures G.55
0.02 mg/mg-day
The 2t
The
kmax
Parameters
Starting day
Ending day
Xo,mg/L
Y, mg/mg
1st
addition
of TCA
26.49
31.54
18.79a
T, mg/mg
0.7
0.15
0.11
1.76
0.37
0.09
0.4
VL,L
0.1
b, day4
kmax,But, mg/mg-day
KS,TCA, mg/L
kmax,TCA, mg/mg-day
addition
of TCA
31.54
95
8.55a
0.7
0.15
0.11
1.76
0.37
0.09
0.4
0.1
0.057
0.057
38.05
38.05
0.55
0.55
ITIcc,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
Vg, L
Hcc,But, mg-L/mg-L
km
= 0.05 mg/mg-day
1st
The
addition
of TCA
26.49
31.54
The 2''
addition
of TCA
31.54
km
= 0.09 mg/mg-day
1st
2nd
The
addition
of TCA
26.49
31.54
The
addition
of TCA
31.54
18.79a
0.7
0.12 mg/mg-day
The
The 2nd
km
1st
addition
of TCA
31.54
95
addition
of TCA
26.49
31.54
400a
18.79a
0.7
0.024a
0.7
1.5
0.11
1.76
0.11
1.76
0.37
0.09
0.4
0.37
0.09
0.4
95
18.79a
95
6.64a
0.7
0.2
0.11
1.76
0.37
0.09
0.4
0.7
0.2
0.11
1.76
0.37
0.09
0.4
0.37
0.09
0.4
0.7
0.3
0.11
1.76
0.37
0.09
0.4
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.3
0.11
1.76
1.5
297
Section F: The first addition of 1,1,1-TCA on day 26
0.02
BRC-2
)0 016
Ks = 0.37
Ks = 2
Ks = 1
0.012
= 1.5
Ks=2
0.008
..o.0o4
Ks = a37
25
26
27
28
30
29
32
31
Time, day
Biomass (X) at starting of model simulation
(mgIL)
KS,TCA = 1
KS,TCA = 1.5
= 0.37
18.79
KS,TCA
=2
18.79
KS,TCA
18.79
18.79
Section G: The second addition of 1,1,1 -TCA on day 31
0.2
BRC-2
Ks = 0.37
0.16
E
Ks = I
U)
U' 012
---Ks = 1.5
- -
0.08
Ks = 2
- 0.04
0
0
20
40
60
80
100
Time, day
KS,TCA
= 0.37
8.55
Biomass (X) at starting of model simulation
(mg/L)
1
KS,TCA
KS,TCA = 1.5
8.54
8.53
KS,TCA
2
8.55
Figure G.55 Simulations of 1,1,1-TCA transformation for microcosm BRC-2
over a range of 1,1,1-TCA half-saturation constants
Table G.44 Parameters for model simulations in Figures G.56
0.02 mg/mg-day
The
The 2nd
kmax
Parameters
Starting day
Ending day
X0, mgIL
Y, mg/mg
1st
addition
of TCA
31.54
38.05
19.26a
addition
of TCA
38.05
95.1
7.1 la
0.05 mg/mg-day
The
The 2'
km
1st
addition
of TCA
31.54
38.05
19.26a
0.00la
0.7
0.7
1.5
0.11
1.76
0.11
1.76
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
T, mg/mg
0.37
0.09
0.6
VL,L
0.1
0.1
0.057
0.057
38.05
38.05
HCC,BUt, mg-L/mg-L
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a = Obtained from the previous simulation
addition
of TCA
38.05
0.1
0.11
1.76
Vg, L
addition
of TCA
31.54
38.05
0.37
0.09
0.6
0.11
1.76
mg/L
kmaxTCA, mg/mg-day
addition
of TCA
38.05
lS
0.7
0.3
0.11
1.76
0.37
0.09
0.6
0.7
0.2
KS,TCA,
addition
of TCA
31.54
38.05
0.12 mg/mg-day
The
The 2'
kmax =
0.7
0.2
0.11
1.76
0.37
0.09
0.6
0.7
0.15
KS,BU,mg/L
kmax,But, mg/mg-day
lS
95.1
2.67a
0.7
0.3
0.11
1.76
0.37
0.09
0.6
19.26a
0.7
0.15
0.11
1.76
0.37
0.09
0.6
b, day'
addition
of TCA
38.05
0.09 mg/mg-day
The
The 2nd
km
95.1
5.13a
19.26a
95.1
1.5
Section F: The first addition of 1,l,1-TCA on day 25
0.04
a,
E 0.03
U,
0.02
0.01
0 422
24
26
28
Time, day
32
30
34
Biomass (X) at starting of model simulation
(mg/L)
= 0.37
19.56
KS,TCA
KS,TCA
1
KS,TCA
=2
19.56
1.5
KS,TCA
19.56
19.56
Section 0: The second addition of 1,1,1-TCA on day 32
BRC-3
a,
E 0.16
Ks = 0.37
Ks = 1
U,
U,
Ks = 1.5
0.12
Ks=2
0.08
.
'- 0.04
0
0
= 0.37
5.52
KS,TCA
20
40
60
Time, day
80
100
Biomass (X) at starting of model simulation
(mg/L)
KS,TCA = 1
KS,TCA = 1.5
5.45
5.43
120
=2
5.44
KS,TCA
Figure G.56 Simulations of 1,1,1-TCA transformation for microcosm BRC-3
over a range of 1,1,1-TCA half-saturation constants
Table G.45 Parameters for model simulations in Figures G.57
0.02 mg/mg-day
The
The 2nid
kmax =
Parameters
Starting day
Ending day
Xo,mg!L
1st
addition
of TCA
31.54
38.05
addition
of TCA
38.05
19.26a
95.1
7.11a
0.7
0.15
= 0.05 mg/mg-day
The 1st
The 2''
km
addition
of TCA
31.54
38.05
addition
of TCA
38.05
19.26a
95.1
5.13a
0.7
0.15
0.7
0.2
0.7
0.2
0.11
1.76
0.11
1.76
0.11
1.76
T, mg/mg
0.37
0.09
0.6
0.37
0.09
0.6
VL,L
0.1
0.1
Y,mglmg
b, day'
KS,BUt, mg/L
kmax,But, mg/mg-day
KS,TCA,
mg!L
kmaxTCA, mg/mg-day
0.057
0.057
38.05
38.05
0.55
0.55
HCC,TCA, mg-L/mg-L
Note a Obtained from the previous simulation
Vg, L
HCC,BUt, mg-L/mg-L
km
= 0.09 mg/mg-day
The
addition
of TCA
31.54
38.05
lS
2nd
The
addition
of TCA
38.05
I
k
0.12 mg/mg-day
The
The 2'
lS
addition
of TCA
31.54
38.05
addition
of TCA
38.05
19.26a
0.00la
19.26a
95.1
2.67a
0.7
0.7
0.7
0.7
0.11
1.76
0.3
0.11
1.76
0.3
0.11
1.76
1.5
0.11
1.76
1.5
0.11
1.76
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.37
0.09
0.6
0.1
0.1
0.1
0.1
0.1
0.1
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0,55
0.057
38.05
0.55
0.057
38.05
0.55
0.057
38.05
0.55
95.1
301
Section F: The first addition of 1,1,1-TCA on day 31
0.02-,-
BRC-4
C)
Ks = 0.37
E 0.015
Ks = 2
Ks=1
U)
Ks = 1.5
0.01
Ks= 2
?-0.005
Ks = 0.37
0
28
30
32
34
Time, day
36
40
38
Biomass (X) at starting of model simulation
(mg/L)
KS,TCA = 0.37
KSTCA
19.26
1
19.26
KS,TCA = 1.5
KS,TCA =2
19.26
19.26
Section G: The second addition of 1,1,1-TCA on day 38
BRC-4
Ks = 0.37
C)
E 0.16
Ks = I
U)
U,
------Ks = 1.5
0.12
Ks = 2
0
'
0.08
,Iii
4
0
0
20
80
40
60
Time, day
100
Biomass (X) at starting of model simulation
(mgIL)
KS.TCA = 0.37
KSTCA = 1
KS,TCA = 1.5
KS,TCA = 2
7.11
7.09
7.09
7.09
Figure G.57 Simulations of 1,1,1-TCA transformation for microcosm BRC-4
over a range of 1,1,1-TCA half-saturation constants