BIOFUEL POTENTIAL, NITROGEN UTILIZATION, AND GROWTH RATES OF
TWO GREEN ALGAE ISOLATED FROM A WASTEWATER TREATMENT
FACILITY
by
Everett O'Brien Eustance
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Chemical Engineering
MONTANA STATE UNIVERSITY
Bozeman, Montana
July 2011
©COPYRIGHT
by
Everett O'Brien Eustance
2011
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Everett O'Brien Eustance
This thesis has been read by each member of the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citation, bibliographic
style, and consistency and is ready for submission to The Graduate School.
Brent Peyton
Approved for the Department of Chemical and Biological Engineering
Ron Larsen
Approved for The Graduate School
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a master’s
degree at Montana State University, I agree that the Library shall make it available to
borrowers under rules of the Library.
If I have indicated my intention to copyright this thesis by including a copyright
notice page, copying is allowable only for scholarly purposes, consistent with “fair use”
as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation
from or reproduction of this thesis in whole or in parts may be granted only by the
copyright holder.
Everett O'Brien Eustance
July 2011
iv
DEDICATION
This thesis is dedicated to my wife who provided support during the research and
writing of this thesis. This thesis is also dedicated to my family who endowed me with a
strong work ethic, and whose personal sacrifices allowed me the chance to follow my
dreams.
v
ACKNOWLEDGEMENTS
Special thanks to Rob Gardner, who initially trained me and constantly provided
feedback and insight into the scientific method.
Special thanks to my advisor Brent Peyton for the opportunity to further my
education while doing research I love.
Special thanks to my advisor Robin Gerlach who provided analytical support and
guidance when things were not working.
Special thanks to Adie Phillips for training on IC and HPLC for nitrogen analysis.
Special thanks to Lauren Franco, Tiza Bell, and Karen Moll for 18S analysis on
Scenedesmus sp. 131 and Kirchneriella sp. 92.
Special thanks to Joseph Menicucci who initially provided me with a job that
allowed me to cultivate my love for algal research.
Special thanks to CTW energy and Gary Chilcott for the opportunity to initially
learn about algae.
Special thanks to the Montana Board of Research and Commercialization
Technology for their funding support.
Special thanks to Deer Lodge, MT where the two strains were isolated and the
reason MBRCT provided funding.
Special thanks to the people in the Algal Biofuels Group, who provided constant
interaction and discussion on various aspects of algae research.
Special thanks to Keith Cooksey who started the Algal Biofuels Group.
Special thanks to the Center for Biofilm Engineering for instrument support.
vi
TABLE OF CONTENTS
1. INTRODUCTION ......................................................................................................... 1
Wastewater ..................................................................................................................... 1
Nutrient Requirements .................................................................................................... 2
Lipid Production ............................................................................................................. 4
2. BACKGROUND ........................................................................................................... 6
Wastewater Treatment .................................................................................................... 6
Nitrogen and Phosphorus Remediation Using Algae ................................................. 6
Biofuel Production from Wastewater Derived Algae ................................................. 8
Carbon - Nitrogen Metabolism ....................................................................................... 9
Carbon ......................................................................................................................... 9
Nitrogen .................................................................................................................... 13
Nitrate ....................................................................................................................... 13
Nitrate Uptake in the Presence of Low CO2 and its pH Effect ................................. 14
Ammonium ............................................................................................................... 15
Effects of Low Carbon with Ammonium on Growth ............................................... 17
Free Ammonia Inhibition.......................................................................................... 17
Interaction of Ammonium with Other Nitrogen Sources ......................................... 19
Summary ....................................................................................................................... 19
3. METHODS .................................................................................................................. 21
Organism Isolation and Culture .................................................................................... 21
Culture Identification .................................................................................................... 22
Experimental Conditions .............................................................................................. 23
Media ........................................................................................................................ 23
Experimental System ................................................................................................ 23
Analysis ........................................................................................................................ 24
pH and Ion Chromatography .................................................................................... 24
Nile Red Staining Protocol ....................................................................................... 26
Biofuel Potential Measured by Gas Chromatography .............................................. 27
Cell Concentrations and Harvesting ............................................................................. 28
Kinetics ......................................................................................................................... 29
Growth Rate .............................................................................................................. 29
vii
TABLE OF CONTENTS - CONTINUED
4. OPTIMIZATION OF GROWTH ON AMMONIUM AND
COMPARISON TO GROWTH ON NITRATE OR UREA ....................................... 31
Comparison of Growth on Different Nitrogen Sources with 5% CO2 ......................... 31
Growth on Air with on Nitrate or Ammonium ............................................................. 35
Growth on Ammonium with Biological Buffers .......................................................... 40
Growth on Ammonium using pH Controllers .............................................................. 46
Analysis of Culture Conditions for Optimal Nitrogen Removal .................................. 49
5. BICARBONATE INJECTION .................................................................................... 55
6. COMPARISON OF SCENEDESMUS SP. 131 AND
KIRCHNERIELLA SP. 92 .......................................................................................... 58
7. CONCLUSIONS.......................................................................................................... 64
8. SUGGESTIONS FOR FUTURE WORK .................................................................... 66
APPENDICES .................................................................................................................. 76
APPENDIX A: Organism Selection ............................................................................. 77
APPENDIX B: Experimental Data ............................................................................... 86
APPENDIX C: HEPES Buffer Interaction on IC ....................................................... 154
APPENDIX D: Bold's Basal Medium ........................................................................ 161
APPENDIX E: Detailed Methods .............................................................................. 163
viii
LIST OF TABLES
Table
Page
1.
Eluent Gradient for HPLC and Urea Analysis. ................................................ 25
2.
Comparison of Nitrogen Source When Grown on 5% CO2.
Reported values include standard deviation of experiments in
triplicate. ........................................................................................................... 35
3.
Comparison of Nitrate or Ammonium Grown on Air. Reported
values include standard deviation of experiments in triplicate. ....................... 39
4.
Comparison of Growth on Ammonium with Biological Buffers.
Reported values include standard deviation of experiments in
triplicate. ........................................................................................................... 45
5.
Comparison of Growth on Ammonium with 5% CO2 Using pH
Control. Reported values include standard deviation of
experiments in duplicate. .................................................................................. 49
6.
Comparison of Time to Remove Nitrogen from Media. .................................. 52
7.
Nitrogen Yield Comparison for Experiments. Reported values
include standard deviation of experiments in triplicate. ................................... 53
8.
Bicarbonate Injection. Reported values include standard
deviation of experiments in triplicate. .............................................................. 57
9.
Comparison of Biofuel Potential Concentrations. Reported with
standard deviation of the biological reactor replicates. .................................... 62
10.
Estimated Biodiesel Productivity for Industrial-Scale Growth. ........................ 63
A.1. Comparison of Initial Isolates. ............................................................................ 78
B.1.
Cell Concentration for Strain 131 on Nitrate and 5% CO2
(cells/mL). ........................................................................................................ 87
B.2.
pH for Strain 131 on Nitrate and 5% CO2. ....................................................... 87
B.3.
Total Nile Red Fluorescence for Strain 131 on Nitrate and 5%
CO2. .................................................................................................................. 87
ix
LIST OF TABLES - CONTINUED
Table
Page
B.4.
Specific Nile Red Fluorescence for Strain 131 on Nitrate and
5% CO2. ............................................................................................................ 88
B.5.
Nitrate Concentration for Strain 131 on Nitrate and 5% CO2
(mg/L). .............................................................................................................. 88
B.6.
Dry Cell Weight for Strain 131 on Nitrate and 5% CO2 (g/L). ........................ 88
B.7.
% Biofuel Potential for Strain 131 on Nitrate and 5% CO2. ............................ 88
B.8.
Cell Concentration for Strain 92 on Nitrate and 5% CO2
(cells/mL). ........................................................................................................ 89
B.9.
pH for Strain 92 on Nitrate and 5% CO2. ......................................................... 89
B.10. Total Nile Red Fluorescence for Strain 92 on Nitrate and 5%
CO2. .................................................................................................................. 90
B.11. Specific Nile Red Fluorescence for Strain 92 on Nitrate and 5%
CO2. .................................................................................................................. 90
B.12. Nitrate Concentration for Strain 92 on Nitrate and 5% CO2
(mg/L). .............................................................................................................. 90
B.13. Dry Cell Weight for Strain 92 on Nitrate and 5% CO2 (g/L). .......................... 91
B.14. % Biofuel Potential for Strain 92 on Nitrate and 5% CO2. .............................. 91
B.15. Cell Concentration for Strain 131 on Nitrate and Air (cells/mL). .................... 92
B.16. pH for Strain 131 on Nitrate and Air. ............................................................... 92
B.17. Total Nile Red Fluorescence for Strain 131 on Nitrate and Air. ...................... 93
B.18. Specific Nile Red Fluorescence for Strain 131 on Nitrate and
Air. .................................................................................................................... 93
B.19. Nitrate Concentration for Strain 131 on Nitrate and Air (mg/L)...................... 94
B.20. Dry Cell Weight for Strain 131 on Nitrate and Air (g/L). ................................ 94
B.21. % Biofuel Potential for Strain 131 on Nitrate and Air. .................................... 94
x
LIST OF TABLES - CONTINUED
Table
Page
B.22. Cell Concentration for Strain 92 on Nitrate and Air (cells/mL). ...................... 95
B.23. pH for Strain 92 on Nitrate and Air. ................................................................. 95
B.24. Total Nile Red Fluorescence for Strain 92 on Nitrate and Air. ........................ 96
B.25. Specific Nile Red Fluorescence for Strain 92 on Nitrate and Air. ................... 96
B.26. Nitrate Concentration for Strain 92 on Nitrate and Air (mg/L)........................ 97
B.27. Dry Cell Weight for Strain 92 on Nitrate and Air (g/L). .................................. 97
B.28. % Biofuel Potential for Strain 92 on Nitrate and Air. ...................................... 97
B.29. Cell Concentration for Strain 131 on Nitrate, 5% CO2 and
Bicarbonate Injection (cells/mL). ..................................................................... 98
B.30. pH for Strain 131 on Nitrate, 5% CO2 and Bicarbonate
Injection. ........................................................................................................... 98
B.31. Total Nile Red Fluorescence for Strain 131 on Nitrate, 5% CO2
and Bicarbonate Injection. ................................................................................ 99
B.32. Specific Nile Red Fluorescence for Strain 131 on Nitrate, 5%
CO2 and Bicarbonate Injection. ........................................................................ 99
B.33. Nitrate Concentration for Strain 131 on Nitrate, 5% CO2 and
Bicarbonate Injection (mg/L). ........................................................................ 100
B.34. Dry Cell Weight for Strain 131 on Nitrate, 5% CO2 and
Bicarbonate Injection (g/L). ........................................................................... 100
B.35. % Biofuel Potential for Strain 131 on Nitrate, 5% CO2 and
Bicarbonate Injection. .................................................................................... 100
B.36. Cell Concentration for Strain 92 on Nitrate, 5% CO2 and
Bicarbonate Injection (cells/mL). ................................................................... 101
B.37. pH for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection. .................. 101
B.38. Total Nile Red Fluorescence for Strain 92 on Nitrate, 5% CO2
and Bicarbonate Injection. .............................................................................. 102
xi
LIST OF TABLES - CONTINUED
Table
Page
B.39. Specific Nile Red Fluorescence for Strain 92 on Nitrate, 5%
CO2 and Bicarbonate Injection. ...................................................................... 102
B.40. Nitrate Concentration for Strain 92 on Nitrate, 5% CO2 and
Bicarbonate Injection (mg/L). ........................................................................ 103
B.41. Dry Cell Weight for Strain 92 on Nitrate, 5% CO2 and
Bicarbonate Injection (g/L). ........................................................................... 103
B.42. % Biofuel Potential for Strain 92 on Nitrate, 5% CO2 and
Bicarbonate Injection. .................................................................................... 103
B.43. Cell Concentration for Strain 131 on Urea and 5% CO2
(cells/mL). ...................................................................................................... 104
B.44. pH for Strain 131 on Urea and 5% CO2. ........................................................ 104
B.45. Total Nile Red Fluorescence for Strain 131 on Urea and 5%
CO2. ................................................................................................................ 105
B.46. Specific Nile Red Fluorescence for Strain 131 on Urea and 5%
CO2. ................................................................................................................ 105
B.47. Urea Concentration for Strain 131 on Urea and 5% CO2 (mg/L). ................. 105
B.48. Dry Cell Weight for Strain 131 on Urea and 5% CO2 (g/L). ......................... 106
B.49. % Biofuel Potential for Strain 131 on Urea and 5% CO2............................... 106
B.50. Cell Concentration for Strain 92 on Urea and 5% CO2
(cells/mL). ...................................................................................................... 107
B.51. pH for Strain 92 on Urea and 5% CO2. .......................................................... 107
B.52. Total Nile Red Fluorescence for Strain 92 on Urea and 5% CO2. ................. 107
B.53. Specific Nile Red Fluorescence for Strain 92 on Urea and 5%
CO2. ................................................................................................................ 108
B.54. Urea Concentration for Strain 92 on Urea and 5% CO2 (mg/L). ................... 108
B.55. Dry Cell Weight for Strain 92 on Urea and 5% CO2 (g/L). ........................... 108
xii
LIST OF TABLES - CONTINUED
Table
Page
B.56. % Biofuel Potential for Strain 92 on Urea and 5% CO2................................. 108
B.57. Cell Concentration for Strain 131 on NH4+, 5% CO2 and No
Buffer (cells/mL). ........................................................................................... 109
B.58. pH for Strain 131 on NH4+, 5% CO2 and No Buffer. ..................................... 109
B.59. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2
and No Buffer ................................................................................................. 109
B.60. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5%
CO2 and No Buffer. ........................................................................................ 110
B.61. Ammonium Concentration for Strain 131 on NH4+, 5% CO2 and
No Buffer (mg/L). .......................................................................................... 110
B.62. Dry Cell Weight for Strain 131 on NH4+, 5% CO2 and No
Buffer (g/L). ................................................................................................... 110
B.63. % Biofuel Potential for Strain 131 on NH4+, 5% CO2 and No
Buffer.............................................................................................................. 110
B.64. Cell Concentration for Strain 92 on NH4+, 5% CO2 and No
Buffer (cells/mL). ........................................................................................... 111
B.65. pH for Strain 92 on NH4+, 5% CO2 and No Buffer. ....................................... 111
B.66. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2
and No Buffer ................................................................................................. 111
B.67. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2
and No Buffer. ................................................................................................ 112
B.68. Ammonium Concentration for Strain 92 on NH4+, 5% CO2 and
No Buffer (mg/L). .......................................................................................... 112
B.69. Dry Cell Weight for Strain 92 on NH4+, 5% CO2 and No Buffer
(g/L). ............................................................................................................... 112
B.70. % Biofuel Potential for Strain 92 on NH4+, 5% CO2 and No
Buffer.............................................................................................................. 112
xiii
LIST OF TABLES - CONTINUED
Table
Page
B.71. Cell Concentration for Strain 131 on NH4+, Air and No Buffer
(cells/mL). ...................................................................................................... 113
B.72. pH for Strain 131 on NH4+, Air and No Buffer. ............................................. 113
B.73. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and
No Buffer. ....................................................................................................... 114
B.74. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and
No Buffer. ....................................................................................................... 114
B.75. Ammonium Concentration for Strain 131 on NH4+, Air and No
Buffer (mg/L). ................................................................................................ 115
B.76. Dry Cell Weight for Strain 131 on NH4+, Air and No Buffer
(g/L). ............................................................................................................... 115
B.77. % Biofuel Potential for Strain 131 on NH4+, Air and No Buffer. .................... 115
B.78. Cell Concentration for Strain 92 on NH4+, Air and No Buffer
(cells/mL). ...................................................................................................... 116
B.79. pH for Strain 92 on NH4+, Air and No Buffer. ............................................... 116
B.80. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and No
Buffer.............................................................................................................. 117
B.81. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and
No Buffer. ....................................................................................................... 117
B.82. Ammonium Concentration for Strain 92 on NH4+, Air and No
Buffer (mg/L). ................................................................................................ 118
B.83. Dry Cell Weight for Strain 92 on NH4+, Air and No Buffer
(g/L). ............................................................................................................... 118
B.84. % Biofuel Potential for Strain 92 on NH4+, Air and No Buffer. .................... 118
B.85. Cell Concentration for Strain 131 on NH4+, 5% CO2 and PIPES
Buffer (cells/mL). ........................................................................................... 119
xiv
LIST OF TABLES - CONTINUED
Table
Page
B.86. pH for Strain 131 on NH4+, 5% CO2 and PIPES Buffer. ............................... 119
B.87. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2
and PIPES Buffer. .......................................................................................... 120
B.88. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5%
CO2 and PIPES Buffer. .................................................................................. 120
B.89. Ammonium Concentration for Strain 131 on NH4+, 5% CO2 and
PIPES Buffer (mg/L). ..................................................................................... 120
B.90. Dry Cell Weight for Strain 131 on NH4+, 5% CO2 and PIPES
Buffer (g/L). ................................................................................................... 121
B.91. % Biofuel Potential for Strain 131 on NH4+, 5% CO2 and PIPES
Buffer.............................................................................................................. 121
B.92. Cell Concentration for Strain 92 on NH4+, 5% CO2 and PIPES
Buffer (cells/mL). ........................................................................................... 122
B.93. pH for Strain 92 on NH4+, 5% CO2 and PIPES Buffer. ................................. 122
B.94. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2
and PIPES Buffer. .......................................................................................... 122
B.95. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2
and PIPES Buffer. .......................................................................................... 123
B.96. Ammonium Concentration for Strain 92 on NH4+, 5% CO2 and
PIPES Buffer (mg/L). ..................................................................................... 123
B.97. Dry Cell Weight for Strain 92 on NH4+, 5% CO2 and PIPES
Buffer (g/L). ................................................................................................... 123
B.98. % Biofuel Potential for Strain 92 on NH4+, 5% CO2 and PIPES
Buffer.............................................................................................................. 123
B.99. Cell Concentration for Strain 131 on NH4+, 5% CO2, PIPES
Buffer and 2 mM KOH (cells/mL). ................................................................ 124
xv
LIST OF TABLES - CONTINUED
Table
Page
B.100. pH for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH. .............................................................................................................. 124
B.101. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2,
PIPES Buffer, and 2 mM KOH. ..................................................................... 125
B.102. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5%
CO2, PIPES Buffer, and 2 mM KOH. ............................................................ 125
B.103. Ammonium Concentration for Strain 131 on NH4+, 5% CO2,
PIPES Buffer, and 2 mM KOH (mg/L). ......................................................... 126
B.104. Dry Cell Weight for Strain 131 on NH4+, 5% CO2, PIPES
Buffer, and 2 mM KOH (g/L). ....................................................................... 126
B.105. % Biofuel Potential for Strain 131 on NH4+, 5% CO2, PIPES
Buffer, and 2 mM KOH. ................................................................................ 126
B.106. Cell Concentration for Strain 92 on NH4+, 5% CO2, PIPES
Buffer, and 2 mM KOH (cells/mL). ............................................................... 127
B.107. pH for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH. .............................................................................................................. 127
B.108. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2,
PIPES Buffer, and 2 mM KOH. ..................................................................... 128
B.109. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2,
PIPES Buffer, and 2 mM KOH. ..................................................................... 128
B.110. Ammonium Concentration for Strain 92 on NH4+, 5% CO2,
PIPES Buffer, and 2 mM KOH(mg/L). .......................................................... 129
B.111. Dry Cell Weight for Strain 92 on NH4+, 5% CO2, PIPES Buffer,
and 2 mM KOH (g/L). .................................................................................... 129
B.112. % Biofuel Potential for Strain 92 on NH4+, 5% CO2, PIPES
Buffer, and 2 mM KOH. ................................................................................ 129
B.113. Cell Concentration for Strain 131 on NH4+, Air and HEPES
Buffer (cells/mL). ........................................................................................... 130
xvi
LIST OF TABLES - CONTINUED
Table
Page
B.114. pH for Strain 131 on NH4+, Air and HEPES Buffer....................................... 130
B.115. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and
HEPES Buffer. ............................................................................................... 131
B.116. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and
HEPES Buffer. ............................................................................................... 131
B.117. Ammonium Concentration for Strain 131 on NH4+, Air and
HEPES Buffer (mg/L). ................................................................................... 132
B.118. Dry Cell Weight for Strain 131 on NH4+, Air and HEPES Buffer
(g/L). ............................................................................................................... 132
B.119. % Biofuel Potential for Strain 131 on NH4+, Air and HEPES
Buffer.............................................................................................................. 132
B.120. Cell Concentration for Strain 92 on NH4+, Air and HEPES
Buffer (cells/mL). ........................................................................................... 133
B.121. pH for Strain 92 on NH4+, Air and HEPES Buffer......................................... 133
B.122. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and
HEPES Buffer. ............................................................................................... 134
B.123. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and
HEPES Buffer. ............................................................................................... 134
B.124. Ammonium Concentration for Strain 92 on NH4+, Air and
HEPES Buffer (mg/L). ................................................................................... 135
B.125. Dry Cell Weight for Strain 92 on NH4+, Air and HEPES Buffer
(g/L). ............................................................................................................... 135
B.126. % Biofuel Potential for Strain 92 on NH4+, Air and HEPES
Buffer.............................................................................................................. 135
B.127. Cell Concentration for Strain 131 on NH4+, Air and PIPES
Buffer (cells/mL). ........................................................................................... 136
B.128. pH for Strain 131 on NH4+, Air and PIPES Buffer. ....................................... 136
xvii
LIST OF TABLES - CONTINUED
Table
Page
B.129. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and
PIPES Buffer. ................................................................................................. 137
B.130. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and
PIPES Buffer. ................................................................................................. 137
B.131. Ammonium Concentration for Strain 131 on NH4+, Air and
PIPES Buffer (mg/L). ..................................................................................... 138
B.132. Dry Cell Weight for Strain 131 on NH4+, Air and PIPES Buffer
(g/L). ............................................................................................................... 138
B.133. % Biofuel Potential for Strain 131 on NH4+, Air and PIPES
Buffer.............................................................................................................. 138
B.134. Cell Concentration for Strain 92 on NH4+, Air and PIPES Buffer
(cells/mL). ...................................................................................................... 139
B.135. pH for Strain 92 on NH4+, Air and PIPES Buffer. ......................................... 139
B.136. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and
PIPES Buffer. ................................................................................................. 140
B.137. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and
PIPES Buffer. ................................................................................................. 140
B.138. Ammonium Concentration for Strain 92 on NH4+, Air and
PIPES Buffer (mg/L). ..................................................................................... 141
B.139. Dry Cell Weight for Strain 92 on NH4+, Air and PIPES Buffer
(g/L). ............................................................................................................... 141
B.140. % Biofuel Potential for Strain 92 on NH4+, Air and PIPES
Buffer.............................................................................................................. 141
B.141. Cell Concentration for Strain 131 on NH4+ and 5% CO2 Using
pH Controller Run 1 (cells/mL). .................................................................... 142
xviii
LIST OF TABLES - CONTINUED
Table
Page
B.142. pH for Strain 131 on NH4+ and 5% CO2 Using pH Controller
Run 1. ............................................................................................................. 143
B.143. Total Nile Red Fluorescence for Strain 131 on NH4+ and 5%
CO2 Using pH Controller Run 1..................................................................... 143
B.144. Specific Nile Red Fluorescence for Strain 131 on NH4+ and 5%
CO2 Using pH Controller Run 1..................................................................... 144
B.145. Ammonium Concentration for Strain 131 on NH4+ and 5% CO2
Using pH Controller Run 1 (mg/L). ............................................................... 144
B.146. Dry Cell Weight for Strain 131 on NH4+ and 5% CO2 Using pH
Controller Run 1 (g/L). ................................................................................... 144
B.147. % Biofuel Potential for Strain 131 on NH4+ and 5% CO2 Using
pH Controller Run 1. ...................................................................................... 144
B.148. Cell Concentration for Strain 131 on NH4+ and 5% CO2 Using
pH Controller Run 2 (cells/mL). .................................................................... 145
B.149. pH for Strain 131 on NH4+ and 5% CO2 Using pH Controller
Run 2. ............................................................................................................. 145
B.150. Total Nile Red Fluorescence for Strain 131 on NH4+ and 5%
CO2 Using pH Controller Run 2..................................................................... 146
B.151. Specific Nile Red Fluorescence for Strain 131 on NH4+ and 5%
CO2 Using pH Controller Run 2..................................................................... 146
B.152. Ammonium Concentration for Strain 131 on NH4+ and 5% CO2
Using pH Controller Run 2 (mg/L). ............................................................... 147
B.153. Dry Cell Weight for Strain 131 on NH4+ and 5% CO2 Using pH
Controller Run 2 (g/L). ................................................................................... 147
B.154. % Biofuel Potential for Strain 131 on NH4+ and 5% CO2 Using
pH Controller Run 2. ...................................................................................... 147
B.155. Cell Concentration for Strain 92 on NH4+ and 5% CO2 Using
pH Controller Run 1 (cells/mL). .................................................................... 148
xix
LIST OF TABLES - CONTINUED
Table
Page
B.156. pH for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run
1. ..................................................................................................................... 148
B.157. Total Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2
Using pH Controller Run 1. ........................................................................... 149
B.158. Specific Nile Red Fluorescence for Strain 92 on NH4+ and 5%
CO2 Using pH Controller Run 1..................................................................... 149
B.159. Ammonium Concentration for Strain 92 on NH4+ and 5% CO2
Using pH Controller Run 1 (mg/L). ............................................................... 150
B.160. Dry Cell Weight for Strain 92 on NH4+ and 5% CO2 Using pH
Controller Run 1 (g/L). ................................................................................... 150
B.161. % Biofuel Potential for Strain 92 on NH4+ and 5% CO2 Using
pH Controller Run 1. ...................................................................................... 150
B.162. Cell Concentration for Strain 92 on NH4+ and 5% CO2 Using
pH Controller Run 2 (cells/mL). .................................................................... 151
B.163. pH for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run
2. ..................................................................................................................... 151
B.164. Total Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2
Using pH Controller Run 2. ........................................................................... 152
B.165. Specific Nile Red Fluorescence for Strain 92 on NH4+ and 5%
CO2 Using pH Controller Run 2..................................................................... 152
B.166. Ammonium Concentration for Strain 92 on NH4+ and 5% CO2
Using pH Controller Run 2 (mg/L). ............................................................... 153
B.167. Dry Cell Weight for Strain 92 on NH4+ and 5% CO2 Using pH
Controller Run 2 (g/L). ................................................................................... 153
B.168. % Biofuel Potential for Strain 92 on NH4+ and 5% CO2 Using
pH Controller Run 2. ...................................................................................... 153
C.1.
Data from Day 4-12-2011 Analyzing HEPES and Ammonium
Interaction. ...................................................................................................... 158
xx
LIST OF TABLES - CONTINUED
Table
Page
C.2.
Comparison of Nanopure to Calculated Value of Ammonium
from 4-12-2011............................................................................................... 158
C.3.
Data from Day 4-13-2011 Analyzing HEPES and Ammonium
Interaction. ...................................................................................................... 159
C.4.
Comparison of Nanopure to Calculated Value of Ammonium
from 4-13-2011............................................................................................... 159
E.1.
Eluent Gradient for HPLC and Urea Analysis. .............................................. 172
xxi
LIST OF FIGURES
Figure
Page
1.
Light micrograph of Scenedesmus sp. 131, and a fluorescence
micrograph stained with Nile Red. ................................................................... 22
2.
Light micrograph of Kirchneriella sp. 92, and a fluorescence
micrograph stained with Nile Red. ................................................................... 22
3.
Total Nile Red fluorescence (solid line) and nitrogen
concentration (dashed line) (a), pH (b) and average cell density
(c) with standard deviation for triplicate reactors (1)
Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on 5%
CO2 using nitrate (□) ammonium (▲) or urea (♦) as the nitrogen
source................................................................................................................ 32
4.
Micrographs for Kirchneriella sp. 92 (top row) and
Scenedesmus sp. 131 (bottome row) grown on ammonium with
5% CO2 over consecutive sampling time points showing the
decrease in chlorophyll content. ....................................................................... 34
5.
Total Nile Red fluorescence (solid line) and nitrogen
concentration (dashed line) (a), pH (b) and average cell density
(c) with standard deviation for triplicate reactors (1)
Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on air
using nitrate (■) or ammonium () as the nitrogen source. ............................ 36
6.
Total Nile Red fluorescence (solid line) and nitrogen
concentration ( dashed line) (a), pH (b) and average cell density
(c) with standard deviation for triplicate reactors (1)
Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on
ammonium as the nitrogen source and growing on air buffered
with 8 mM HEPES (●) growing on air with PIPES. (○) growing
on CO2 with PIPES (■) and growing on CO2 with PIPES plus 2
mM KOH injection (final concentration) (□). .................................................. 42
7.
Total Nile Red fluorescence (a), pH (b) and average cell density
(c) with standard deviation for duplicate reactors (1)
Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on
ammonium with pH control run 1 (■) run 2 (○). .............................................. 47
xxii
LIST OF FIGURES
Figure
Page
8.
Average nitrogen concentration in mM nitrogen with standard
deviation for (1) Scenedesmus sp. 131 and (2) Kirchneriella sp.
92 grown on CO2 (a) using nitrate (□), urea (♦), unbuffered
ammonium (▲), bicarbonate addition (○), ammonium with
PIPES (▼), ammonium with PIPES plus 2 mM KOH spike ()
and ammonium with pH controllers run 1 () run 2 () . The
strains were also grown on air (b) with nitrate (■), unbuffered
ammonium (), ammonium with HEPES (►), and ammonium
with PIPES (◄). ............................................................................................... 51
9.
Total Nile Red fluorescence (a), pH (b) and average cell density
(c) with standard deviation for (1) Scenedesmus sp. 131 and (2)
Kirchneriella sp. 92 grown on nitrate and utilizing the
bicarbonate addition grown on 5% CO2 and switched to air (○)
growing on 5% CO2 (□) and growing on air (■). (↓) Represents
the point at which 50 mM sodium bicarbonate (final
concentration) was injected into the Scenedesmus media................................ 56
10.
Micrographs of strain 92 growing a) on nitrate with air b) and c)
on ammonium buffered with PIPES on 5% CO2. d) is a
fluorescence micrograph of c) stained with Nile Red. ..................................... 58
11.
Micrographs of strain 131 growing a) on ammonium buffered
with PIPES on 5% CO2, b) on nitrate with air, and c) is a
fluorescence micrograph of b) stained with Nile Red. ..................................... 59
12.
Total Nile Red fluorescence (a), pH (b) and average cell density
(c) with standard deviation for triplicate reactors (○)
Scenedesmus sp. 131 grown on nitrate as the nitrogen source
and utilizing the bicarbonate addition and (■) Kirchneriella sp.
92 grown on ammonium with PIPES on 5% CO2. (↓)
Represents the point at which 50 mM sodium bicarbonate (final
concentration) was injected into the Scenedesmus media. ............................... 61
A.1
Micrograph of strain 123 as a single cell and as a pair grown on
nitrate and 5% CO2. .......................................................................................... 79
A.2
Average growth curve for strain 123 grown in triplicate on 5%
CO2 and nitrate. ................................................................................................ 80
A.3
Micrograph of strain 131 growing in a 4 cell grouping. ................................... 80
xxiii
LIST OF FIGURES
Figure
Page
A.4
Average growth curve for strain 131 grown in triplicate on 5%
CO2 and nitrate. ................................................................................................ 81
A.5
Micrograph of strain 111 growing in a 4 cell grouping. ................................... 81
A.6
Average growth curve for strain 111 grown in duplicate on 5%
CO2 and nitrate. ................................................................................................ 82
A.7
Micrograph of strain 112 growing in 4 cell groupings with large
spines and visible lipid vacuoles. ..................................................................... 83
A.8
Average growth curve for strain 112 grown in duplicate on 5%
CO2 and nitrate. ................................................................................................ 83
A.9
Micrograph of strain 71T growing in a pair on 5% CO2 and
nitrate. ............................................................................................................... 84
A.10 Average growth curve for strain 71T grown in triplicate on 5%
CO2 and nitrate. ................................................................................................ 84
A.11 Micrograph of strain 92 growing on 5% C02 and nitrate. ................................. 85
A.12 Average growth curve for strain 92 grown in triplicate on 5%
CO2 and nitrate. ................................................................................................ 85
C.1
Bolds Basal Medium with 50 mg/L of NH4+, which shows good
peak separation. Ammonium peak is at approximately 6
minutes. .......................................................................................................... 156
C. 2
This is the same chromatogram as Figure C.1 zoomed in for a
better view of the separation between the sodium peak (5.3
minutes) and the ammonium peak (6 minutes). ............................................. 156
C.3
Chromatogram of HEPES buffer in nanopure water showing a
large peak that comes out just before 6 minutes and has the
potential to mask the ammonium peak. .......................................................... 157
C.4
Chromatogram of HEPES buffer with 50 mg/L NH4+. It is
evident by the peak size and shape that the two peaks are
combined. ....................................................................................................... 157
xxiv
LIST OF FIGURES
Figure
Page
C.5
Chromatogram of 8 mM HEPES buffer with 100 mg/L NH4+ . ...................... 158
C.6
Sample with 10 μL injection had overlap of the sodium peak
with the HEPES peak making analysis impossible. ....................................... 160
C.7
When the samples were run with a 5 μL injection to essentially
dilute the sample by 2 allowed for peak separation between the
sodium and HEPES. ....................................................................................... 160
E.1.
Light micrograph of Scenedesmus sp. 131, along with a
fluorescence micrograph stained with Nile Red............................................. 165
E.2.
Light micrograph of Kirchneriella sp. 92, along with a
fluorescence micrograph stained with Nile Red. ............................................ 165
E.3.
3D schematic of tube reactor system, where the aquarium acts as
a water bath to increase temperature stability. ............................................... 170
xxv
ABSTRACT
Nitrogen removal from wastewater by algae provides the additional benefit of
producing lipids for biofuel and biomass for anaerobic digestion. As ammonium is the
renewable form of nitrogen produced during anaerobic digestion and one of the main
nitrogen sources associated with wastewater, experiments focused on the optimization of
growth and lipid production when grown on ammonium were evaluated. Scenedesmus
sp. 131 and Kirchneriella sp. 92 were grown in a 14:10 light/dark cycle on ammonium,
nitrate or urea in the presence of 5% CO2 and ammonium and nitrate in the presence of
air. Growth on nitrate and urea showed similar growth rates, and provided knowledge on
the target growth rate for optimizing growth on ammonium. Results showed the pH
decreased during exponential growth on ammonium in both 5% CO2 and air, causing
chlorophyll degradation. Growth on nitrate and air increased the pH of the medium and
produced an increase in Nile Red fluorescence and biofuel potential for strain 131, but
not for strain 92. Biological buffers were implemented to counteract the change in pH to
prevent growth inhibition.
Cultures were grown on 5% CO2 or air, which showed that increased levels of
CO2 are required for increased growth, biofuel potential, and ammonium utilization. This
increased the growth rates from 0.26 d-1 to 1.04 d-1 for strain 131 and 0.45 d-1 to 1.31 d-1
for strain 92. pH-controllers using 0.1 M KOH were used in experiments with 5%
CO2with the understanding that buffers are limited to lab scale experiments and pH
control would bridge the gap to industrial processes. The growth rate while utilizing pHcontrollers showed similar growth rates to buffered experiments. Growth on nitrate, urea,
and buffered ammonium with 5% CO2 showed an increase in the biofuel potential for
strain 92 in comparison to growth with air. Strain 131 had a decrease in biofuel potential
when grown on ammonium compared to growth on nitrate or urea. Both strains showed
increased levels of CO2 is required to increase biofuel productivity.
1
1. INTRODUCTION
Wastewater
Municipal wastewater treatment is an unwanted but necessary burden that is both
inefficient and expensive. Wastewater treatment occurs in some manner in all
communities. Furthermore, many of the facilities within the United States are out of date
and are quickly falling behind new EPA regulations limiting nitrogen and phosphorus
output, which affect downstream ecosystems. To meet newer EPA regulations for
nitrogen and phosphorus removal, smaller facilities are required to invest money in
additional treatment stages using physical or chemical methods. Both methods increase
cost due to either chemical or energy use. In many cases, the chemical methods cause
contamination of the wastewater sludge, which requires additional treatment steps
(Hoffmann 1998).
Since the late 1960's, wastewater treatment by algae has been proposed as an
alternative to standard mechanical and chemical processes by providing cheaper nitrogen
and phosphorus bioremediation that occurs naturally in wastewater (Woertz et al. 2009b).
One of the most beneficial aspects to using algae for bioremediation of wastewater in
conjunction with bacterial degradation of organic matter is the increased nitrogen and
phosphorus removal (Cabije et al. 2009; Hoffmann 1998). Additionally, algal technology
is currently being designed in order to retrofit existing smaller municipal wastewater
facilities (Cabije et al. 2009). The reason for this is to increase nitrogen and phosphorus
removal, which reduces the required capital cost to meet newer EPA standards. Finally,
2
algae provide the potential to reduce total operational costs if the algae are harvested and
processed for energy and other products (Greenwell et al. 2010; Hoffmann 1998). Lipids
produced by algae can be removed, if economically feasible, for the production of
biodiesel (Sialve et al. 2009). If it is not economically feasible to remove the lipids, the
entire algal cell can be processed in anaerobic digestion that will increase the energy
produced compared to anaerobic digestion of the cell wall and proteins (Sialve et al.
2009). The process of anaerobic digestion provides three essential factors for algal
growth: energy, CO2, and nitrogen and phosphorus-rich fertilizer. These factors have
been identified by several researchers as reasons why anaerobic digestion should be a
quintessential part of industrial scale algal biofuel production (Lundquist et al. 2010;
Sialve et al. 2009). Overall, the incorporation of algae can help ease the financial
burdens of processing municipal wastewater.
Nutrient Requirements
The basic requirements for algal growth are CO2, light, nitrogen and phosphorus.
Current research in industrial-scale processes focus on two important factors for
industrial viability of algae 1) decreasing the light attenuation, increasing biomass
density, and 2) CO2 abatement, increasing algal growth rate (Brennan and Owende 2010;
Chisti 2008; Huntley and Redalje 2007; Woertz et al. 2009b). Even though many experts
have dismissed the limitations of nitrogen as minor in comparison to other requirements
for growth, the limitation does not take into consideration several key points. First, the
nitrogen utilization of algae compared to terrestrial based crops is between 55 and 111
3
times greater than for rapeseed (ha-1 year-1) (Sialve et al. 2009). However, it is important
to note that this value does not take into account the increased biomass and lipid
productivity for the same area and timeframe. When comparing the molecular formulas
of algae, CO0.48H1.83N0.11, to rapeseed oil cake, CO0.63H1.79N0.11, or a general biomass
stoichiometric formula, CO0.5H1.83N0.2, the nitrogen content is very similar (Chisti 2007;
Doran 1995; Pstrowska et al. 2010). This means that the nitrogen requirement per ton of
biomass for most crops would be very similar. Algae uptake more nitrogen on a per acre
per time basis, because they reproduce more quickly than terrestrial crops. Using
wastewater as a nitrogen and phosphorus source at an average concentration of 40 mgN*L-1 and 3 mg-P*L-1 will require 2.5 m3 of wastewater to produce 1 kg of dried algal
biomass, where nitrogen is the limiting factor (Lundquist et al. 2010; Sedlak 1991).
At this time, many researchers have identified ammonium as the only legitimate
nitrogen source for large-scale production of algae (Lundquist et al. 2010; Sialve et al.
2009). This is due to several key factors. First, municipal wastewater is composed of
urea and ammonium, which account for approximately 60% and 40%, respectively, of
the total nitrogen available at approximately 40 mg-N*L-1 (Sedlak 1991). Urea is an
acceptable nitrogen source for algal growth; however, ammonium partially inhibits urea
utilization, and urea is rapidly degraded by bacteria, which use urease to catalyze the
hydrolytic cleavage of urea into ammonium and bicarbonate (Solomon et al. 2010).
Furthermore, large-scale algal facilities will need to use anaerobic digestion or other
methods to process the algal biomass. This will generate onsite CO2, energy and a
method to recycle nutrients. In methods that recycle nitrogen, ammonium is produced
4
from the degraded proteins (Sialve et al. 2009). Therefore, industrial-scale algal facilities
will need to utilize ammonium for growth to create a closed loop to reduce the cost
associated with nutrient input.
The most common nitrogen source in algal research is nitrate, which is in stark
contrast to the most abundant source of nitrogen in the form ammonium. This is because
nitrate is considered to be the most stable form of nitrogen used (in terms of pH and
temperature) and research has shown that lipid concentration increases with respect to an
increase in the pH of the medium (Gardner et al. 2010; Guckert and Cooksey 1990).
However, using nitrate in a large-scale system would make the entire process cost
prohibitive (Lundquist et al. 2010).
Because nitrate, urea, and ammonium have different effects on the carbonnitrogen metabolism, all three nitrogen sources were compared to determine the
advantages and disadvantages for growth and lipid accumulation. Ammonium was the
main focus, and methods were employed to determine the growth conditions that could
be applied to industrial-scale systems.
Lipid Production
The increased biofuel potential of algae is associated with the increased areal
productivity, which leads to an estimated oil yield of 1,190 L*ha-1 for rapeseed and
58,700 L*ha-1 for algae (Chisti 2007). For algae, the value is based upon a lipid content
of 30 % (w/w) and theoretical calculations for growth rate and culture density. It is
important to note that lipid content of algal strains can be artificially inflated due to
5
uncertainty in the extraction and analysis protocols. Strains have been reported to have
between 25 and 75 percent lipid by weight (Chisti 2007). However, in large-scale
production facilities with open ponds, raceways, or even photobioreactors, the actual lipid
content will be significantly lower than ideal conditions in lab-scale experiments (Woertz
et al. 2009a). Furthermore, open systems will be dealing with both algal and bacterial
communities, which may out-compete the selected algal strain for nutrients and will most
likely have lower lipid content with a higher growth rate.
The purpose of this thesis was to compare growth rate, biofuel potential and
nitrogen utilization of two green algae Scenedesmus sp. 131 and Kirchneriella sp. 92
isolated from a wastewater treatment facility in Deer Lodge, Montana. Both algae belong
to different families within the Chlorophyta. The two strains were grown on nitrate, urea,
or ammonium as the nitrogen source with 5% CO2 to allow for maximum growth rate.
Both strains were also grown on nitrate or ammonium in the presence of air to determine
the effects of carbon limitation. To improve growth on ammonium both strains were
grown in buffered media and in the presence of 5% CO2 or air. Furthermore, to increase
the lipid accumulation of the strains, a bicarbonate injection protocol was used (Gardner
et al. 2011). The final experiments used pH controllers with ammonium and 5% CO2
after determining pH control was required for scalability.
6
2. BACKGROUND
Wastewater Treatment
Nitrogen and Phosphorus Remediation Using Algae
The concept of using algae to help treat wastewater has been around since the
1950’s when the raceway pond was first developed (Ludwig and Oswald 1952; Ludwig
et al. 1951; Oswald et al. 1957). During the late 1980’s and early 1990’s algal research
was booming due to the Aquatic Species Program, a DOE funded project designated to
look at renewable energy from algae (Sheehan et al. 1998). The predominant research of
the time was focused on the use of suspended algae in large settling ponds or in HighRate Algal Ponds (HRAP), which were designed to be shallow and were constructed
using the raceway design (Garcia et al. 2000; Hoffmann 1998).
There are several advantages to using algae as a secondary or tertiary step in
wastewater treatment. The main advantage over the classical secondary treatment
methods is the increased efficiency for removal of nitrogen and phosphorus. The
removal of ammonium and phosphate occurs by two distinct mechanisms 1) biological
assimilation by algae for proteins (nitrogen) and DNA (phosphorus) and 2) the increase
in the medium pH from around 8 to around 11. This increase in pH is caused by
photosynthesis, which consumes dissolved CO2 and bicarbonate (HCO3-) and shifts the
dissolved inorganic carbon (DIC) equilibrium towards carbonate (CO3-2) (Schumacher et
al. 2003). The pKa of ammonium/ammonia is 9.25; therefore, when the pH increases
above 9.25, a majority of ammonia is in the volatile form (NH3) and volatilization occurs
7
(Heggemann et al. 2001). In addition, at a higher pH range phosphate precipitates with
cations such as calcium and magnesium, which can account for a large portion of the
reduced phosphorus levels (Cao and Harris 2008; Jarvie et al. 2002; Schumacher and
Sekoulov 2002).
There are two approaches to using algae for wastewater treatment. Algae can be
in the suspended form in either open ponds or HRAP, or algae can be utilized in a biofilm
(Hoffmann 1998; Roeselers et al. 2008; Wei et al. 2008). The use of an algal biofilm
removed up to 76 percent of the phosphorus in the medium and essentially all of the
nitrogen, while providing a method that decreased harvesting costs (Bush et al. 1963).
Algal biofilms have been shown to create the same type of environmental conditions as
planktonic cultures by increasing the pH, increasing dissolved oxygen, and increasing
nutrient uptake. Furthermore, utilizing an algal biofilm allows for easier phosphate
removal. This is because the precipitated phosphate is incorporated into the extracellular
polymeric substance (EPS) layer and is harvested along with the biofilm. In planktonic
growth, the phosphate precipitates to the bottom of the ponds, due to pH increase, and
reenters solution at night or when the algal bloom dies off, both of which causes the pH
of the medium to decrease and reduces the phosphate removal efficiency (Craggs et al.
1996; Hoffmann 1998; Wei et al. 2008). However, the main disadvantage to biofilm use
is a decreased growth rate that is attributed to the limited diffusion of nutrients and light
into the biofilm (Liehr et al. 1989, 1990). This will decrease the overall nitrogen and
phosphorus removal.
8
The capacity of algae to remove nitrogen from the medium is based on both the
nitrogen source and the DIC concentration, which indicates that the carbon-nitrogen
metabolism plays a significant role in nitrogen remediation. In wastewater, the main
nutrient required to be removed is nitrogen, based on its large concentration (40 mg-N*L1
) compared to phosphorus (3 mg-P*L-1) (Lundquist et al. 2010; Sedlak 1991). The
generic molecular formula for algal biomass is CO0.48H1.83N0.11P0.01, which has a molar
ratio of approximately 10:1 for N:P (Chisti 2007; Doran 1995). However, Scenedesmus
has shown to have an optimal molar ratio for N:P around 30:1 (Rhee 1978), which is
very close to the average molar ratio found in wastewater based on the 40 mg-N*L-1 and
3 mg-P*L-1. Nitrogen is the limiting nutrient in wastewater and nitrogen utilization plays
an integral part in growth, medium pH, and biofuel potential, and therefore it is
advantageous to study its effects (Dvořáková-Hladká 1971; Fernández et al. 2004; Li et
al. 2008; Nunez et al. 2001; Schumacher and Sekoulov 2002; Voltolina et al. 2005; Fuggi
et al. 1981).
Biofuel Production from Wastewater Derived Algae
The use of algae in wastewater treatment has the capability to offsetting
wastewater treatment costs by the production of algal biofuels. The algal cell can be
processed to 1) make biodiesel from lipids, 2) use anaerobic digestion to produce
methane gas from the remaining cell biomass and 3) create a rich fertilizer (Sialve et al.
2009; U.S.DOE 2010). Higher levels of lipid production in the cell allow for processing
the lipids into biodiesel; however, if the lipid content of the cell is low, the entire cell can
be processed using anaerobic digestion. This is because lipids increase the caloric value
9
of the cell and provide a better input to anaerobic digestion (Illman et al. 2000; Sialve et
al. 2009).
Carbon - Nitrogen Metabolism
The carbon-nitrogen metabolism in algae and higher plants is a complex
interaction that depends on both the nitrogen source and the concentration of carbon
dioxide in the medium, both in the aeration (high and low CO2 levels) and in the
dissolved inorganic carbon (DIC) content. The three main nitrogen sources studied for
algal growth are nitrate, ammonium, and urea (Bongers 1956; Fernández et al. 2004;
Hodson and Thompson 1969; Kristiansen 1983; Li et al. 2008; Ludwig 1938; Toetz et al.
1977). Nitrate has been the primary nitrogen source used in research labs, based on the
number of nitrate based media (Andersen 2005). This may be due to nitrate being the
most stable form of nitrogen in terms of handling broad pH and temperature ranges.
However, in industrial scale, both ammonium and urea will be required for economic
viability of algal growth (Lundquist et al. 2010).
Carbon
In general, algae are capable of growing on a broad range of CO2 concentrations
from air (0.04%) to high CO2 concentrations from flue gas at over 60% CO2; however,
the optimal CO2 for growth of the strains from previous research was between 5% and
10% CO2 (Hanagata et al. 1992; Nakano et al. 1996). The main reason algae are capable
of growing at low concentrations of CO2 is the carbon concentrating mechanism (CCM),
which is activated when the concentration of CO2 in the medium is low (Thielmann et al.
10
1990). The concentration of DIC to activate the CCM is based on the growth rate and the
cell concentration, which directly affects the DIC concentration of the medium. The
green algae utilize the C3 carbon pathway, which is better known as the Calvin cycle. In
the Calvin cycle, CO2 is incorporated into ribulose-1,5-bisphosphate using the enzyme
ribulose bisphosphate carboxylase oxygenase (RUBISCO). The fact that RUBISCO is a
carboxylase and an oxygenase means that the organism can undergo two different
reactions, one with CO2 and the other with O2 as shown in Equations 1 and 2,
respectively (Giordano et al. 2005).
ribulose - 1,5 - bisphosphate + CO 2 + H 2 O → (2) glycerate - 3 - P
(Equation 1)
ribulose - 1,5 - bisphosphate + O 2 → glycerate - 3 - P + glycolate - 2 - P (Equation 2)
Phosphoglycolate produced by the oxygenase activity inhibits the carboxylase activity of
RUBISCO; however, dephosphorylation of phosphoglycolate alleviates the inhibition,
and the glycolate is then usable in the photorespiration metabolism (Giordano et al.
2005). It is important to note that at standard concentrations of CO2 in equilibrium with
air, RUBISCO is typically less than half-saturated, which reduces the efficiency of
photosynthesis and allows for the oxygenase activity to interfere with the carboxylase
activity of RUBISCO (Giordano et al. 2005). Therefore, to help increase the kinetics for
CO2 reactions with RUBISCO, the CCM increases the internal concentration of CO2,
boosting the photosynthetic efficiency (Badger et al. 1998). Depending on the pH of the
medium and the algal strain, there are different types of CCM processes involved. The
standard CCM process used in many green algae uses multiple types of carbonic
11
anhydrase (CA), an enzyme utilized in the interconversion of bicarbonate and CO2.
Many green algae have an external CA attached to the periplasmic membrane for the
conversion of bicarbonate in the medium to CO2 for diffusion into the cell (Sultemeyer
1998). In addition to the external CA, the cell also contains different isotypes of CA in
the periplasmic space, the cytosol, the chloroplast, and the pyrenoid, which is the final
destination of carbon. One of the main reasons the cell has an extensive collection of
CAs is to allow the CO2 to diffuse across each membrane and then convert it back into
bicarbonate. This prevents diffusion back across the membrane to keep higher
concentrations of carbon in the cell than in the external medium. The pyrenoid resides
within the chloroplast and contains high concentrations of RUBISCO to react with
incoming carbon utilized in the Calvin cycle, and becomes prominent in the presence of
low CO2 (Sultemeyer 1998). In addition to this type of CCM, a few algal species have a
secondary CCM that allows for direct uptake of bicarbonate. In the genus Scenedesmus
and in Chlamydomonas, the ability to uptake bicarbonate through the use of a bicarbonate
pump occurs in alkaline media (Shiraiwa et al. 1993; Spalding 2008). The cells will
utilize the standard CCM at neutral pH values, but when the pH of the medium increases
above 9 and therefore increases the concentration of DIC in the medium, the cell utilizes
the bicarbonate pump (Shiraiwa et al. 1993). The process of directly removing
bicarbonate from the medium causes an increase in the pH to above 10. This is caused by
two factors, 1) the removal of bicarbonate requires the cell to expel hydroxyl ions to
remain neutral, and 2) the removal of carbon from the medium causes an increase in pH
due to the shifting carbonate equilibrium (Shiraiwa et al. 1993). The bicarbonate pump is
12
one of the key features associated with Scenedesmus and its increased lipid production
during bicarbonate injections (Gardner et al. 2011). Bicarbonate is required for the
conversion of acetyl-CoA to malonyl-CoA, which utilizes the carboxyl group from
bicarbonate in an ATP-dependent reaction (Sukenik and Livne 1991).
The concentration of CO2 in the aeration line directly affects the concentration of
DIC by increasing the concentration of dissolved gaseous CO2 in equilibrium with the
gas phase based on Henry's Law in Equation 3. Therefore, bicarbonate and carbonate will
also increase depending on the pH of the medium and its equilibrium point to the
dissolved CO2. However, increasing CO2 in the medium causes acidification by creating
carbonic acid as shown in Equation 4 (Shiraiwa et al. 1993).
p = k H [CO 2 ]
(Equation 3)
Where p is the partial pressure of CO2 in the gas, kH is Henry's constant, which is equal to
29.4 L*atm*mol-1 at standard temperature and pressure, and [CO2] is the concentration of
CO2 in the medium.
CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+ ↔ CO32- + H+
(Equation 4)
The two forms of carbon that are considered bioavailable in the medium are CO2
and bicarbonate. There are multiple methods of transporting carbon into the cell in low
DIC conditions for utilization with the CCM. CA is responsible for bicarbonate transport
at neutral pH, and some strains have a bicarbonate pump that is active at alkaline pH
because of the increased fraction of total DIC in the form of bicarbonate. The CCM is
not active under increased levels of CO2.
13
Nitrogen
The three nitrogen sources analyzed in this paper were nitrate, ammonium, and to
some extent, urea. These nitrogen sources utilize different metabolic pathways to be
converted in amino acids, which provide different metabolic control points and have
diverse affects on the algal strain and its environment. Nitrogen is the backbone of
proteins, which provides cellular functions for growth and lipid production. The lack of
nitrogen in the medium causes cell-cycle inhibition and allows the cell to store carbon in
the form of TAG molecules for future growth (Gardner et al. 2010). In addition to
metabolic differences associated with the nitrogen sources, nitrate and ammonium cause
changes to the pH of the medium during uptake (Fuggi et al. 1981).
Nitrate
The nitrate metabolism starts with transport across the cellular membrane and has
biphasic kinetics with high and low affinity nitrate transporters. Low affinity transport
occurs in the millimolar range and high affinity transport occurs in the micromolar range
(Fernández et al. 2004).
The reduction of nitrate occurs within the chloroplast and is mainly associated
with the pyrenoid. Nitrate is reduced to nitrite in the pyrenoid, using nitrate reductase
(Equation 5) and NADPH (Nicotinamide adenine dinucleotide phosphate, a reducing
agent) to catalyze the two electron transfer (Lopez-Ruiz et al. 1985). Nitrite is then
reduced to ammonium using nitrite reductase and ferrodoxin (Eqn. 6).
NO3- + 2e - + 2H + → NO-2 + H 2 O
(Equation 5)
NO-2 + 6e - + 8H + → NH +4 + 2H 2 O
(Equation 6)
14
The conversion of nitrate to ammonium occurs in the chloroplast and is most likely due
to the large interaction of the carbon and nitrogen metabolisms (Turpin 1991). The
reducing power for nitrate assimilation comes from the photosynthetically generated
electron flow, and utilizes approximately 20% of the total electron flow, while the rest is
directed towards photosynthetic CO2 fixation (Turpin 1991).
Nitrate Uptake in the Presence of Low CO2 and its pH Effect
As with other nitrogen sources in the presence of low CO2, nitrate uptake is
reduced due to a co-transport requirement (Turpin 1991). However, the process of nitrate
uptake releases hydroxyl ions to maintain cell neutrality, which increases the pH of the
medium (Fuggi et al. 1981). The pH increase allows for an increase in DIC equilibrium
of the medium, which would potentially allow for a higher cell concentration. In
addition, increased pH has shown to increase the lipid content of Scenedesmus sp. WC-1
and Coelastrella sp. PC-3 by preventing cell-cycling (Gardner et al. 2010).
Urea
Urea is hydrolyzed to ammonium using the enzyme complex ATP:urea
amidolyase or hydrolytically cleaved using urease (Bekheet and Syrett 1977; Leftley and
Syrett 1973). The enzyme complex completes two enzymatic activities. The first, shown
in Equation 7, is urea carboxylase, which catalyzes the ATP-dependent condensation of
urea and bicarbonate to produce allophanate, and requires Mg2+ and K+ for the enzyme
to operate (Fernández et al. 2004). Allophanate is then converted to ammonium using
15
allophanate lyase, the second part of the enzyme complex (Equation 8) (Hodson et al.
1975; Leftley and Syrett 1973)
Urea + ATP + HCO3 - → Allophanate + ADP + Pi
(Equation 7)
Allophanate + 3H2O + H+ → 2NH4+ + 2HCO3-
(Equation 8)
Urease uses a single step to hydrolytically cleave urea as shown in Equation 9.
Urea + H 2 O → CO 2 + 2NH3
(Equation 9)
The potential benefit associated with utilizing urea is the intracellular bicarbonate
production during the enzymatic degradation to ammonium.
Ammonium
Ammonium is the base unit required for the production of amino acids and is the
final form produced when other nitrogenous sources are transported into the cell and
processed for assimilation (Fernández et al. 2004). Ammonium is assimilated using the
glutamine synthetase/glutamate synthase (GS/GOGAT) cycle (Equations 10 and 11,
respectively), which combines ammonium and glutamate in an ATP-dependent reaction
to form glutamine for production of amino acids and the regeneration of glutamate
(Fernández et al. 2004).
Glutamate + NH+4 + ATP → Glutamine + ADP + Pi
2 - Oxoglutara te + Glutamine + NADPH → 2Glutamate NADP+
(Equation 10)
(Equation 11)
16
The main concern with growing algae on ammonium is the potential for decreased
or inhibited growth that has been shown to be species specific (Bongers 1956;
Dvořáková-Hladká 1971; Elrifi et al. 1988; Guy et al. 1989; Ludwig 1938; Thacker and
Syrett 1972). The growth inhibition appears to be related to the carbon-nitrogen
metabolism and the difference between growth on ammonium and nitrate or urea. When
algae are grown on ammonium, the cells increase anaplerotic reactions to replace TCA
cycle intermediates that are used for the increase in amino acid synthesis (Elrifi et al.
1988; Norici et al. 2002; Turpin et al. 1991; Vanlerberghe et al. 1990). This is
accomplished by increasing the production of phosphoenolpyruvate carboxylase (PEPC),
the main enzyme utilized by Chlorophytes in anaplerotic reactions (Norici et al. 2002).
PEPC is a well known enzyme documented in various photosynthetic organisms that
utilize the C4 pathway, including diatoms and certain plants (Beardall et al. 1976;
Reinfelder et al. 2004). The C4 pathway utilizes bicarbonate to react with
phosphoenolpyruvate (PEP) in the presence of PEPC to produce oxaloacetic acid (OAA)
(Turpin 1991). In Chlorophyta, the C4 mechanism is limited to the replenishment of TCA
cycle intermediates. In many cases, the carbon requirements associated with anaplerotic
reactions compete with carbon utilized for photosynthetic reactions. With the increased
presence of PEPC, there is an increase in carbon requirements for anaplerotic reactions of
approximately 0.3 moles of carbon per mole of nitrogen (Turpin et al. 1991). Because of
this, to prevent a reduction in growth rate, algal cultures growing with ammonium must
be provided with CO2 to overcome carbon limitations. However, when the algal cell is
17
grown in a low CO2 environment (i.e. air), these pathways compete for the incoming
carbon based on the half-saturation kinetics of RUBISCO and PEPC.
Effects of Low Carbon with Ammonium on Growth
For algae in low CO2 environments, grown on ammonium, the enzymatic activity
of RUBISCO and PEPC is extremely important and dictates how carbon in the cell is
utilized. Growth on ammonium up regulates PEPC and increases the anaplerotic
reactions used in replacing TCA cycle intermediates (Schuller et al. 1990; Vanlerberghe
et al. 1990). The algal cell will preferentially utilize carbon towards anaplerotic reactions
over photosynthetic reactions for growth. This was shown using carbon isotope studies
where PEPC had a higher affinity for bicarbonate than RUBISCO. However, because
PEPC utilizes bicarbonate, the cell may be rapidly using the bicarbonate before
conversion to CO2 occurs (Amory et al. 1991; Elrifi et al. 1988; Fernández et al. 2004;
Guy et al. 1989; Norici and Giordano 2002). Therefore, in a low carbon environment, the
algal cell will become growth inhibited due to carbon limitation at a lower cell
concentration in the presence of ammonium than in other nitrogen sources due to the
increased anaplerotic reaction. Low carbon can also inhibit ammonium uptake into the
cell, and as was the case with nitrate, CO2 must be present for uptake to occur (Amory et
al. 1991).
Free Ammonia Inhibition
The pKa of ammonium to ammonia is 9.24 (Equation 12) (Heggemann et al.
2001).
18
NH3 + H 2O ↔ NH4+ + OH-
(Equation 12)
High concentrations of ammonium have been linked to algal growth inhibition,
because of the increased presence of free ammonia within the medium (Abeliovich and
Azov 1976; Azov and Goldman 1982). This inhibition can be avoided by keeping the pH
below 7.5, which equates to approximately 1.5% of the ammonium being in the free
ammonia form. If the algae are growing on very high concentrations of ammonium, the
pH of the medium should be decreased to below 7 to decrease the concentration of free
ammonia to approximately 0.5% of the total ammonium concentration. The inhibitory
effect of ammonia is strain specific, and most strains show a 50% reduction in growth
rate at millimolar concentrations; however, it has been shown that some algal strains are
sensitive to even 0.5 mM free ammonia (Abeliovich and Azov 1976; Azov and Goldman
1982). Maintaining a pH of 7 during photosynthetic activity is obtainable through
acidification of the medium with CO2, which provides additional carbon required for
growth on ammonium and reduces ammonia inhibition.
Another major issue associated with algal growth on ammonium is the decrease in
pH associated with the translocation of protons out of the cell to maintain cell neutrality
during ammonium utilization (Fuggi et al. 1981). Acidification reduces algal growth by
decreasing the DIC of the medium. Therefore, algal growth on ammonium has a limited
pH window for optimum growth rate between an expected pH of 6.35, the point at which
bicarbonate becomes the dominant form of carbon in the medium, and a pH value of 7.0
to 7.5 (depending on ammonium concentration) (Abeliovich and Azov 1976; Norici and
Giordano 2002).
19
Interaction of Ammonium with Other Nitrogen Sources
Ammonium is the foundation for building amino acids and proteins, and
organisms will preferentially utilize ammonium over other sources of nitrogen. It has
been shown that ammonium has an inhibitory effect on the utilization of both nitrate and
urea through multiple mechanisms (Dortch 1990; Fernández et al. 2004; Molloy and
Syrett 1988). One way ammonium can inhibit nitrate and urea utilization is to limit the
uptake of the other nitrogen sources (Dortch 1990; Ingemarsson et al. 1987; Larsson et al.
1985; Molloy and Syrett 1988). In addition to inhibiting uptake, the conversion of
ammonium and glutamate to glutamine in the GS/GOGAT cycle appears to inhibit nitrate
reductase (Smith and Thompson 1971). Theory suggests that nitrate reductase is
dependent on an active permease of nitrate into the cell, which is inhibited in the presence
of ammonium (Florencio and Vega 1983; Laeuchli and Bieleski 1983). However, with
urea, it appears that ammonium represses the induction of urea amidolyase (Hodson et al.
1975).
Summary
The incorporation of algae into wastewater treatment achieves two beneficial
goals 1) The removal of nitrogen and phosphorus (Hoffmann 1998) and 2) the potential
to produce biofuels (Woertz et al. 2009a). In order to effectively remove nitrogen and
increase the total biofuel potential, an understanding of two factors is required 1) carbon
concentration plays a large role in growth rates and therefore nitrogen removal rates
20
(Turpin et al. 1991) and 2) The type of nitrogen can influence growth and biofuel
potential by changing the metabolic pathways (Lourenco et al. 2002; Turpin 1991).
Growth on nitrate and urea require conversion to ammonium prior to entering the
GS/GOGAT cycle. This indicates that growth on ammonium can inhibit utilization and
uptake of nitrate and urea in a mixed nitrogen source medium (Dortch 1990; Fernández et
al. 2004; Molloy and Syrett 1988). In wastewater, ammonium is often the primary
nitrogen source and therefore is important to optimize growth and biofuel potential for
algae grown on ammonium. The two main considerations for growth on ammonium is
the CO2 concentration to provide enough carbon for photosynthetic and anaplerotic
reactions, and the pH of the medium effected by ammonium uptake (Fuggi et al. 1981;
Norici and Giordano 2002).
21
3. METHODS
For complete detailed methods, see Appendix E.
Organism Isolation and Culture
The two green algae Scenedesmus sp. 131 (strain 131) and Kirchneriella sp. 92
(strain 92) (Figure 1 and 2, respectively) were isolated from wastewater settling ponds in
Deer Lodge, Montana, and identified through screening experiments as the two primary
candidates to continue with for further studies. Comparison of strains during initial
testing can be found in Appendix A. The strains were isolated using Bold's Basal
Medium (BBM) agar plates (2% w/w). Using a standard stereoscope, single colonies
were picked using sterile glass pipettes, and transferred into 1 mL of liquid medium.
This method reduced the number of strains that were isolated because 1) not all strains
can grow on solid agar medium (Andersen 2005) and 2) BBM is a basic medium that
does not have added vitamins some strains may require. Once the colonies had grown in
the 1 mL volumes, the 1mL was added to 5 mL of BBM and bacteria check broth. The
bacteria check broth was made by adding 0.5% (w/v) dextrose to BBM prior to
autoclaving. If the bacteria check broth was positive for bacterial growth and/or
microscopy showed multiple algal strains, the culture was streaked on a new plate for
further isolation. The strains were streaked a total of five times to remove bacteria and
provide a unialgal culture.
22
Figure 1. Light micrograph of Scenedesmus sp. 131, and a fluorescence micrograph
stained with Nile Red.
Figure 2. Light micrograph of Kirchneriella sp. 92, and a fluorescence micrograph
stained with Nile Red.
Culture Identification
The strains were first identified as unialgal by morphology and genus level
identification was made using past research papers, which provided key morphological
markers to identify Scenedesmus sp. 131 and Kirchneriella sp. 92 (Marvan et al. 1984;
23
Prescott 1978; Trainor et al. 1976). Furthermore, Scenedesmus sp. 131 was identified
based on sequencing of the SSU 18S RNA gene. Molecular sequencing of the SSU 18S
region of Scenedesmus sp. 131 shows > 99% alignment to Scenedesmus communis.
Experimental Conditions
Media
All strains were grown in Bold's Basal Medium with the pH adjusted to 7.8 with
KOH (Bischoff and Bold 1963). This medium was modified for different nitrogen source
experiments for Scenedesmus sp. 131 and Kirchneriella sp. 92, including modification by
replacing the sodium nitrate (2.94 mM) with either urea (1.47 mM) or ammonium
chloride (2.94 mM), both of which were filter sterilized into the autoclaved medium after
it reached room temperature. The concentration of nitrogen in BBM is with the
concentration range found in wastewater. The range varies between 2.5 and 3.5 mM, but
depending on the facility can vary outside of this range (Sedlak 1991; Woertz et al.
2009a). Experiments with ammonium were run unbuffered or buffered with 8 mM
Piperazine-1,4-bis(2-ethanesulfonic Acid)] (PIPES, pKa 6.8) or N-(2hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES, pKa 7.5) depending on the
test parameters. The 8 mM concentration was chosen over 10 mM due to precipitant
after autoclaving and because it provided adequate buffering to minimize pH change.
Experimental System
The cultures were grown in a 14:10 light/dark cycle at room temperature (24 ±
1°C). Biological triplicate cultures were grown in vertical, tubular photobioreactors
24
(diameter = 70 mm, height = 500mm) filled with 1 L of BBM. Reactors were inserted
into a circulating water bath (aquarium) to increase temperature control, and were
illuminated by 12 T5 4 ft fluorescent lights for a total illumination of 350 µmoles m-2 s-1,
measured using a photosynthetically active radiation (PAR) meter (LI-COR). Each
reactor utilized a rubber stopper with three ports for sampling, aeration, and ventilation.
The reactors were also modified for pH control by the addition of an inlet port for base
and a slot for an autoclavable pH probe (Cole-Parmer). The probe was attached to a pH
controller (HANNA Instruments BL 931700-1) with a minimum pH set point, to activate
a dosing pump (HANNA Instrument BL 1.5-1) with 0.1 M KOH. The aeration and
ventilation ports were equipped with 0.2 μm filters (Millipore) to prevent contamination
and release of algae. Incoming air was bubbled through water prior to entering the
reactors to saturate the incoming gas with water and reduce the rate of evaporation in the
reactors. The reactors were aerated with either 400 mL*min-1 of air or air with 5% CO2.
The CO2 tank was mixed with compressed house air, run through a water/oil trap, and
utilized an automated on/off controller set to turn the CO2 on with the lights.
Analysis
pH and Ion Chromatography
Medium pH was measured on samples using a pH meter (AB15pH, Accumet).
Concentrations of phosphate, sulfate, and nitrate were measured by ion chromatography
(IC) using an IonPac AS9-HC Anion-Exchange Column (Dionex) with a 9.0 mM sodium
carbonate buffer set at a flow rate of 1.0 mL*min-1. Detection was performed using a
25
CD20 conductivity detector (Dionex) at 21°C, and IC data were analyzed on Dionex
PeakNet 5.2 software. Phosphate and sulfate concentrations were measured by IC to
confirm they were in excess during experimentation.
Concentrations of ammonium were measured by IC using a Metrohm Metrosep
C4 150/4.0 mm IC column (Metrohm) controlled to 25 °C on an Agilent 1200 series
column compartment with dipicolinic acid solution at a flow rate of 0.9 mL*min-1.
Dipicolinic acid solution was made dissolving 0.1170 g of Fluka 2,6-pyridinedicarboxylic
acid >99.5% purity for ion chromatography (Sigma-Aldrich) and 0.1011 ml of 67-70%
trace metal grade nitric acid (Fisher) into 1 L of nanopure water. The detection was
performed using a Metrohm 732 IC detector. Data were analyzed using HP ChemStation
software.
Concentrations of urea were measured using an Agilent HPLC with a Zorbax
Eclipse XDB-C18 column (Agilent Technologies) controlled to 35 °C with 20 mM
sodium acetate ACS reagent (Sigma-Aldrich) adjusted to pH 7.20 and 10% acetonitrile
added (100% HPLC Grade acetonitrile, Burdick and Jackson). Eluent was delivered
using an Agilent 1100 series pump programmed to deliver gradient of 2 solutions (20
mM sodium acetate with 10% ACN and 100% ACN) at 1.0 ml*min-1 as shown in Table
1.
Table 1. Eluent Gradient for HPLC and Urea Analysis.
Time
(min)
0
12.6
13.6
22.6
20 mM Sodium Acetate with 10% ACN
(% of 1.0 mL/min)
88.9
55.5
1.1
88.9
100 % ACN
(% of 1.0 mL/min)
11.1
44.5
98.9
11.1
26
Detection was performed using an Agilent 1100 series UV-visisble diode-array detector
programmed to record results at 230 nm. Samples were run using an Agilent 1100 series
autosampler programmed to perform autosampler derivatization (xanthydrol
derivatization) (Clark et al. 2007). Data was analyzed using HP ChemStation software.
Nile Red Staining Protocol
Algae were stained with Nile Red (9-diethylamino-5H-benzo(α)phenoxazine-5one) (Kodak) at a concentration (4 µL of Nile Red for 1 mL sample from a 250 µg/mL in
acetone stock solution) to monitor TAG accumulation over time by using the method
developed by Cooksey et al. (1987). The correlation between Nile Red fluorescence and
TAG was recently reconfirmed by Gardner et al. (2010). Aliquots of 1 mL of sample
were removed from cultures and, depending on the cell density, were either stained
without dilution, diluted with 4 mL ultrapure H2O, or diluted with 0.5 mL of sample with
4.5 mL ultrapure H2O before assaying for Nile Red fluorescence. Dilution was
determined using two methods. The first method was a standard dilution curve to
determine the linear range of the instrument, which is strain specific. This was
accomplished by running a serial dilution on a culture with high cell concentration and
nitrogen depleted to increase the TAG content. The second method was done using realtime analysis of each experiment by running two dilutions of each sample to verify that
the fluorescence values were equivalent. This was necessary due to variations in cell size
and lipid content at given cell concentrations, which changed the linear range. Nile Red
fluorescence was quantified on a microplate reader (Bio-Tek instruments Inc.) utilizing
480/580 nm excitation/emission filters. A baseline sensitivity setting of 75 was
27
experimentally determined to maximize the signal-to-noise ratio while ensuring a range
that accommodated fluorescent level changes over 10,000 units. To minimize
fluorescence spillover, black walled 96-well plates were loaded with 200 µL of sample.
Unstained samples were used for background medium and cellular autofluorescence
correction. It has been shown by Cooksey et al. that the Nile Red intensity shifts for
different algal strains over time (1987). This was recently reconfirmed (Elsey et al.
2007). Measurement times of 60 min after staining were optimal for both strain 131 and
strain 92.
Biofuel Potential Measured by Gas Chromatography
Biofuel Potential of the cell was analyzed using direct transesterification based on
the protocol designed by Griffiths (2010). For Griffiths' alkali-acid method, 10 mg of
lyophilized biomass was added to 5 mL glass serum vials along with 1.0 mL toluene.
Sodium methoxide (2 ml) (Pure, titrated, ACROS) was added to the mixture prior to
crimp sealing with Teflon-lined septa and vortexing. Samples were incubated at 80 oC
for 30 min with intermittent vortexing and cooled for 10 min. 2 mL 14% boron
trifluoride methanol (BF3-methanol, Thermo Scientific) was added before repeating the
incubation. The bottles were cooled to room temperature for 10 min before 0.8 mL H2O
and 0.8 mL hexane were added and vortexed. Samples were centrifuged at 3,000 x g for
5 min and the upper hexane/toluene layer, containing the fatty acid methyl esters (FAME)
extract, was transferred to vials for appropriate dilution with triple solvent (chloroform
(HPLC grade, EMD)/tetrahydrofuran (HPLC grade, Fisher Scientific)/hexanes (GC
grade, Fisher Scientific), 1:1:1 by volume) and analysis by GC.
28
In preparation for analysis by GC, extracts were diluted to the appropriate
concentration range using triple solvent. Prior to analysis, 10 L of 10 mg/mL octacosane
was added as an internal standard to 1 mL of diluted sample in a 1.5 mL GC autosampler
vial capped with a Teflon lined septum. Samples (1 L) were injected into an Agilent
6890N GC and quantified with a flame ionization detector using a 15 m Restek
RTX65TG column (fused silica with a film thickness of 0.1 μm; Restek, Bellefonte, PA).
The oven temperature increased from 60-370 oC at a rate of 10 oC/min using helium as
the carrier gas at a pressure of 61.2 kPa and a flow rate of 1.3 mL/min. A split mode with
a ratio of 1:30.8 was used for analysis of FAMEs from direct transesterification reactions.
Palmitic acid methyl ester, nonadecanoic acid methyl ester, glyceryl tripalmitin and
glyceryl tristearin were used as calibration standards at a concentration range of 0.001
mg/mL to 2 mg/mL. Peak area for standards were calculated and using a linear
correlation allowed for calculation of sample concentrations. The concentration in the
samples run on GC were not the concentration of the initial samples. The concentration
analyzed on the GC was multiplied by the volume of the organic phase (1.8 mL),
multiplied by the dilution factor (3), and divided by the biomass (10 mg). This provided
a final answer in % FAME/Biofuel Potential (w/w).
Cell Concentrations and Harvesting
Algal cells were counted directly using a hemocytometer with a minimum of 400
cells counted for statistical reliability (Andersen 2005). Micrographs of cell morphology
were taken using a transmitted light microscope (Nikon Eclipse E800) with an Infinity 2
29
color camera equipped with fluorescence capabilities to record Nile Red stained samples
for comparison between consecutive experiments. Cells were harvested at the end of the
experiment by centrifugation (4000x g for 10 min) of 750 mL of the reactor contents (3 x
250 mL bottles), washed once, and frozen for lyophilization at -80 °C. An additional 25
mL was collected from each reactor and filtered, using 0.7 µm glass fiber filters (Fischer
Scientific), to determine dry cell weight (DCW). The filters with algae were dried in an
oven at 60 °C until the weight of the biomass and filter remained constant between
weight measurements. Biomass yields were calculated by subtracting the dry weight of
the clean filter from the oven-dried weight of the filter with biomass, and cell density was
determined by dividing by the volume of sample filtered.
Kinetics
Growth Rate
The specific growth rate for each experiment was calculated using the exponential
growth equation (Equation 13). Even though algae do not always follow the standard
growth pattern of one parent cell producing two daughter cells (Trainor et al. 1976), the
basic equation for exponential growth given in Equation 13 provides a method for
determining the differences between different strains and environmental conditions.
X 2 = X 1e
μt
ln(
⇒ μ=
X2
t
X1 )
(Equation 13)
The specific growth rate for each experiment was analyzed using data from the
exponential growth excluding the lag phase and the first data point of stationary phase.
30
The reason for excluding the first point of the stationary phase is the lack of knowledge
of the precise time the system reached stationary phase.
31
4. OPTIMIZATION OF GROWTH ON AMMONIUM AND COMPARISON TO
GROWTH ON NITRATE OR UREA
Comparison of Growth on Different Nitrogen Sources with 5% CO2
The comparison of three different nitrogen sources (nitrate, ammonium, and urea)
for both Scenedesmus sp. 131 and Kirchneriella sp. 92 was carried out using Bold's Basal
medium with either the standard concentration of sodium nitrate (2.94 mM), or with the
molar nitrogen equivalent of ammonium (2.94 mM) or urea (1.47 mM). In addition, to
ensure that there were no carbon limitations during exponential growth, the strains were
grown on 5% CO2. The growth curves for both strains are shown in Figure 3 along with
pH of the medium when grown on 5% CO2, the total Nile Red fluorescence, and nitrogen
concentration. When the strains were grown on nitrate, acidification of the medium, by
adding 5% CO2, prevented an increase in pH. Without an increase in pH, strain 131 did
not produce its maximum Nile Red fluorescence, as previously shown by Gardner et al.
for the similar organism Scenedesmus sp. WC-1 (2010). Furthermore, when grown on
urea, the pH of the medium remained constant because the transport of a neutral molecule
does not require the release of hydroxyl ions or protons to maintain cell neutrality
(Fernández et al. 2004; Hodson and Thompson 1969). Therefore, strain 131 grown on
urea showed similar Nile Red fluorescence values to when grown on nitrate. Figure 3
also shows that strain 92 had a large increase in Nile Red fluorescence when grown on
urea compared to nitrate, and that strain 92 had varying levels of Nile Red fluorescence
depending on the nitrogen source, which in all cases was greater than that for strain 131.
32
Figure 3. Total Nile Red fluorescence (solid line) and nitrogen concentration (dashed
line) (a), pH (b) and average cell density (c) with standard deviation for triplicate reactors
(1) Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on 5% CO2 using nitrate (□)
ammonium (▲) or urea (♦) as the nitrogen source.
33
When grown on 5% CO2 and ammonium, Scenedesmus sp. 131, had large error bars
associated with the pH because one tube had a pH that was below 4 while the other tubes
had a pH = 5. Figure 3 shows that growth on ammonium without pH buffer caused a
significant decrease in the medium pH, which caused cell-cycle inhibition and
chlorophyll degradation (Fig. 4). Figure 3 also shows that strain 131 was not as
adaptable to low pH as strain 92, based on a shorter exposure time for chlorophyll
degradation to occur. The degradation of chlorophyll was assumed to indicate a loss of
viability, in the medium used for growth, and is shown in Figure 3 as a decrease in viable
cell concentration. The growth inhibition on ammonium prevented complete utilization
of ammonium for both strain 131 and strain 92 as shown in Figure 3. This is in contrast
to growth on nitrate and urea, which was utilized in approximately 6 days. The only
exception was strain 131 grown on urea and showed complete utilization in 5 days.
Chlorophyll degradation caused the reactors to change from bright green to
brown/yellow and finally a white color as the cells decreased in chlorophyll content. The
reason for this is unknown, but has been documented in algae and cyanobacteria (Kallas
and Castenholz 1982). Kallas and Castenholz showed that the cytosolic pH of the cell
remains constant in both prokaryotic and eukaryotic cells during the decrease in medium
pH (1982). This eliminates intracellular pH as the cause of chlorophyll degradation and
suggests that chlorophyll degradation is not the cause of decreased cell viability, but is a
resulting effect. Figure 4 shows micrographs of cells at consecutive sampling points,
which indicates chlorophyll degradation into pheophytins by the loss of the bright green
color.
34
Figure 4. Micrographs for Kirchneriella sp. 92 (top row) and Scenedesmus sp. 131
(bottome row) grown on ammonium with 5% CO2 over consecutive sampling time points
showing the decrease in chlorophyll content.
Previous research associated with growth on ammonium did not take into account
the change in pH, because it was either not monitored or reported, and therefore was not
considered as a factor for reduced growth (Bongers 1956; Li et al. 2008; Ludwig 1938).
Table 2 shows little difference in the average growth rate between species and the
different nitrogen conditions. Table 2 also indicates that both strains had a lower DCW
when grown on urea in comparison to nitrate. The final DCW on ammonium was
significantly lower due to chlorophyll degradation preventing further growth. The
biofuel potential as shown in Table 2 indicates that strain 131 had the highest biofuel
35
potential when grown on urea compared to nitrate; whereas, strain 92 has a higher biofuel
potential when grown on nitrate.
Table 2. Comparison of Nitrogen Source When Grown on 5% CO2. Reported values
include standard deviation of experiments in triplicate.
Experimental Condition
Nitrate on 5% CO2
Urea on 5% CO2
Ammonium on 5% CO2
Experimental Condition
Nitrate on 5% CO2
Urea on 5% CO2
Ammonium on 5% CO2
Scenedesmus sp. 131
Specific
Final DCW
Growth
(g DW/L)
Rate (d-1)
1.12 ± 0.07 2.41 ± 0.04
1.02 ± 0.02 1.89 ± 0.06
0.90 ± 0.04 0.34 ± 0.07
Kirchneriella sp. 92
Specific
Final DCW
Growth
(g DW/L)
Rate (d-1)
1.09 ± 0.07 1.89 ± 0.06
1.27 ± 0.05 1.30 ± 0.30
1.13 ± 0.01 0.51 ± 0.01
Final Cell
Concentration
(cells/mL)
2.17 ± 0.07 x 107
1.64 ± 0.26 x 107
2.20 ± 0.61 x 104
Biofuel
Content
(% w/w)
25.6 ± 1.0
30.2 ± 1.0
8.9 ± 1.3
Final Cell
Concentration
(cells/mL)
3.39 ± 0.52 x 107
3.15 ± 1.26 x 107
1.03 ± 0.11 x 106
Biofuel
Content
(% w/w)
38.4 ± 1.9
33.9 ± 2.8
23.80 ± 10.9
Growth on Air with on Nitrate or Ammonium
To analyze the change in pH and effects of carbon concentration, strain 131 and
strain 92 were grown on nitrate or ammonium in the presence of air to decrease the
buffering effect of the carbonate equilibrium. Growth on either nitrate or ammonium
caused a flux of hydroxyl ions or protons, respectively, into the medium (Fuggi et al.
1981). This is shown in Figure 5 by the significant pH change. In nitrate, the pH steadily
increased, due to the release of hydroxyl ions during nitrate uptake and the utilization of
carbonic acid and bicarbonate in the medium, which shifted the carbonate equilibrium to
a pH above 11 where carbonate ions were the predominant form (Fig. 5). Growth on
nitrate alone increased the pH above 9 based on the introduction of hydroxyl ions in to
36
Figure 5. Total Nile Red fluorescence (solid line) and nitrogen concentration (dashed
line) (a), pH (b) and average cell density (c) with standard deviation for triplicate reactors
(1) Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on air using nitrate (■) or
ammonium () as the nitrogen source.
37
the medium during nitrate uptake (Fuggi et al. 1981). This is in contrast to ammonium
based experiments, where pH decreased even with the lack of DIC in the medium
(Schumacher et al. 2003). This is because the increase in protons due to ammonium
uptake creates a stronger buffer than the carbonate equilibrium. Previous research has
discussed carbon limitation and PEPC having a higher affinity for carbon than
RUBISCO. However, the decrease and shift in the DIC equilibrium and availability due
to decreasing pH has not been discussed with growth on ammonium in unbuffered
medium (Elrifi et al. 1988; Guy et al. 1989; Schuller et al. 1990). The DIC equilibrium
of the medium is primarily influenced by the pH in this instance. DIC availability is
associated with the carbon flux into the medium, and due to constant aeration rate and
bubble size, is dictated by the driving force. The driving force is dependent upon the
shift in the actual concentration of DIC compared to equilibrium. Figure 5 shows that the
growth of both strain 131 and strain 92 on nitrate and ammonium was limited at higher
cell concentrations and assumed to be due to the DIC limitation in the medium, based on
the change in specific growth rate. Further evidence for DIC limitation is shown in
Figure 7 where the pH increased to above 11 for growth on nitrate. It is important to note
that the large standard deviation for the cell concentration of strain 131 grown on
ammonium was due to the degradation of chlorophyll in one tube prior to the other two,
and therefore a lower viable cell count for growth in the given medium. Figure 5 also
shows that when grown on ammonium both strain 131 and strain 92 did not fully utilize
ammonium due to pH stress. However, both strain 131 and strain 92 utilized nitrate
within 8 days and 12 days, respectively. The increase in Nile Red fluorescence for both
38
strains grown on nitrate correlates with nitrogen depletion. Additionally, the reason for
the large standard deviation for Nile Red fluorescence values for strain 92 and strain 131
when grown on nitrate with air is due to the lack of an increase in fluorescence for one of
the three tubes in each experiment.
With Scenedesmus, the increased pH has been shown to limit cell cycling and
allow for an increase in Nile Red fluorescence and TAG accumulation (Gardner et al.
2010). The increase in pH appears to allow the cell to continue the influx of carbon, but
instead of directing the carbon towards photosynthetic growth, the pH stress causes the
cell to convert fixed carbon into lipid storage. As shown in Figure 5, strain 131 growing
on nitrate had an increase in Nile Red fluorescence that was greater than when grown on
5% CO2; however, to increase the Nile Red fluorescence by growing the algae on air
required 21 days instead of 10 to 12 days. For strain 92 growing on nitrate, the total Nile
Red fluorescence of the system was similar for the strain grown on 5% CO2 or air, with
the exception that growth on air required twice the time to reach the same Nile Red
fluorescence. Figure 5 also shows that when either strain 131 or strain 92 were grown on
air with ammonium, the cultures grew for approximately 16 days before cell
concentrations decreased due to chlorophyll degradation.
As shown in Table 3, the strains reached a lower cell concentration and DCW
when grown on nitrate with air in comparison to 5% CO2, which is attributed to the
increase in pH and carbon limitation. Table 3 also shows that strain 131 grown on nitrate
with air had an increased biofuel potential compared to nitrate with 5% CO2.
Furthermore, Table 3 also shows that there is a decrease in DCW when grown on
39
ammonium with air compared to ammonium with 5% CO2 for strain 92, which is may be
attributed to the lower concentration of DIC.
Table 3. Comparison of Nitrate or Ammonium Grown on Air. Reported values include
standard deviation of experiments in triplicate.
Scenedesmus sp. 131
Experimental Condition
Nitrate on Air
Ammonium on Air
Experimental Condition
Nitrate on Air
Ammonium on Air
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
0.43 ± 0.03 1.23 ± 0.23
1.00 ± 0.18 x 107
0.55 ± 0.05 0.26 ± 0.02
3.29 ± 3.03 x 105
Kirchneriella sp. 92
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
0.43 ± 0.05 1.13 ± 0.21
2.69 ± 0.59 x 107
0.49 ± 0.01 0.37 ± 0.02
4.35 ± 0.72 x 106
Biofuel Content
(% w/w)
33.4± 5.9
8.2 ± 2.4
Biofuel Content
(% w/w)
24.1 ± 1.5
14.1 ± 0.3
The decrease in final DCW for growth on unbuffered ammonium with air
compared to nitrate with air (Table 3) is attributed to two major differences associated
with the nitrogen source and the strains' metabolism: 1) The increase in anaplerotic
reactions associated with growth on ammonium (Norici and Giordano 2002), and 2) The
change in pH, which affects the DIC equilibrium and availability. Previous papers have
reported growth after inoculation in ammonium based medium; however, growth stopped
during the middle of the experiments (Bongers 1956; Dvořáková-Hladká 1971). These
experiments were grown without the addition of CO2. Therefore, without DIC being
measured, it is possible that DIC became limiting and could account for the lack of
growth at higher cell concentrations. Table 3 also shows that growth on ammonium had
40
lower biofuel potential, which may be attributed to growth limitations associated with pH
and potentially a decreased DIC equilibrium and availability.
Growth on Ammonium with Biological Buffers
The abundance of ammonium in wastewater and its ability to inhibit utilization of
other nitrogen sources in mixed medium indicates the importance for studying and
understanding methods to increase bioremediation and lipid production of algal growth
on ammonium. Research has documented high ammonium removal rates from
wastewater using algae (Aslan and Kapdan 2006; Nunez et al. 2001; Voltolina et al.
1999). However, few studies have attempted to determine lipid accumulation with
ammonium as the sole nitrogen source. Recently lipid accumulation on ammonium in
comparison to nitrate was reported by Li et al. (2008), but the paper did not take into
account the decrease in the medium pH, which can inhibit cell growth and is the most
likely cause for a decreased cell yield and a decreased lipid accumulation. Additionally,
previous papers on algal ammonium removal also did not consider the effects of pH and
CO2 concentration on the rates of ammonium removal (Garcia et al. 2000; Voltolina et al.
1999). When cultures were grown on ammonium in the presence of CO2 or air, the pH
dropped quickly (Fig. 3 and Fig. 5), causing the strains to become inhibited and the
chlorophyll to degrade, based on visual observation. The increase in anaplerotic
reactions for growth on ammonium, in the presence of low DIC concentrations, may
prevent complete utilization of ammonium from the medium (Amory et al. 1991; Elrifi et
al. 1988; Norici and Giordano 2002; Vanlerberghe et al. 1990). Higher DIC
41
concentrations are required to prevent a reduction in specific growth rate on ammonium
during exponential growth, which is dependent on the flux of carbon into the medium.
When grown on ammonium, algae require approximately 0.3 moles of carbon per mole
of nitrogen for utilization in anaplerotic reactions required to replace TCA intermediates
(Guy et al. 1989; Turpin 1990). Furthermore, these reactions have been shown to have a
greater affinity for carbon and therefore reduce the amount of carbon available to provide
photosynthetic CO2 fixation for growth (Elrifi et al. 1988; Norici et al. 2002;
Vanlerberghe et al. 1990).
To prevent the decrease in pH and the resultant drop in DIC associated with
growth on ammonium, biological buffers PIPES (pKa 6.76) and HEPES (pKa 7.5) were
added to the medium for different experiments. The experiments used a concentration of
8 mM buffer, which was more than double the concentration of protons capable of being
released from the cell. As shown in Figure 8, the pH of the medium still dropped.
However when buffered, the change in pH was less than one pH unit (on air or 5% CO2),
whereas previously, the medium dropped several pH units and the ammonium was not
fully utilized (Fig. 3 and Fig. 5).
Figure 6 shows Scenedesmus sp. 131 had very slow growth on air when buffered
with HEPES and likely became carbon limited after 4 days at a cell concentration of
approximately 6.3x105 cells/mL. This was assumed because of the linear increase in cell
concentration after day 4, which showed growth was most likely limited by the carbon
flux into the medium. Kirchneriella sp. 92 showed similar results (Fig. 6) and likely
became carbon limited after 4 days at a cell concentration of approximately 3.6x106
42
Figure 6. Total Nile Red fluorescence (solid line) and nitrogen concentration ( dashed
line) (a), pH (b) and average cell density (c) with standard deviation for triplicate reactors
(1) Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on ammonium as the
nitrogen source and growing on air buffered with 8 mM HEPES (●) growing on air with
PIPES. (○) growing on CO2 with PIPES (■) and growing on CO2 with PIPES plus 2 mM
KOH injection (final concentration) (□).
43
cells/mL. Both cultures were run for 26 days to monitor ammonium uptake and Nile Red
fluorescence. Using Nessler's assay (Roon and Levenberg 1968), ammonium was
monitored during the last 10 days of the experiment on strain 131 showing a decrease in
ammonium concentration to approximately 1 mg-NH4+/L, the limit of quantification,
which remained in the medium until the time of harvesting. Strain 92 showed greater
ammonium removal and ammonium decreased below detection limits by day 16. These
experiments also emphasize the increased carbon requirement for strain 131.
Strains were also grown on air with ammonium using an 8 mM PIPES buffer
(pKa 6.8). Figure 8 shows that strain 131 became carbon limited after 4 days at a cell
concentration of approximately 8.3x105 cells/mL and strain 92 after 4 days at a cell
concentration of approximately 2.1x106 cells/mL. This was assumed because of the
linear increase in cell concentration, which showed growth was most likely limited by the
carbon flux into the medium.
Figure 6 also shows that ammonium utilization when the cultures were grown on
air was slow, and that only strain 92 when grown in HEPES buffer completely utilized
the ammonium during the 26 day experiments. For that culture, ammonium was removed
in 16 days. The slow utilization of ammonium was due to the slow growth associated
with low carbon conditions. This shows that for efficient ammonium removal, inorganic
carbon must be added by increasing the CO2 concentration. To increase the DIC
concentration, the strains were grown on 5% CO2 and buffered with PIPES. This allowed
for evaluation of the maximum growth rate, DCW, Nile Red fluorescence, and biofuel
potential.
44
In addition to growth on air, Figure 6 also shows cultures grown on ammonium
with 5% CO2 buffered with PIPES, which had an initial pH of 6.6 and a final pH of 6.1,
due to acidification of the medium. The decrease in the pH decreases the DIC
equilibrium and availability, which could decrease the growth and biofuel potential in
dense cultures. To assess what would occur if the pH remained higher, 2 mM KOH (final
concentration) was added once the pH dropped below 6.4 to increase the pH to 6.8. The
change in DIC equilibrium, based on pH of 6.1 and 6.8 is 2.65 mM and 6.47 mM,
respectively, and is an approximate 2.5 fold increase in the DIC concentration at
equilibrium (STP). Figure 6 shows the pH adjustment had little effect on growth for
either strain. However, the increase in Nile Red fluorescence in Scenedesmus when
buffered with 8 mM PIPES and spiked with 2 mM KOH was unexpected with the small
increase in pH, as previous work has shown that high pH is required to limit cell cycling
and increase Nile Red fluorescence with nitrate as the nitrogen source (Gardner et al.
2010). The potential reason for this change in Nile Red fluorescence may be due to the
difference in DIC equilibrium and availability at a pH of 6.8 and 6.1 or the addition of
KOH created an ionic stress. Figure 6 also shows that the strains efficiently removed the
ammonium from the medium in 5 days for strain 92 and 6 days for strain 131 in either
experiment with 5% CO2, and that the addition of 2 mM KOH did not affect ammonium
utilization.
Table 4 shows the average specific growth rate, DCW, and biofuel potential for
both strains. When grown on 5% CO2 both strains showed growth rates and final DCWs
similar to cultures grown on urea or nitrate with 5% CO2. Strain 92 also showed similar
45
biofuel potential to cultures grown on urea or nitrate with 5% CO2. However, strain 131
showed a decrease in biofuel potential, which may be attributed to an increase in protein
content of the cells (Lourenco et al. 2002).
Table 4. Comparison of Growth on Ammonium with Biological Buffers. Reported
values include standard deviation of experiments in triplicate.
Scenedesmus sp. 131
Experimental Condition
Ammonium on Air
HEPES Buffer
Ammonium on Air
Pipes Buffer
Ammonium on 5% CO2
Pipes Buffer
Ammonium on 5% CO2
Pipes Buffer and 2 mM
KOH
Experimental Condition
Ammonium on Air
HEPES Buffer
Ammonium on Air
Pipes Buffer
Ammonium on 5% CO2
Pipes Buffer
Ammonium on 5% CO2
Pipes Buffer and 2 mM
KOH
Specific
Growth
Rate (d-1)
Final DCW
(g DW/L)
Final Cell
Concentration
(cells/mL)
Biofuel Content
(% w/w)
0.22 ± 0.02
0.70 ± 0.13
6.84 ± 0.27 x 106
18.4 ± 1.0
0.26 ± 0.01
0.58 ± 0.10
4.17 ± 0.12 x 106
12.4 ± 4.1
1.04 ± 0.04
1.64 ± 0.01
1.75 ± 0.08 x 107
19.2 ± 0.7
1.18 ± 0.02
2.09 ± 0.09
1.48 ± 0.10 x 107
19.0 ± 0.3
Kirchneriella sp. 92
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
Biofuel Content
(% w/w)
0.45 ± 0.01
0.93 ± 0.03
2.82 ± 0.28 x 107
21.9 ± 2.7
0.46 ± 0.02
0.88 ± 0.06
2.18 ± 0.11 x 107
13.8 ± 0.7
1.31 ± 0.05
1.70 ± 0.03
2.81 ± 0.11 x 107
35.4 ± 4.3
1.58 ± 0.03
1.26 ± 0.23
1.94 ± 0.92 x 107
33.8 ± 1.9
Table 4 also shows that the change in pH, for growth on air with HEPES or
PIPES, had little effect on the specific growth rate and the cell concentration at which the
medium became carbon limited. However, based on equilibrium concentrations at STP
46
for an open system, the change in pH from 7.3 to 6.5 decreases the DIC equilibrium from
0.135 mM to 0.033 mM; this change in DIC concentration may increase the DIC
availability and may account for the increased biofuel potential of both strains as shown
in Table 4. However, it is important to note that experiments with buffers are limited to
lab scale, and can only be used as an initial step prior to using pH-controlled reactors.
Growth on Ammonium using pH Control
The next set of experiments used active pH control, which added 0.1 M KOH to
maintain near-constant pH. The strains were inoculated into BBM with ammonium and
grown on 5% CO2. The cultures were grown in duplicate in two separate experiments for
a total of four cultures as shown in Figure 7. The pH of the medium prior to inoculation
was 6.2. Over the first two days, the pH was increased in a stepwise manner. The pH
controller was set at 6.2 for day zero. For day one, the set point was increased to 6.4, and
finally set at 6.55 on day two. The reason the pH was increased incrementally was to
allow the carbonate equilibrium shift due to algal growth to aid in the pH increase and
reduce the amount of KOH required to achieve the final pH. The pH was set to 6.55 ±
0.05, because it was the highest pH allowable with 5% CO2, without the addition of
enough KOH to significantly change the volume of the reactors.
The two main problems associated with growing algae with ammonium, as the
nitrogen source, are the inability to maintain growth due to the significant decrease in pH,
and the decrease in biofuel potential for strain 131. When the pH is controlled through
the use of a pH controller or a pH buffer, these negatives effects can be reduced or
47
Figure 7. Total Nile Red fluorescence (a), pH (b) and average cell density (c) with
standard deviation for duplicate reactors (1) Scenedesmus sp. 131 and (2) Kirchneriella
sp. 92 grown on ammonium with pH control run 1 (■) run 2 (○).
48
eliminated. Figure 7 shows when algae are grown on 5% CO2 and pH controlled, the
algal growth rate is comparable to growth on buffered ammonium with 5% CO2, nitrate
on 5% CO2, and urea on 5% CO2. However, when the algae were grown on 5% CO2, the
Nile Red fluorescence was lower in the pH controlled reactors than when buffered with 8
mM PIPES. In the first run, strain 131 had a significant lag phase that differed between
the two tubes. Tube 1 had a lag period of three days, while tube 2 had a lag of two days
and accounts for the large error bars in Figure 7. Figure 7 also shows that Kirchneriella
sp. 92 reached higher cell concentrations of 4.5 x 107 cells/mL on ammonium compared
to growth on buffered ammonium with 5% CO2, nitrate on 5% CO2, and urea on 5% CO2,
which reached between 2 and 3 x 107 cells/mL. However, Table 5 shows that strain 92
did not achieve a higher DCW, indicating that the cells most likely decreased in size.
Table 5 also shows that the biofuel potential, final DCW, and specific growth rate for
strain 131 and strain 92 were comparable to growth on ammonium with PIPES and 5%
CO2.
49
Table 5. Comparison of Growth on Ammonium with 5% CO2 Using pH Control.
Reported values include standard deviation of experiments in duplicate.
Experimental Condition
Ammonium on 5%
CO2 with pH
Controller Run 1
Ammonium on 5%
CO2 with pH
Controller Run 2
Experimental Condition
Ammonium on 5%
CO2 with pH
Controller Run 1
Ammonium on 5%
CO2 with pH
Controller Run 2
Scenedesmus sp. 131
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
Biofuel Content
(% w/w)
1.16 ± 0.03
2.25 ± 0.17
1.25 ± 0.24 x 107
19.8 ± 1.2
1.05 ± 0.04
1.87 ± 0.03
8.32 ± 0.12 x 106
19.0 ± 0.6
Kirchneriella sp. 92
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
Biofuel Content
(% w/w)
1.42 ± 0.01
1.46 ± 0.08
4.64 ± 0.07 x 107
28.6 ± 2.5
1.40 ± 0.09
1.71 ± 0.07
4.58 ± 0.39 x 107
31.8 ± 3.2
Analysis of Culture Conditions for Optimal Nitrogen Removal
Culture conditions had a significant impact on the strains' abilities to remove
nitrogen from the media. The main factor that affects growth and therefore nitrogen
remediation is the DIC available. Therefore, high concentrations of CO2 must be present
for the algae to efficiently remove nitrogen from the medium (Amory et al. 1991). When
the strains were grown on ammonium with air, compared to nitrate with air, the nitrogen
removal rate for both strains was decreased. This is associated with the increased carbon
requirements for anaplerotic reactions for growth on ammonium over nitrate (Elrifi et al.
1988), and the change in pH. This change in pH due to translocation of either a proton or
50
hydroxyl ion during the uptake of ammonium or nitrate, respectively, which influenced
pH that controls the DIC equilibrium and availability. The change in ammonium
concentration was monitored in both HEPES and PIPES buffered experiments grown on
air, which showed that the nitrogen removal time for HEPES buffered samples, with a
final pH of 7.2 to 7.4 (Fig. 6), was greater than that for samples grown in PIPES, with a
final pH of 6.4 to 6.5 (Fig. 6), as shown in Table 6 and Figure 8. This is most likely due
to the increased DIC equilibrium and availability, which provided additional carbon for
ammonium uptake.
As shown in Table 6 and Figure 8, the time required for removal of nitrogen
when grown on 5% CO2 was between 5 and 6 days for both strains, and the rate of
nitrogen utilization was very similar. This was expected as the 5% CO2 concentration
provided an environment where fast growth could utilize nitrogen quickly. The only time
the strains did not fully utilize ammonium when grown on 5% CO2, was when the
medium had no pH buffer and the pH decrease prevented further growth (Fig. 3). When
the strains were grown on air, the time required for nitrogen depletion was somewhat
variable; from 8 days when grown on nitrate to incomplete utilization when grown on
unbuffered ammonium (Table 6). Table 6 shows that growth on unbuffered ammonium
in either air or 5% CO2 provided the highest nitrogen removal rates, however this is an
artifact due the low DCW and the method of calculating nitrogen removal rates.
Nitrogen removal rates were calculated by taking the amount of nitrogen used during
growth and eliminated nitrogen uptake during the initial lag phase, and dividing it by the
time during growth and the final DCW as shown in Equation 14.
51
N - Removal Rate =
mg - N
d * g - Biomass
(Equation 14)
Figure 8. Average nitrogen concentration in mM nitrogen with standard deviation for (1)
Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on CO2 (a) using nitrate (□),
urea (♦), unbuffered ammonium (▲), bicarbonate addition (○), ammonium with PIPES
(▼), ammonium with PIPES plus 2 mM KOH spike () and ammonium with pH
controllers run 1 () run 2 () . The strains were also grown on air (b) with nitrate (■),
unbuffered ammonium (), ammonium with HEPES (►), and ammonium with PIPES
(◄).
52
Table 6. Comparison of Time to Remove Nitrogen from Media.
Nitrogen
Source
Air/5%
CO2
pH Buffer
Nitrate
Nitrate
Urea
Air
5% CO2
5% CO2
-
Ammonium
Air
-
Ammonium
5% CO2
-
Ammonium
Air
8 mM HEPES
Ammonium
Air
8 mM PIPES
Ammonium
5% CO2
Ammonium
5% CO2
Ammonium
5% CO2
Ammonium
5% CO2
8 mM PIPES
8 mM PIPES
+ 2 mM KOH
pH Controlled
run 1
pH Controlled
run 2
Scenedesmus sp. 131
Removal
Removal
Time
Rate
mg-N/d/gdays
Biomass
8
6.51 ± 1.29
6
4.48 ± 0.03
5
5.66 ± 0.42
10 (55%)*
6.68 ± 0.21
17 (71%)*
5 (71%)*
17.7 ± 1.56
14 (95%)*
2.73 ± 0.56
26 (98%)*
20 (91%)*
2.29 ± 0.45
26 (98%)*
6
7.54 ± 0.09
Kirchneriella sp. 92
Removal
Removal
Time
Rate
mg-N/d/gdays
Biomass
12
4.13 ± 0.75
6
5.30 ± 0.34
6
8.10 ± 2.27
10 (57%)*
4.64 ± 0.10
18 (68%)*
7 (80%)*
12.52 ± 0.03
16
18 (95%)*
26 (97%)*
5
3.05 ± 0.19
1.98 ± 0.10
7.42 ± 0.36
5
6.89 ± 0.24
5
11.18 ± 1.51
6
4.82 ± 0.26
6
6.63 ± 0.66
5
4.17 ± 0.04
5
5.53 ± 0.33
*If the experiment did not utilize all of the nitrogen in the medium, the total time the
experiment was run was placed in the table along with the percent of the nitrogen used in
parenthesis. Experiments where two values are given, indicate the time point when a
majority of the nitrogen was removed and the total duration of the experiment.
Strain 92 showed little variation in growth rate and DCW between growth on
ammonium with air and buffered with PIPES or HEPES values (Table 3). However, the
change in pH affected the uptake of ammonium where, during growth with HEPES,
ammonium depleted after 16 days (strain 92), but during growth in PIPES, ammonium
was not fully utilized after 26 days. Strain 92 also had a difference in ammonium
utilization rate, as shown in Table 6. This indicates that the small increase in DIC
equilibrium and availability between PIPES and HEPES slightly increased ammonium
53
utilization. Table 6 also shows that growth with HEPES buffer had an improved nitrogen
utilization time, and the rate of ammonium utilization was increased for strain 92.
As shown in Table 7, cell yields with respect to nitrogen (YX/N) for both strain 92
and strain 131 were highly variable depending on the experimental conditions. This is
associated with the variation in DIC available for growth during the experiment. The
YX/N was calculated using equation 15, which used the final DCW and the nitrogen
consumed during the course of the experiment.
Yield XN = -
(final - inoculum) g Biomass
(final - initial) g Nitrogen
(Equation 15)
Table 7. Nitrogen Yield Comparison for Experiments. Reported values include standard
deviation of experiments in triplicate.
Nitrogen
Source
Nitrate
Nitrate
Urea
Ammonium
Ammonium
Ammonium
Ammonium
Ammonium
Air/5%
CO2
Air
5% CO2
5% CO2
Air
5% CO2
Air
Air
5% CO2
Ammonium
5% CO2
Ammonium
run 1
Ammonium
run 2
pH Buffer
8 mM HEPES
8 mM PIPES
8 mM PIPES
8 mM PIPES
+ 2 mM KOH
Scenedesmus sp. 131
(g-biomass/g-nitrogen)
25.8 ± 4.1
55.0 ± 2.3
42.1 ± 2.5
8.8 ± 0.3
11.4 ± 1.1
17.3 ± 3.4
14.3 ± 2.6
42.8 ± 0.5
Kirchneriella sp. 92
(g-biomass/g-nitrogen)
24.4 ± 4.3
42.9 ± 1.6
29.9 ± 7.4
12.9 ± 0.6
15.2 ± 0.1
22.5 ± 1.6
19.1 ± 1.6
42.3 ± 3.5
45.3 ± 1.1
29.0 ± 4.3
5% CO2
pH Controlled
51.1 ± 1.6
36.4 ± 2.8
5% CO2
pH Controlled
47.9 ± 0.4
41.4 ± 1.4
It can be seen in Table 7 that growth on 5% CO2 with nitrate, urea, buffered
ammonium, or pH-controlled ammonium had the greatest nitrogen yields, which
54
indicates that the strains likely had sufficient carbon for growth, and were not inhibited
by pH. Table 7 also shows that among these conditions growth on nitrate with 5% CO2
and growth on ammonium with pH controllers provided the highest nitrogen yields.
Nitrogen yields for growth on ammonium or nitrate on air, were lower than growth on
5% CO2, specifically the yield for growth on nitrate with air was approximately half that
of the yield for growth on nitrate with 5% CO2. Growth on buffered ammonium with air
provided lower nitrogen yields than growth on nitrate with air. Overall, Table 7 shows
that for high nitrogen yields, 5% CO2 is required.
55
5. BICARBONATE INJECTION
Even though there was little difference in lipid content for Kirchneriella sp. 92
when grown on air or CO2 with nitrate as the nitrogen source, there was a difference in
Scenedesmus sp. 131 (Fig. 9). Recent work has shown that increased pH at the time of
nitrogen depletion can increase Nile Red Fluorescence (Gardner et al. 2010). Therefore,
both strains were tested with the bicarbonate injection prior to nitrogen stress as
described by Gardner et al. (2011). The basis of the addition is to grow the strains on 5%
CO2 during exponential growth and, prior to nitrogen stress, switch the reactor to air and
add 50 mM sodium bicarbonate (final concentration) to increase the DIC of the medium.
This method is designed to provide a sudden large increase in DIC, as well as create a pH
increase related to the algal production of hydroxyl ions from bicarbonate use (Shiraiwa
et al. 1993). Figure 9 shows the result of the bicarbonate addition for strain 131 and
strain 92, along with growth on nitrate in air or 5% CO2 for comparison.
The experiment showed that strain 131 responded to the bicarbonate spike and
increased to its maximum lipid concentration in 11 days instead of 21 days when grown
on air. This was expected due to the presence of the bicarbonate pump in Scenedesmus,
which allows for direct transport of bicarbonate ions across the cell membrane (Shiraiwa
et al. 1993). The experiment also showed that for strain 92 the sudden increase in pH
prevented a large increase in Nile Red Fluorescence (Fig. 9), and is most likely due to
variation in the growth mechanisms of strain 92 compared to strain 131. Kirchneriella
56
Figure 9. Total Nile Red fluorescence (a), pH (b) and average cell density (c) with
standard deviation for (1) Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on
nitrate and utilizing the bicarbonate addition grown on 5% CO2 and switched to air (○)
growing on 5% CO2 (□) and growing on air (■). (↓) Represents the point at which 50
mM sodium bicarbonate (final concentration) was injected into the Scenedesmus media.
57
utilizes autospore formation to split into 3 or 4 individual cells, whereas Scenedesmus
grows through a cellular division into 2, 4, or 8 cells (Pickett-Heaps and Staehelin 1975;
Pickett-Heaps 1970; Trainor et al. 1976). It appears that Kirchneriella has lipid
associated with the autospores independent of environmental conditions, and once the pH
increases, the cell-cycling delay did not increase the overall lipid content of the strain as
extensively as nitrogen depletion.
Table 8 shows the growth rate, final DCW and the lipid content of the strains. The
biofuel potential for strain 131 was similar to growth on 5% CO2 and lower than growth
on air. Table 8 also shows that strain 131 produced a similar DCW to growth on nitrate
with air, which is less than half the DCW produced during growth on nitrate with 5%
CO2. Strain 92 produced a lower biofuel potential and DCW compared to growth on 5%
CO2, which indicates that the strain requires 5% CO2 during the entire experiment for
increased DCW.
Table 8. Bicarbonate Injection. Reported values include standard deviation of
experiments in triplicate.
Experimental Condition
Scenedesmus sp. 131
Specific
Final Cell
Final DCW
Growth
Concentration
(g DW/L)
Rate (d-1)
(cells/mL)
Nitrate on 5% CO2
+ 50mM bicarbonate
1.44 ± 0.10
Experimental Condition
Specific
Growth
Rate (d-1)
Nitrate on 5% CO2
+ 50mM bicarbonate
1.30 ± 0.06
Biofuel Content
(% w/w)
1.09 ± 0.06 x 107
24.1 ± 2.4
Final DCW
(g DW/L)
Final Cell
Concentration
(cells/mL)
Biofuel Content
(% w/w)
1.11 ± 0.10
2.05 ± 0.23 x 107
26.7 ± 0.3
1.11 ± 0.02
Kirchneriella sp. 92
58
6. COMPARISON OF SCENEDESMUS SP. 131 AND KIRCHNERIELLA SP. 92
Figure 10a and Figure 10b shows that strain 92 is capable of decreasing the
cellular size in the presence of low carbon to allow it to continue to grow to higher cell
concentrations. Under optimal conditions when grown on ammonium, buffered with
PIPES, and 5% CO2 the strain produces high Nile Red fluorescence and what is assumed
to be lipid vacuoles as seen in Figure 10c. Figure 10d shows the same cell using Nile
Red fluorescence, which indicates high concentrations of lipids as indicated by the bright
yellow color.
Figure 10. Micrographs of strain 92 growing a) on nitrate with air b) and c) on
ammonium buffered with PIPES on 5% CO2. d) is a fluorescence micrograph of c)
stained with Nile Red.
59
Figure 11a also shows when strain 131 was grown on 5% CO2 the cells were
larger and did not produce a pyrenoid. Strain Figure 11b shows strain 131 growing with
air on nitrate, and the presence of a large visible pyrenoid, indicative of carbon limitation
(Giordano et al. 2005). This is in stark comparison to strain 92, which has a naked
pyrenoid, meaning that it does not produce a visible spot (Marvan et al. 1984; PickettHeaps 1970). As Figure 11c shows, when strain 131 is grown on nitrate and air, the Nile
Red Fluorescence is optimal, and mainly produces lipids in two vacuoles at each end of
the cell. In every condition tested, strain 92 had a consistently higher Nile Red
fluorescence. Both strains had a doubling time that ranged from 10 to 13 hours during
mid exponential phase; however, in a few experiments, strain 92 showed a maximum
doubling time close to 9 hours.
Figure 11. Micrographs of strain 131 growing a) on ammonium buffered with PIPES on
5% CO2, b) on nitrate with air, and c) is a fluorescence micrograph of b) stained with Nile
Red.
60
Figure 12 shows the optimal Nile Red fluorescence for both strain 131 and strain
92. Strain 131 produced optimal Nile Red fluorescence in 11 days when utilizing the
bicarbonate injection method, whereas strain 92 produced optimal Nile Red fluorescence
in 7 days when grown on ammonium buffered with PIPES using 5% CO2. The Nile Red
fluorescence of strain 92 was approximately 6 to 7 times greater than strain 131, and was
accomplished in less time. The two strains require significantly different conditions to
increase Nile Red Fluorescence. Strain 131 requires high pH to increase cell-cycle
inhibition combined with nitrogen stress to achieve optimal Nile Red fluorescence,
whereas strain 92 requires a neutral pH accompanied by nitrogen stress for optimal Nile
Red fluorescence.
Table 9 provides a comparison of the concentration of total biofuel potential in gFAME/L-medium, and was calculated by multiplying the % biofuel potential (w/w) by
the final DCW. Table 9 shows that strain 131 had a lower total biofuel potential when
grown on ammonium compared to growth on nitrate and urea. Furthermore, strain 131
also shows that, in order to achieve a high total biofuel potential, growth needs to be on
5% CO2. Table 9 also shows that strain 92 was not affected by the nitrogen source, but
provides further evidence that 5% CO2 is required to increase the total biofuel potential.
To assess the overall biodiesel productivity for these experiments, the data was
extrapolated using three assumptions: 1) Cultures grown on 5% CO2 have a 2.5 week
growth and harvest period, 2) Cultures grown on air have a 3.5 week growth and harvest
period due to the slower growth rate, and 3) A 1 hectare pond is 0.3 m deep, providing a
total volume of 3 million liters. These calculations provide the results shown in Table 10,
61
Figure 12. Total Nile Red fluorescence (a), pH (b) and average cell density (c) with
standard deviation for triplicate reactors (○) Scenedesmus sp. 131 grown on nitrate as the
nitrogen source and utilizing the bicarbonate addition and (■) Kirchneriella sp. 92 grown
on ammonium with PIPES on 5% CO2. (↓) Represents the point at which 50 mM sodium
bicarbonate (final concentration) was injected into the Scenedesmus media.
62
Table 9. Comparison of Biofuel Potential Concentrations. Reported with standard
deviation of the biological reactor replicates.
Nitrogen Source
Air/5%
CO2
Nitrate
Nitrate
Air
5% CO2
Nitrate
5% CO2
Urea
Ammonium
Ammonium
Ammonium
Ammonium
Ammonium
5% CO2
Air
5% CO2
Air
Air
5% CO2
Ammonium
5% CO2
Ammonium run 1
Ammonium run 2
5% CO2
5% CO2
pH Buffer
Bicarbonate
Injection
8 mM HEPES
8 mM PIPES
8 mM PIPES
8 mM PIPES
+ 2 mM KOH
pH Controlled
pH Controlled
Scenedesmus sp. 131
Biofuel Potential
(g/L)
0.42 ± 0.14
0.62 ± 0.03
Kirchneriella sp. 92
Biofuel Potential
(g/L)
0.27 ± 0.05
0.72 ± 0.03
0.27 ± 0.03
0.28 ± 0.03
0.58 ± 0.04
0.02 ± 0.00
0.03 ± 0.01
0.14 ± 0.01
0.08 ± 0.03
0.31 ± 0.01
0.45 ± 0.14
0.04 ± 0.00
0.12 ± 0.05
0.20 ± 0.03
0.12 ± 0.01
0.59 ± 0.08
0.40 ± 0.02
0.42 ± 0.06
0.44 ± 0.05
0.35 ± 0.06
0.42 ± 0.05
0.54 ± 0.03
which exacerbate the trends for difference in growth on 5% CO2 and air. This is due to
the difference in growth periods made during the initial assumptions. Table 10 shows
that growth on nitrate with 5% CO2 provides the largest amount of biodiesel production
for both strain 131 and strain 92. Growth on ammonium with pH controllers had a lower
productivity than growth on nitrate, but the range of productivity is on the same order of
magnitude as Chisti (2007).
63
Table 10. Estimated Biodiesel Productivity for Industrial-Scale Growth.
Nitrogen
Source
Air/5%
CO2
Nitrate
Air
Nitrate 5% CO2
Nitrate 5% CO2
Urea
5% CO2
Ammonium Air
Ammonium 5% CO2
Ammonium Air
Ammonium Air
Ammonium 5% CO2
Ammonium 5% CO2
Ammonium 5% CO2
Ammonium 5% CO2
Scenedesmus sp. 131 Kirchneriella sp. 92
Biodiesel
Biodiesel
(L/ha/year)
(L/ha/year)
21,200
13,800
43,800
51,400
Bicarbonate Injection
19,000
19,900
41,000
31,700
1,500
1,900
2,200
8,600
8 mM HEPES
7,000
10,400
8 mM PIPES
3,800
6,200
8 mM PIPES
22,300
42,200
8 mM PIPES + 2 mM KOH
28,600
30,100
pH Controlled run 1
31,000
30,000
pH Controlled run 2
17,800
38,500
pH Buffer
64
7. CONCLUSIONS
Scenedesmus sp. 131 and Kirchneriella sp. 92 can effectively remove nitrogen
from their environment for application in wastewater treatment. However, the main
requirement for efficient nitrogen removal to occur is increased CO2 concentrations. This
improves growth and therefore nitrogen utilization.

Strains 131 and 92 can grow on ammonium, but the pH of the medium must be
maintained between 6.5 and 7.5 for continued growth.

pH was initially maintained using the biological buffers PIPES (pKa 6.76) and
HEPES (pKa 7.5) with air to assess growth and nitrogen removal in the presence
of low DIC concentrations. This reduced the growth rate and nitrogen utilization
rate, indicating growth on ammonium requires increased concentrations of CO2.

Experiments showed that increasing the pH from 6.7 to 7.5, by using HEPES in
place of PIPES, improved the rate at which nitrogen was removed from the
medium when grown on air.

Strains grown on ammonium with PIPES and 5% CO2 had nitrogen removal rates
equivalent to algae grown on nitrate or urea with 5% CO2, and greater than when
grown on air.

The growth of stains 131 and 92 on ammonium with pH controllers and 5% CO2
was similar to growth on ammonium with buffers and 5% CO2, nitrate with 5%
CO2, and urea with 5% CO2.
65

Strain 131 had a lower % biofuel potential when grown on ammonium compared
to growth on nitrate or urea with 5% CO2. Strain 92 was not affected by the
nitrogen source.

Strain 131 and strain 92 required 5% CO2 to increase the total biofuel potential.

Overall, it was determined that when grown on ammonium, the pH of the medium
must be controlled and 5% CO2 is required to achieve higher DCW, biofuel
potential, and increased nitrogen removal rates.
66
8. SUGGESTIONS FOR FUTURE WORK
There are several areas that could be enhanced by future work. One of the most
interesting areas would be to investigate strains grown on a mixture of ammonium and
nitrate to analyze uptake rates, pH of the medium, and biofuel potential. The mixture of
ammonium and nitrate would decrease the pH of the medium at first when ammonium is
taken up. The pH of the medium would increase when nitrate is taken up, and would
increase the DIC equilibrium and availability to allow for better growth in dense cultures.
In addition, future work could examine scaling-up the strains growing on
ammonium using raceway ponds with pH-controlled reactors. This would allow for
testing pH and DIC gradients in the medium to analyze the distribution of base injection
points and bubblers for CO2. Growth in small-scale raceway ponds would provide
information on environmental and physiological change during scale-up.
Current tube reactors produce between 1 and 3 g/L of biomass. Experiments
could be completed with increased light intensity and higher nitrogen content to produce
cultures with DCWs greater than 3 g/L to assess the biofuel potential and growth rate of
dense cultures.
67
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Wei Q, Hu Z, Li G, Xiao B, Sun H, Tao M (2008) Removing Nitrogen and Phosphorus
from Dimulated Wastewater Using Algal Biofilm Technique. Frontiers of
Environmental Science & Engineering in China 2 (4):446-451
Woertz I, Feffer A, Lundquist T, Nelson Y (2009a) Algae Grown on Dairy and
Municipal Wastewater for Simultaneous Nutrient Removal and Lipid Production
for Biofuel Feedstock. Journal of Environmental Engineering-Asce 135
(11):1115-1122
Woertz I, Fulton L, Lundquist T Nutrient Removal & Greenhouse Gas Abatement with
CO2 Supplemented Algal High Rate Ponds. In: WEFTEC Annual Conference,
Orlando, FL, 2009b.
76
APPENDICES
77
APPENDIX A
ORGANISM SELECTION
78
Table A.1 presents the key features of each strain during initial screening on 5%
CO2 and nitrate. The main features of the algal species required by the grant for in-depth
study are to have a good growth rate and maximum lipid content. The preliminary data
show very low specific fluorescence associated with suboptimal environmental
conditions. An algal strain having high lipid accumulation should have a total
fluorescence of more than 10,000 or a specific fluorescence greater than 20 (based on
specific fluorescence values from other species with known lipid content). Table A.1
shows three values for growth of the organism: growth rate, doubling time and peak
doubling time. The peak doubling time looks at the data points during the exponential
growth phase.
Table A.1. Comparison of Initial Isolates.
Strain
-
Growth Rate (h
1
)
Doubling Time
(h)
Peak Doubling
Time (h)
Final Cell
Density
Final Dry
Weight (g/L)
Maximum Nile
Red
Maximum
Specific Nile
Red
Cultivation
Time at max
specific Nile
Red (days)
Light/Dark
Cycle
123
131
111
112
71T
92
0.041
0.046
0.039
0.045
0.039
0.045
16.7
14.9
17.6
15.5
17.7
15.3
12.6
14.1
16.3
11.7
15.8
13.4
1.94E+07
2.17E+07
6.72E+07
2.25E+07
2.78E+07
3.32E+07
2.41
2.25
3.04
2.38
1.89
2010
3470
2160
3025
1835
16075
1.03
1.60
0.51
1.52
0.70
5.26
14
10
6
8
10
9
14:10
14:10
14:10
14:10
14:10
14:10
79
A.1 Selected Strains
The two strains that were selected for continued investigation are 92 and strain
131. These strains were chosen based on good growth rates and were the best lipid
producing strains isolated.
A.2 Strain 123 Description
Strain 123 is a Scenedesmus sp. shown in Figure A.1. It is a green alga that grows
as a single cell or in groups of 2, 4, or 8. The grouping of this organism along with most
Scenedesmus species depend on the presence of predators, through the use of chemicals
such as urea, and bacteria, which increases the appearance of 4 and 8 cell groupings. The
organism was axenic during testing and was present in single cells and pairs. The large
error during exponential phase shown in Figure A.2 is due to a lag phase in one of the
tubes.
Figure A.1 Micrograph of strain 123 as a single cell and as a pair grown on nitrate and
5% CO2.
80
NO3 Average Growth Curve
Concentration (cells/ml)
1.0E+08
1.0E+07
1.0E+06
1.0E+05
Average
1.0E+04
0
5
Time (day)
10
15
Figure A.2 Average growth curve for strain 123 grown in triplicate on 5% CO2 and
nitrate.
A.3 Strain 131 Description
Strain 131 is a Scenedesmus sp. and was the second organism tested (Fig. A.3).
As shown in Table A.1, Strain 131 had the smallest overall doubling time and had the
third highest specific fluorescence. Figure A.4 shows the growth curve.
Figure A.3 Micrograph of strain 131 growing in a 4 cell grouping.
81
NO3 Average Growth Curve
Concentration (cells/ml)
1.0E+08
1.0E+07
1.0E+06
1.0E+05
Average
1.0E+04
0
5
10
Time (day)
15
20
Figure A.4 Average growth curve for strain 131 grown in triplicate on 5% CO2 and
nitrate.
A.4 Strain 111 Description
Strain 111 is another Scenedesmus sp. and reached the highest cell density (Fig.
A.5). This strain was smaller in comparison to most of the other strains and is probably
why it was capable of reaching a higher cell density. Figure A.6 shows the growth curve.
Figure A.5 Micrograph of strain 111 growing in a 4 cell grouping.
82
NO3 Average Growth Curve
Concentration (cells/ml)
1.0E+08
1.0E+07
1.0E+06
1.0E+05
Average
1.0E+04
0
5
10
Time (day)
15
20
Figure A.6 Average growth curve for strain 111 grown in duplicate on 5% CO2 and
nitrate.
A.5 Strain 112 Description
Strain 112 is another Scenedesmus sp. and has very long spines when in groups of
4. The culture was not axenic during the experiment. As shown in Figure A.7 this
organism has lipid vacuoles accumulating in the ends, and given proper conditions may
produce large amounts of lipid. Figure A.8 shows the growth curve noting the presence
of lag at the beginning and became nitrate depleted before day 6 causing the average
doubling time to be higher.
83
Figure A.7 Micrograph of strain 112 growing in 4 cell groupings with large spines and
visible lipid vacuoles.
NO3 Average Growth Curve
Concentration (cells/ml)
1.0E+08
1.0E+07
1.0E+06
1.0E+05
Average
1.0E+04
0
5
10
Time (day)
15
20
Figure A.8 Average growth curve for strain 112 grown in duplicate on 5% CO2 and
nitrate.
84
A.6 Strain 71T Description
Strain 71T is a Scenedesmus that was first isolated in a modified Bristol’s medium
(Fig. A.9). The strain was then transferred to Bold’s to compare with the other
Scenedesmus isolates due to the high plasticity of the genus. The key morphological
difference is the lack of spines on the cell. Figure A.10 shows the growth curve and the
initial lag
Figure A.9 Micrograph of strain 71T growing in a pair on 5% CO2 and nitrate.
Concentration (cells/ml)
1.0E+08
NO3 Average Growth Curve
1.0E+07
1.0E+06
1.0E+05
Average
1.0E+04
0
5
10
Time (day)
15
20
Figure A.10 Average growth curve for strain 71T grown in triplicate on 5% CO2 and
nitrate.
85
A.7 Strain 92 Description
Strain 92 is the only algal strain isolated that was not a Scenedesmus, and belongs
to the Selenastraceae family (Fig. A.11). Taxonomic identification has proven difficult,
but has been preliminarily identified as the genus Kirchneriella or Raphidocelis. Out of
all isolated strains, 92 has the best lipid production based on a total Nile Red fluorescence
of 16075 and a doubling time of 15.3 h. Figure A.12 shows the growth curve and the
error during exponential growth was due to small differences in initial cell
concentrations.
Figure A.11 Micrograph of strain 92 growing on 5% C02 and nitrate.
NO3 Average Growth Curves
Concentration (cells/ml)
1.00E+08
1.00E+07
1.00E+06
1.00E+05
Average
1.00E+04
0.00
5.00
10.00
15.00
Time (day)
20.00
25.00
Figure A.12 Average growth curve for strain 92 grown in triplicate on 5% CO2 and
nitrate.
86
APPENDIX B
EXPERIMENTAL DATA
87
B.1 Growth on Nitrate and CO2
The two Strains Scendesmus sp. 131 and Kirchneriella sp. 92 were both grown in triplicate in
Bold's Basal Medium with the standard nitrate concentration and 5% CO2.
Scenedesmus sp. 131
Table B.1. Cell Concentration for Strain 131 on Nitrate and 5% CO2 (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
2.90E+04
Average Cell
Count
2.87E+04
2.60E+04
3.10E+04
2.52E+03
1.99
1.02E+05
6.00E+04
4.60E+04
6.93E+04
2.91E+04
3.99
6.32E+05
6.30E+05
4.28E+05
5.63E+05
1.17E+05
5.99
6.67E+06
6.48E+06
4.70E+06
5.95E+06
1.09E+06
7.99
1.79E+07
1.46E+07
1.56E+07
1.60E+07
1.69E+06
9.95
1.92E+07
2.10E+07
1.53E+07
1.85E+07
2.91E+06
11.99
1.92E+07
2.26E+07
2.24E+07
2.14E+07
1.91E+06
15.96
2.17E+07
2.24E+07
2.11E+07
2.17E+07
6.51E+05
St Dev
Table B.2. pH for Strain 131 on Nitrate and 5% CO2.
Time
(days)
0.00
Tube
1
7.59
Tube
2
7.70
Tube
3
7.76
Average
pH
7.68
1.99
6.46
6.44
6.46
6.45
0.01
3.99
6.55
6.55
6.49
6.53
0.03
5.99
7.04
6.98
6.9
6.97
0.07
7.99
7.03
7.02
6.99
7.01
0.02
9.95
6.93
6.98
6.97
6.96
0.03
11.99
6.75
6.74
6.73
6.74
0.01
15.96
6.88
6.92
6.92
6.91
0.02
St Dev
0.09
Table B.3. Total Nile Red Fluorescence for Strain 131 on Nitrate and 5% CO2.
1.99
Tube
1
355
Tube
2
565
Tube
3
200
Average Nile Red
Fluorescence
373
3.99
275
210
140
208
68
5.99
1050
915
380
782
354
7.99
2140
2795
1940
2292
447
Time (days)
St Dev
183
9.95
3235
3725
2610
3190
559
11.99
3295
3080
3080
3152
124
15.96
3875
3100
3435
3470
389
88
Table B.4. Specific Nile Red Fluorescence for Strain 131 on Nitrate and 5% CO2.
1.99
Tube
1
34.8
Tube
2
94.2
Tube
3
43.5
Average Specific
Fluorescence
57.5
3.99
4.4
3.3
3.3
3.7
0.6
5.99
1.6
1.4
0.8
1.3
0.4
7.99
1.2
1.9
1.2
1.5
0.4
9.95
1.7
1.8
1.7
1.7
0.0
11.99
1.7
1.4
1.4
1.5
0.2
15.96
1.8
1.4
1.6
1.6
0.2
Time (days)
St Dev
32.1
Table B.5. Nitrate Concentration for Strain 131 on Nitrate and 5% CO2 (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
0.00
184.8
206.5
192.4
Average NO3Concentration
194.6
1.99
189.7
193.5
193.0
192.0
2.0
3.99
154.5
162.6
167.2
161.4
6.4
5.99
0.53
0.02
19.1
6.56
10.9
7.99
0.04
0.01
0.00
0.02
0.02
9.95
0
0
0
0
0
St Dev
11.0
Table B.6. Dry Cell Weight for Strain 131 on Nitrate and 5% CO2 (g/L).
Time
(days)
15.96
Tube 1
Tube 2
Tube 3
2.36
2.44
2.43
Average Dry
Weight
2.41
St Dev
0.04
Table B.7. % Biofuel Potential for Strain 131 on Nitrate and 5% CO2.
Time
(days)
15.96
Tube 1
Tube 2
Tube 3
24.53
25.74
26.59
Average Biofuel
Potential
25.62
St Dev
1.04
89
Kirchneriella sp. 92
Table B.8. Cell Concentration for Strain 92 on Nitrate and 5% CO2 (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
1.10E+05
Average Cell
Count
9.12E+04
6.16E+04
1.02E+05
2.59E+04
1.99
6.08E+05
7.75E+05
1.29E+06
8.91E+05
3.55E+05
3.99
5.78E+06
5.83E+06
9.47E+06
7.03E+06
2.12E+06
6.00
2.90E+07
2.97E+07
2.94E+07
2.93E+07
3.25E+05
8.99
3.01E+07
3.08E+07
3.07E+07
3.05E+07
3.79E+05
11.01
3.27E+07
2.76E+07
3.20E+07
3.08E+07
2.76E+06
12.98
2.66E+07
3.29E+07
2.83E+07
2.93E+07
3.26E+06
15.00
2.82E+07
3.57E+07
3.58E+07
3.32E+07
4.36E+06
16.98
3.02E+07
2.64E+07
3.28E+07
2.98E+07
3.22E+06
18.97
2.99E+07
3.03E+07
3.25E+07
3.09E+07
1.40E+06
21.98
2.91E+07
3.94E+07
3.31E+07
3.39E+07
5.19E+06
Table B.9. pH for Strain 92 on Nitrate and 5% CO2.
Time
(days)
0.00
Tube
1
6.71
Tube
2
6.66
Tube
3
6.66
Average
pH
6.68
1.99
6.55
6.55
6.45
6.52
0.06
3.99
6.61
6.8
6.83
6.75
0.12
6.00
6.94
6.99
7.02
6.98
0.04
8.99
6.82
6.88
6.9
6.87
0.04
11.01
6.84
6.8
6.85
6.83
0.03
12.98
6.74
6.77
6.79
6.77
0.03
15.00
6.94
6.88
6.88
6.90
0.03
16.98
6.79
6.80
6.83
6.81
0.02
18.97
6.78
6.71
6.75
6.75
0.04
21.98
6.76
6.77
6.79
6.77
0.02
St Dev
0.03
St Dev
90
Table B.10. Total Nile Red Fluorescence for Strain 92 on Nitrate and 5% CO2.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
-400
255
415
Average Nile Red
Fluorescence
90
1.99
-120
670
-125
142
458
3.99
2245
2795
3585
2875
674
6.00
7495
11795
14835
11375
3688
8.99
12515
14360
21350
16075
4660
11.01
8420
14130
12695
11748
2970
12.98
11445
11300
12345
11697
566
15.00
8485
10910
13195
10863
2355
16.98
9860
10545
12465
10957
1350
18.97
10375
11935
11345
11218
788
21.98
9125
9200
12005
10110
1642
St Dev
432
Table B.11. Specific Nile Red Fluorescence for Strain 92 on Nitrate and 5% CO2.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
-64.94
25.00
37.73
Average Specific
Fluorescence
-0.74
1.99
-1.97
8.65
-0.97
1.90
5.86
3.99
3.88
4.79
3.79
4.15
0.56
6.00
2.58
3.98
5.05
3.87
1.24
8.99
4.16
4.66
6.95
5.26
1.49
11.01
2.57
5.12
3.97
3.89
1.27
12.98
4.30
3.43
4.36
4.03
0.52
15.00
3.01
3.06
3.69
3.25
0.38
16.98
3.26
3.99
3.80
3.69
0.38
18.97
3.47
3.94
3.49
3.63
0.26
21.98
3.14
2.34
3.63
3.03
0.65
St Dev
55.96
Table B.12. Nitrate Concentration for Strain 92 on Nitrate and 5% CO2 (mg/L).
Tube 1
Tube 2
Tube 3
195.4
196.3
194.5
Average NO3Concentration
195.4
1.99
181.9
180.9
171.4
178.1
5.79
3.99
80.6
21.3
0.54
34.1
41.5
6.00
0.37
0.02
0.03
0.14
0.20
8.99
0.00
0.00
0.00
0.00
0.00
Time
(days)
0.00
St Dev
0.92
91
Table B.13. Dry Cell Weight for Strain 92 on Nitrate and 5% CO2 (g/L).
Time
(days)
21.98
Tube 1
Tube 2
Tube 3
1.86
1.85
1.96
Average Dry
Weight
1.89
Table B.14. % Biofuel Potential for Strain 92 on Nitrate and 5% CO2.
Time
Average Biofuel
Tube 1
Tube 2
Tube 3
(days)
Potential
21.98
37.59
40.60
36.96
38.38
St Dev
0.06
St Dev
1.95
92
B.2 Growth on Nitrate and Air
The experiment was run using Bold's Basal Medium with the standard nitrate concentration and
grown on compressed air.
Scenedesmus sp. 131
Table B.15. Cell Concentration for Strain 131 on Nitrate and Air (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
9.56E+04
Average Cell
Count
8.48E+04
8.00E+04
7.89E+04
9.34E+03
2.00
2.01E+05
2.05E+05
2.53E+05
2.20E+05
2.89E+04
4.00
6.50E+05
7.48E+05
9.20E+05
7.73E+05
1.37E+05
6.00
2.06E+06
2.76E+06
3.09E+06
2.64E+06
5.26E+05
St Dev
8.00
2.37E+06
3.03E+06
3.76E+06
3.05E+06
6.95E+05
10.00
4.23E+06
6.77E+06
8.65E+06
6.55E+06
2.22E+06
12.00
6.30E+06
9.66E+06
1.01E+07
8.69E+06
2.08E+06
14.00
6.63E+06
7.50E+06
1.07E+07
8.28E+06
2.14E+06
16.00
8.50E+06
9.98E+06
1.18E+07
1.01E+07
1.65E+06
18.00
1.03E+07
9.10E+06
9.95E+06
9.78E+06
6.17E+05
21.00
7.42E+06
9.48E+06
1.14E+07
9.43E+06
1.99E+06
23.00
8.05E+06
1.14E+07
1.07E+07
1.00E+07
1.75E+06
Table B.16. pH for Strain 131 on Nitrate and Air.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
Average pH
St Dev
8.33
8.45
8.48
8.42
0.08
2.00
9.39
9.35
9.35
9.36
0.02
4.00
10.2
10.5
10.54
10.41
0.19
6.00
10.63
10.88
10.93
10.81
0.16
8.00
10.87
11
11.03
10.97
0.09
10.00
11.09
11.28
11.28
11.22
0.11
12.00
11.02
11.02
11.03
11.02
0.01
14.00
11.27
11.30
11.34
11.30
0.04
16.00
11.07
11.09
11.10
11.09
0.02
18.00
11.03
11.11
11.13
11.09
0.05
21.00
11.15
11.06
10.83
11.01
0.17
23.00
11.14
10.64
10.16
10.65
0.49
93
Table B.17. Total Nile Red Fluorescence for Strain 131 on Nitrate and Air.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
37
-55
-21
Average Nile Red
Fluorescence
-13
2.00
95
180
97
124
49
4.00
192
245
156
198
45
6.00
260
168
241
223
49
8.00
595
385
595
525
121
10.00
430
785
1025
747
299
12.00
855
1450
2195
1500
671
14.00
1415
2165
3235
2272
915
16.00
1675
3910
3690
3092
1232
18.00
2320
4165
4655
3713
1231
21.00
3830
6730
6195
5585
1543
23.00
4425
5020
5505
4983
541
St Dev
47
Table B.18. Specific Nile Red Fluorescence for Strain 131 on Nitrate and Air.
Time
(days)
0.00
Tube
1
4.63
Tube 2
Tube 3
-6.97
-2.20
Average Specific
Fluorescence
-1.51
2.00
4.73
8.78
3.83
5.78
2.64
4.00
2.95
3.28
1.70
2.64
0.83
6.00
1.26
0.61
0.78
0.88
0.34
St Dev
5.83
8.00
2.51
1.27
1.58
1.79
0.64
10.00
1.02
1.16
1.18
1.12
0.09
12.00
1.36
1.50
2.17
1.68
0.44
14.00
2.13
2.89
3.02
2.68
0.48
16.00
1.97
3.92
3.13
3.01
0.98
18.00
2.25
4.58
4.68
3.84
1.37
21.00
5.16
7.10
5.43
5.90
1.05
23.00
5.50
4.42
5.14
5.02
0.55
94
Table B.19. Nitrate Concentration for Strain 131 on Nitrate and Air (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
0
199.4
222.8
207.5
Average NO3Concentration
209.9
2
204.7
208.7
208.2
207.2
2.20
4
166.7
175.4
180.4
174.2
6.92
6
0.57
0.02
20.64
7.08
11.8
8
0.04
0.01
0.00
0.02
0.02
10
0.03
0.01
0.02
0.02
0.01
St Dev
11.9
Table B.20. Dry Cell Weight for Strain 131 on Nitrate and Air (g/L).
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
0.96
1.33
1.39
Average Dry
Weight
1.23
St Dev
0.23
Table B.21. % Biofuel Potential for Strain 131 on Nitrate and Air.
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
26.65
36.23
37.30
Average Biofuel
Potential
33.40
St Dev
5.87
95
Kirchneriella sp. 92
Table B.22. Cell Concentration for Strain 92 on Nitrate and Air (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
2.02E+05
Average Cell
Count
1.99E+05
1.81E+05
2.13E+05
1.63E+04
2.00
4.98E+05
5.44E+05
5.78E+05
5.40E+05
4.01E+04
4.00
4.03E+06
3.64E+06
4.59E+06
4.09E+06
4.78E+05
6.00
9.38E+06
7.18E+06
7.52E+06
8.03E+06
1.18E+06
8.00
1.36E+07
8.95E+06
1.22E+07
1.16E+07
2.37E+06
10.00
2.13E+07
1.11E+07
2.02E+07
1.75E+07
5.60E+06
12.00
2.34E+07
2.34E+07
2.50E+07
2.39E+07
9.24E+05
14.00
2.48E+07
2.15E+07
2.31E+07
2.31E+07
1.65E+06
16.00
2.56E+07
2.64E+07
2.42E+07
2.54E+07
1.11E+06
18.00
2.55E+07
2.06E+07
2.56E+07
2.39E+07
2.86E+06
21.00
2.57E+07
2.30E+07
2.63E+07
2.50E+07
1.76E+06
23.00
3.16E+07
2.03E+07
2.88E+07
2.69E+07
5.88E+06
Table B.23. pH for Strain 92 on Nitrate and Air.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
8.38
8.40
8.43
Average
pH
8.40
8.42
8.44
8.34
8.40
0.05
4.00
9.96
10.05
10.02
10.01
0.05
6.00
10.63
10.63
10.67
10.64
0.02
8.00
10.86
10.77
10.79
10.81
0.05
10.00
11.07
10.97
11
11.01
0.05
12.00
10.64
10.74
10.71
10.70
0.05
14.00
10.82
11.02
10.91
10.92
0.10
16.00
10.60
10.70
10.56
10.62
0.07
18.00
10.47
10.59
10.38
10.48
0.11
21.00
9.76
10.34
9.86
9.99
0.31
23.00
9.49
9.95
9.68
9.71
0.23
2.00
St Dev
0.03
St Dev
96
Table B.24. Total Nile Red Fluorescence for Strain 92 on Nitrate and Air.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
150
21
3
Average Nile Red
Fluorescence
58
2.00
140
159
98
132
31
4.00
223
116
239
193
67
6.00
401
238
281
307
84
8.00
1270
625
455
783
430
10.00
1405
395
1420
1073
588
12.00
2655
1645
2415
2238
528
14.00
5155
1115
4410
3560
2150
16.00
7555
2010
6090
5218
2873
18.00
13200
3095
8555
8283
5058
21.00
21540
6575
13780
13965
7484
23.00
18405
4745
12180
11777
6839
St Dev
80
Table B.25. Specific Nile Red Fluorescence for Strain 92 on Nitrate and Air.
Time (days)
Tube 1
Tube 2
Tube 3
0.00
8.29
0.99
0.15
Average Specific
Fluorescence
3.14
2.00
2.81
2.92
1.70
2.48
0.68
4.00
0.55
0.32
0.52
0.46
0.13
6.00
0.43
0.33
0.37
0.38
0.05
8.00
0.94
0.70
0.37
0.67
0.28
10.00
0.66
0.36
0.70
0.57
0.19
12.00
1.13
0.70
0.97
0.93
0.22
14.00
2.08
0.52
1.91
1.50
0.86
16.00
2.95
0.76
2.52
2.08
1.16
18.00
5.18
1.50
3.34
3.34
1.84
21.00
8.38
2.86
5.24
5.49
2.77
23.00
5.82
2.34
4.23
4.13
1.75
St Dev
4.48
97
Table B.26. Nitrate Concentration for Strain 92 on Nitrate and Air (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
0
199.9
204.2
208.0
Average NO3Concentration
204.0
2
199.4
200.8
205.6
201.9
3.24
4
167.3
171.4
167.3
168.6
2.39
6
112.5
123.8
116.7
117.7
5.73
8
54.24
81.02
78.33
71.20
14.75
10
0.02
36.31
1.86
12.73
20.44
12
0.06
0.27
0.02
0.12
0.13
14
0.00
0.00
0.00
0.00
0.00
St Dev
4.06
Table B.27. Dry Cell Weight for Strain 92 on Nitrate and Air (g/L).
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
1.14
0.91
1.32
1.13
0.21
Table B.28. % Biofuel Potential for Strain 92 on Nitrate and Air.
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
Average Biofuel
Potential
St Dev
22.70
24.36
26.13
24.08
1.54
98
B.3 Growth on Nitrate, CO2 and Bicarbonate Injection
The experiment was run using Bold's Basal Medium with the standard nitrate concentration and
grown on 5% CO2 prior to nitrogen depletion. Once nitrate was near depletion, the system was injected
with 50 mM sodium bicarbonate (final concentration), and the system was switched to compressed air.
Scenedesmus sp. 131
Table B.29. Cell Concentration for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
7.83E+04
9.50E+04
7.50E+04
8.28E+04
1.07E+04
2.00
2.17E+05
1.71E+05
1.97E+05
1.95E+05
2.31E+04
3.00
5.66E+05
6.09E+05
5.75E+05
5.83E+05
2.27E+04
4.00
2.77E+06
2.10E+06
2.34E+06
2.40E+06
3.39E+05
4.69*
6.14E+06
6.06E+06
7.86E+06
6.69E+06
1.02E+06
5.00
5.89E+06
5.63E+06
7.10E+06
6.21E+06
7.84E+05
6.00
7.00E+06
6.78E+06
7.70E+06
7.16E+06
4.80E+05
7.00
1.06E+07
1.22E+07
1.14E+07
1.14E+07
8.01E+05
8.00
9.60E+06
9.98E+06
8.93E+06
9.50E+06
5.32E+05
9.00
1.05E+07
1.03E+07
1.05E+07
1.04E+07
1.15E+05
11.00
1.05E+07
1.06E+07
1.15E+07
* T = 4.69 days is the point of bicarbonate injection
1.09E+07
5.51E+05
Table B.30. pH for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
Tube 1
Tube 2
Tube 3
Average pH
St Dev
0.00
6.83
6.79
6.72
6.78
0.06
2.00
6.80
6.79
6.71
6.77
0.05
3.00
6.86
6.89
6.90
6.88
0.02
4.00
7.06
7.03
6.95
7.01
0.06
4.69*
7.05
7.07
7.09
7.07
0.02
4.69**
8.59
8.62
8.74
8.65
0.08
5.00
9.50
9.54
9.59
9.54
0.05
6.00
10.30
10.37
10.44
10.37
0.07
7.00
10.73
10.75
10.91
10.80
0.10
8.00
11.16
11.16
11.20
11.17
0.02
9.00
11.39
11.32
11.18
11.30
0.11
11.00
11.42
*pH prior to bicarbonate injection
**pH following bicarbonate injection
11.30
10.92
11.21
0.26
99
Table B.31. Total Nile Red Fluorescence for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
48
-42
-18
Average Nile Red
Fluorescence
-4
2.00
-9
101
171
88
91
3.00
147
126
132
135
11
4.00
323
403
381
369
41
4.69*
595
595
260
483
193
5.00
900
1465
1115
1160
285
6.00
1970
1740
2170
1960
215
7.00
3525
4490
4455
4157
547
8.00
3785
4275
3770
3943
287
9.00
4790
5190
4805
4928
227
11.00
5660
7130
6335
6375
736
St Dev
47
Table B.32. Specific Nile Red Fluorescence for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
Tube
1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
6.13
-4.42
-2.40
-0.23
5.60
2.00
-0.41
5.91
8.68
4.72
4.66
3.00
2.60
2.07
2.30
2.32
0.26
4.00
1.17
1.92
1.63
1.57
0.38
4.69*
0.97
0.98
0.33
0.76
0.37
5.00
1.53
2.60
1.57
1.90
0.61
6.00
2.81
2.57
2.82
2.73
0.14
7.00
3.33
3.68
3.93
3.64
0.30
8.00
3.94
4.28
4.22
4.15
0.18
9.00
4.56
5.04
4.58
4.73
0.27
11.00
5.39
6.73
5.51
5.88
0.74
100
Table B.33. Nitrate Concentration for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
Average NO3Concentration
St Dev
0.00
207.25
205.68
206.20
206.37
0.80
2.00
204.91
202.07
197.32
201.44
3.83
3.00
171.66
172.65
167.47
170.60
2.75
4.00
78.28
87.89
90.70
85.62
6.51
4.69*
21.80
18.12
8.49
16.14
6.87
5.00
0.05
0.05
0.00
0.03
0.03
6.00
0.00
0.00
0.00
0.00
0.00
Table B.34. Dry Cell Weight for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
11.00
1.11
1.09
1.13
1.11
0.23
Table B.35. % Biofuel Potential for Strain 131 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
22.32
22.24
26.83
Average Biofuel
Potential
24.13
St Dev
2.40
101
Kirchneriella sp. 92
Table B.36. Cell Concentration for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
1.91E+05
2.09E+05
1.82E+05
1.94E+05
1.37E+04
2.00
9.60E+05
1.12E+06
1.03E+06
1.04E+06
8.02E+04
3.00
5.22E+06
4.15E+06
4.97E+06
4.78E+06
5.60E+05
3.83*
1.17E+07
1.12E+07
1.10E+07
1.13E+07
3.61E+05
4.00
1.14E+07
1.00E+07
1.29E+07
1.14E+07
1.45E+06
5.00
1.97E+07
1.48E+07
1.98E+07
1.81E+07
2.86E+06
6.00
1.96E+07
1.97E+07
1.90E+07
1.94E+07
4.07E+05
7.00
2.38E+07
2.50E+07
2.33E+07
2.40E+07
8.46E+05
8.00
2.04E+07
1.74E+07
2.32E+07
2.03E+07
2.90E+06
9.00
2.18E+07
1.96E+07
2.01E+07
2.05E+07
1.15E+06
2.12E+07
2.05E+07
2.33E+06
11.00
2.24E+07
1.79E+07
* T = 3.83 days is the point of bicarbonate injection
Table B.37. pH for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
Tube 1
Tube 2
Tube 3
Average
pH
St Dev
0.00
6.74
6.74
6.77
6.75
0.02
2.00
6.73
6.61
6.61
6.65
0.07
3.00
6.99
7.04
7.04
7.02
0.03
3.83*
7.16
7.18
7.20
7.18
0.02
3.83**
8.30
8.22
8.28
8.27
0.04
4.00
9.40
9.30
9.35
9.35
0.05
5.00
10.27
10.26
10.29
10.27
0.02
6.00
10.48
10.47
10.47
10.47
0.01
7.00
10.49
10.47
10.47
10.48
0.01
8.00
10.50
10.50
10.50
10.50
0.00
9.00
10.46
10.46
10.46
10.46
0.00
11.00
10.33
*pH prior to bicarbonate injection
**pH following bicarbonate injection
10.34
10.32
10.33
0.01
102
Table B.38. Total Nile Red Fluorescence for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
74
-27
6
18
52
2.00
199
244
306
250
54
3.00
479
589
659
576
91
3.83*
1390
1785
1525
1567
201
4.00
2365
1465
2060
1963
458
5.00
3170
3280
3575
3342
209
6.00
4305
3890
4195
4130
215
7.00
7130
6120
5705
6318
733
8.00
6855
4335
6855
6015
1455
9.00
6500
5710
8700
6970
1549
11.00
7400
5510
10070
7660
2291
Table B.39. Specific Nile Red Fluorescence for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time (days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
3.87
-1.29
0.33
0.97
2.64
2.00
2.07
2.18
2.97
2.41
0.49
3.00
0.92
1.42
1.33
1.22
0.27
3.83*
1.19
1.59
1.39
1.39
0.20
4.00
2.07
1.47
1.60
1.71
0.32
5.00
1.61
2.22
1.81
1.88
0.31
6.00
2.20
1.97
2.21
2.13
0.13
7.00
3.00
2.45
2.45
2.63
0.31
8.00
3.36
2.49
2.95
2.94
0.43
9.00
2.98
2.91
4.33
3.41
0.80
11.00
3.30
3.08
4.75
3.71
0.91
103
Table B.40. Nitrate Concentration for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
Average NO3Concentration
St Dev
0.00
209.71
216.53
214.50
213.58
3.50
2.00
196.42
204.41
202.06
200.97
4.11
3.00
147.56
150.54
144.99
147.70
2.78
3.83*
48.13
65.75
49.18
54.35
9.88
4.00
18.17
31.82
18.68
22.89
7.74
5.00
0.00
0.00
0.00
0.00
0.00
Table B.41. Dry Cell Weight for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
11.00
1.08
0.94
1.14
1.05
0.10
Table B.42. % Biofuel Potential for Strain 92 on Nitrate, 5% CO2 and Bicarbonate Injection.
Time
(days)
23.00
Tube 1
Tube 2
Tube 3
26.36
26.96
26.69
Average Biofuel
Potential
26.67
St Dev
0.30
104
B.4 Growth on Urea and 5% CO2
The experiment was run using Bold's Basal Medium with urea substituted for nitrate at a
concentration of 1.47 mM to compensate for the two nitrogen atoms per molecule and 5% CO2.
Scenedesmus sp. 131
Table B.43. Cell Concentration for Strain 131 on Urea and 5% CO2 (cells/mL).
4.39E+04
Average Cell
Count
4.52E+04
2.84E+03
1.58E+05
1.44E+05
1.37E+05
2.52E+04
1.46E+06
1.58E+06
1.51E+06
1.52E+06
6.03E+04
5.98
7.35E+06
9.35E+06
8.35E+06
8.35E+06
1.00E+06
7.98
1.38E+07
1.42E+07
1.48E+07
1.43E+07
5.27E+05
Time (days)
Tube 1
Tube 2
Tube 3
0.00
4.85E+04
4.33E+04
1.96
1.09E+05
4.00
St Dev
9.98
1.53E+07
1.51E+07
1.76E+07
1.60E+07
1.39E+06
11.98
1.62E+07
1.52E+07
1.75E+07
1.63E+07
1.13E+06
14.00
1.22E+07
1.57E+07
1.71E+07
1.50E+07
2.53E+06
16.02
1.34E+07
1.82E+07
1.76E+07
1.64E+07
2.60E+06
Table B.44. pH for Strain 131 on Urea and 5% CO2.
0.00
Tube
1
6.7
Tube
2
6.75
Tube
3
6.55
1.96
6.72
6.78
4.00
6.59
5.98
8.44
7.98
Time (days)
Average pH
St Dev
6.67
0.10
6.62
6.71
0.08
6.65
6.61
6.62
0.03
8.53
8.78
8.58
0.18
6.56
6.57
6.53
6.55
0.02
9.98
6.42
6.47
6.46
6.45
0.03
11.98
6.46
6.81
6.53
6.60
0.19
14.00
6.71
6.70
6.69
6.70
0.01
16.02
6.56
6.63
6.67
6.62
Spike on day 6 is due to short term loss of CO2 because of empty CO2 tank
0.06
105
Table B.45. Total Nile Red Fluorescence for Strain 131 on Urea and 5% CO2.
Time
(days)
0.00
Tube
1
230
Tube
2
-75
Tube
3
-370
Average Nile Red
Fluorescence
-72
St
Dev
300
1.96
155
335
595
362
221
4.00
275
185
275
245
52
5.98
1910
2320
1940
2057
229
7.98
1815
2380
2185
2127
287
9.98
2565
2990
3265
2940
353
11.98
3370
4090
4180
3880
444
14.00
3905
5125
4670
4567
617
16.02
2655
2945
3465
3022
410
Table B.46. Specific Nile Red Fluorescence for Strain 131 on Urea and 5% CO2.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
47.42
-17.32
-84.28
Average Specific
Fluorescence
-18.06
1.96
14.22
21.20
41.32
25.58
14.07
4.00
1.88
1.17
1.82
1.63
0.39
5.98
2.60
2.48
2.32
2.47
0.14
7.98
1.32
1.68
1.48
1.49
0.18
9.98
1.68
1.98
1.86
1.84
0.15
11.98
2.08
2.69
2.40
2.39
0.31
14.00
3.20
3.26
2.73
3.06
0.29
16.02
1.98
1.62
1.97
1.86
0.20
St Dev
65.86
Table B.47. Urea Concentration for Strain 131 on Urea and 5% CO2 (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
0.00
99.78
95.46
93.96
Average Urea
Concentration
96.40
1.96
96.56
92.07
88.08
92.24
4.24
4.00
48.08
46.15
43.43
45.89
2.34
5.98
0.00
0.00
0.00
0.00
0.00
St Dev
3.02
106
Table B.48. Dry Cell Weight for Strain 131 on Urea and 5% CO2 (g/L).
Time
(days)
16.02
Tube 1
Tube 2
Tube 3
1.86
1.85
1.96
Average Dry
Weight
1.89
St Dev
0.06
Table B.49. % Biofuel Potential for Strain 131 on Urea and 5% CO2.
Time
(days)
16.02
Tube 1
Tube 2
Tube 3
29.50
30.23
30.97
Average Biofuel
Potential
30.23
St Dev
1.04
107
Kirchneriella sp. 92
Table B.50. Cell Concentration for Strain 92 on Urea and 5% CO2 (cells/mL).
5.56E+04
Average Cell
Count
4.80E+04
1.41E+04
4.14E+05
4.21E+05
4.28E+05
1.91E+04
7.78E+06
6.26E+06
7.50E+06
7.18E+06
8.09E+05
6.02
1.77E+07
1.85E+07
1.06E+07
1.56E+07
4.35E+06
8.00
2.94E+07
3.53E+07
1.90E+07
2.79E+07
8.28E+06
Time (days)
Tube 1
Tube 2
Tube 3
0.00
5.67E+04
3.17E+04
1.98
4.50E+05
3.96
St Dev
9.98
2.52E+07
3.51E+07
2.07E+07
2.70E+07
7.39E+06
12.00
2.87E+07
3.17E+07
1.90E+07
2.64E+07
6.66E+06
13.97
3.14E+07
3.51E+07
2.09E+07
2.91E+07
7.37E+06
15.98
3.17E+07
4.39E+07
1.88E+07
3.15E+07
1.26E+07
Table B.51. pH for Strain 92 on Urea and 5% CO2.
Time
(days)
0.00
Tube
1
6.73
Tube
2
6.72
Tube
3
6.77
Average
pH
6.74
1.98
6.72
6.77
6.57
6.69
0.10
3.96
6.58
6.54
6.56
6.56
0.02
6.02
6.76
6.84
6.75
6.78
0.05
8.00
6.67
6.62
6.56
6.62
0.06
9.98
6.76
6.73
6.73
6.74
0.02
12.00
6.96
6.97
6.92
6.95
0.03
13.97
6.59
6.55
6.55
6.56
0.02
15.98
7.13
7.10
6.95
7.06
0.10
St Dev
0.03
Table B.52. Total Nile Red Fluorescence for Strain 92 on Urea and 5% CO2.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
95
-165
415
Average Nile Red
Fluorescence
115
1.98
455
75
595
375
269
3.96
1390
2475
2460
2108
622
6.02
15485
17290
12390
15055
2478
8.00
24215
22915
20950
22693
1644
9.98
20615
23870
26110
23532
2763
12.00
22535
19275
25285
22365
3009
13.97
22125
20540
21270
21312
793
15.98
19820
19485
18310
19205
793
St Dev
291
108
Table B.53. Specific Nile Red Fluorescence for Strain 92 on Urea and 5% CO2.
Time (days)
Tube 1
Tube 2
Tube 3
0.00
16.75
-52.05
74.64
Average Specific
Fluorescence
13.11
1.98
10.11
1.81
14.13
8.69
6.28
3.96
1.79
3.95
3.28
3.01
1.11
6.02
8.75
9.35
11.69
9.93
1.55
8.00
8.24
6.49
11.06
8.59
2.30
9.98
8.18
6.80
12.64
9.21
3.05
12.00
7.87
6.08
13.34
9.10
3.78
13.97
7.05
5.85
10.18
7.69
2.23
15.98
6.25
4.44
9.74
6.81
2.69
St Dev
63.42
Table B.54. Urea Concentration for Strain 92 on Urea and 5% CO2 (mg/L).
Time (days)
Tube 1
Tube 2
Tube 3
0.00
87.38
94.61
98.95
Average Urea
Concentration
93.65
1.98
82.11
86.88
93.49
87.49
5.72
3.96
46.72
47.86
39.00
44.53
4.82
6.02
0.00
0.00
0.00
0.00
0.00
St Dev
5.84
Table B.55. Dry Cell Weight for Strain 92 on Urea and 5% CO2 (g/L).
Time
(days)
15.98
Tube 1
Tube 2
Tube 3
1.26
1.62
1.02
Average Dry
Weight
1.30
St Dev
0.30
Table B.56. % Biofuel Potential for Strain 92 on Urea and 5% CO2
Time
(days)
15.98
Tube 1
Tube 2
Tube 3
34.27
36.52
30.94
Average Biofuel
Potential
33.91
St Dev
2.81
109
B.5 Growth on Ammonium and CO2 with no buffer
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the system. The system was grown on
5% CO2.
Scenedesmus sp. 131
Table B.57. Cell Concentration for Strain 131 on NH4+, 5% CO2 and No Buffer (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
1.07E+05
Average Cell
Count
1.05E+05
1.11E+05
9.56E+04
7.99E+03
2.00
3.85E+05
3.79E+05
3.81E+05
3.82E+05
3.06E+03
3.00
1.47E+06
1.44E+06
1.84E+06
1.58E+06
2.23E+05
4.00
1.57E+06
1.44E+06
1.81E+06
1.60E+06
1.88E+05
St Dev
5.00
1.50E+04
2.60E+04
2.50E+04
2.20E+04
6.08E+03
total population did Not change, cells were only counted if alive based on healthy chlorophyll
Table B.58. pH for Strain 131 on NH4+, 5% CO2 and No Buffer.
Time
(days)
0.00
Tube
1
6.4
Tube
2
6.37
Tube
3
6.37
Average
pH
6.38
2.00
6.4
6.39
6.39
6.39
0.01
3.00
5.48
5.45
3.96
4.96
0.87
4.00
5.2
4.9
3.69
4.60
0.80
5.00
4.96
4.77
3.81
4.51
0.62
St Dev
0.02
Table B.59. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2 and No Buffer
Time
(days)
0.00
Tube
1
24
Tube
2
82
Tube
3
58
Average Nile Red
Fluorescence
55
2.00
107
113
144
121
20
3.00
81
257
257
198
102
4.00
101
68
198
122
68
5.00
299
300
684
428
222
St Dev
29
110
Table B.60. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2 and No Buffer.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
2.16
8.58
5.42
Average Specific
Fluorescence
5.39
2.00
2.78
2.98
3.78
3.18
0.53
3.00
0.55
1.78
1.40
1.24
0.63
4.00
0.65
0.47
1.10
0.74
0.32
273.60*
196.11*
79.16
5.00
199.33*
115.38*
*False increase due to decrease in live cells
St Dev
3.21
Table B.61. Ammonium Concentration for Strain 131 on NH4+, 5% CO2 and No Buffer (mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
Tube
1
53
46
22
18
18
Tube
2
53
47
20
17
17
Tube
3
53
44
13
10
11
Average NH4+
Concentration
53
46
18
15
15
St Dev
0.42
1.46
4.50
3.98
4.01
Table B.62. Dry Cell Weight for Strain 131 on NH4+, 5% CO2 and No Buffer (g/L).
Time
(days)
5.00
Tube 1
Tube 2
Tube 3
0.288
0.304
0.416
Average Dry
Weight
0.336
St Dev
0.07
Table B.63. % Biofuel Potential for Strain 131 on NH4+, 5% CO2 and No Buffer.
Time
(days)
5.00
Tube 1
Tube 2
Tube 3
8.03
8.37
10.37
Average Biofuel
Potential
8.92
St Dev
1.26
111
Kirchneriella sp. 92
Table B.64. Cell Concentration for Strain 92 on NH4+, 5% CO2 and No Buffer (cells/mL).
1.33E+05
Average Cell
Count
1.39E+05
1.93E+04
4.65E+05
5.05E+05
4.84E+05
2.00E+04
2.85E+06
3.11E+06
4.31E+06
3.42E+06
7.79E+05
4.00
4.65E+06
4.53E+06
4.83E+06
4.67E+06
1.51E+05
5.00
3.53E+06
3.36E+06
3.51E+06
3.47E+06
9.29E+04
6.00
1.86E+06
1.94E+06
2.71E+06
2.17E+06
4.69E+05
Time (days)
Tube 1
Tube 2
Tube 3
0.00
1.24E+05
1.61E+05
2.00
4.83E+05
3.00
St Dev
6.88
9.13E+05
1.09E+06
1.10E+06
1.03E+06
1.05E+05
total population did Not change, cells were only counted if alive based on healthy chlorophyll
Table B.65. pH for Strain 92 on NH4+, 5% CO2 and No Buffer.
Time
(days)
0.00
Tube
1
6.38
Tube
2
6.37
Tube
3
6.35
Average
pH
6.37
2.00
6.47
6.48
6.49
6.48
0.01
3.00
6.16
6.29
6.31
6.25
0.08
4.00
4.63
4.63
4.67
4.64
0.02
5.00
3.99
3.96
4
3.98
0.02
6.00
3.93
3.95
3.98
3.95
0.03
6.88
3.98
4.01
4.00
4.00
0.02
St Dev
0.02
Table B.66. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2 and No Buffer
Time (days)
Tube 1
Tube 2
Tube 3
0.00
40
73
39
Average Nile Red
Fluorescence
51
2.00
626
650
620
632
16
3.00
1516
1392
613
1174
489
4.00
5585
6045
4900
5510
576
5.00
7175
4395
6565
6045
1461
6.00
5675
5095
5950
5573
436
6.88
5575
5140
5475
5397
228
St Dev
19
112
Table B.67. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2 and No Buffer.
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
3.23
4.53
2.93
Average Specific
Fluorescence
3.56
2.00
12.96
13.98
12.28
13.07
0.86
3.00
5.32
4.48
1.42
3.74
2.05
4.00
12.01
13.34
10.14
11.83
1.61
5.00
20.33*
13.08*
18.70*
17.37*
3.80
6.00
30.51*
26.26*
21.96*
26.24*
4.28
6.88
61.06*
47.16*
*False increase due to decrease in live cells
49.77*
52.66*
7.39
St Dev
0.85
Table B.68. Ammonium Concentration for Strain 92 on NH4+, 5% CO2 and No Buffer (mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
6.00
6.88
Tube
1
53
50
34
18
14
11
11
Tube
2
53
50
32
16
12
10
10
Tube
3
54
51
33
17
13
11
11
Average NH4+
Concentration
53.39
50.17
32.68
17.40
12.73
10.81
10.54
St Dev
0.47
0.49
0.91
0.89
0.94
0.85
0.61
Table B.69. Dry Cell Weight for Strain 92 on NH4+, 5% CO2 and No Buffer (g/L).
Time
(days)
6.88
Tube 1
Tube 2
Tube 3
0.50
0.51
0.52
Average Dry
Weight
0.51
St Dev
0.01
Table B.70. % Biofuel Potential for Strain 92 on NH4+, 5% CO2 and No Buffer.
Time
(days)
6.88
Tube 1
Tube 2
Tube 3
35.51
21.83
14.06
Average Biofuel
Potential
23.80
St Dev
10.86
113
B.6 Growth on Ammonium and air with No Buffer
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the medium. The reactors were grown
on compressed air.
Scenedesmus sp. 131
Table B.71. Cell Concentration for Strain 131 on NH4+, Air and No Buffer (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
4.50E+04
Average Cell
Count
4.76E+04
5.11E+04
4.67E+04
2.00
1.34E+05
1.89E+05
3.15E+03
1.39E+05
1.54E+05
3.04E+04
4.00
4.54E+05
5.40E+05
5.35E+05
5.10E+05
4.83E+04
6.00
1.02E+06
1.52E+06
1.43E+06
1.32E+06
2.67E+05
8.00
1.08E+06
1.48E+06
1.39E+06
1.32E+06
2.10E+05
10.00
1.52E+06
1.48E+06
1.72E+06
1.57E+06
1.29E+05
12.00
1.91E+06
1.82E+06
1.94E+06
1.89E+06
6.24E+04
14.00
1.78E+06
2.56E+06
2.32E+06
2.22E+06
3.99E+05
16.00
1.41E+06
3.84E+05
2.69E+05
6.89E+05
6.30E+05
St Dev
17.00
6.78E+05
1.50E+05
1.58E+05
3.29E+05
3.03E+05
total population did Not change, cells were only counted if alive based on healthy chlorophyll
Table B.72. pH for Strain 131 on NH4+, Air and No Buffer.
Time
(days)
0.00
Tube
1
7.29
Tube
2
7.41
Tube
3
7.44
Average
pH
7.38
2.00
7.25
7.34
7.34
7.31
0.05
4.00
7.05
7.00
7.03
7.03
0.03
6.00
6.72
6.69
6.67
6.69
0.03
8.00
6.63
6.36
6.28
6.42
0.18
10.00
6.37
5.93
5.74
6.01
0.32
12.00
5.98
4.88
4.73
5.20
0.68
14.00
5.73
4.36
4.28
4.79
0.82
16.00
5.34
4.11
4.11
4.52
0.71
17.00
4.81
4.10
4.10
4.34
0.41
St Dev
0.08
114
Table B.73. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and No Buffer.
Time
(days)
0.00
Tube
1
-6
Tube 2
Tube 3
77
6
Average Nile Red
Fluorescence
26
2.00
131
73
65
90
36
4.00
137
199
155
164
32
6.00
192
181
67
147
69
8.00
98
165
99
121
38
10.00
202
190
244
212
28
12.00
141
265
254
220
69
14.00
284
821
909
671
338
16.00
216
1548
1746
1170
832
17.00
327
1358
1858
1181
781
St Dev
45
Table B.74. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and No Buffer.
Time (days)
Tube 1
Tube 2
Tube 3
0.00
-1.17
16.49
1.33
Average Specific
Fluorescence
5.55
2.00
9.78
3.86
4.68
6.10
3.21
4.00
3.02
3.69
2.90
3.20
0.42
6.00
1.88
1.19
0.47
1.18
0.71
8.00
0.91
1.11
0.71
0.91
0.20
10.00
1.33
1.28
1.42
1.34
0.07
12.00
0.74
1.46
1.31
1.17
0.38
14.00
1.60*
3.21
3.92
2.91*
1.19
16.00
1.53*
40.31*
64.91*
35.58*
31.95
17.00
4.82*
90.53*
*False increase due to decrease in live cells
117.59*
70.98*
58.87
St Dev
9.56
115
Table B.75. Ammonium Concentration for Strain 131 on NH4+, Air and No Buffer (mg/L).
Time
(days)
0.00
Tube
1
Tube
2
Tube
3
Average NH4+
Concentration
St Dev
54
54
55
54.28
0.48
2.00
50
51
51
50.44
0.38
4.00
45
43
43
43.61
0.85
6.00
39
35
36
36.70
2.40
8.00
33
28
28
29.66
3.01
10.00
29
23
23
24.66
3.51
12.00
25
19
19
21.15
3.25
14.00
21
16
17
18.02
2.51
16.00
19
15
16
16.41
2.18
17.00
17
14
16
15.82
1.49
Table B.76. Dry Cell Weight for Strain 131 on NH4+, Air and No Buffer (g/L).
Time
(days)
17.00
Tube 1
Tube 2
Tube 3
0.24
0.27
0.28
Average Dry
Weight
0.26
St Dev
0.02
Table B.77. % Biofuel Potential for Strain 131 on NH4+, Air and No Buffer.
Time
(days)
17.00
Tube 1
Tube 2
Tube 3
10.97
7.14
6.57
Average Biofuel
Potential
8.23
St Dev
2.39
116
Kirchneriella sp. 92
Table B.78. Cell Concentration for Strain 92 on NH4+, Air and No Buffer (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
Tube 3
1.42E+05
Average Cell
Count
1.44E+05
1.48E+05
1.41E+05
3.79E+03
2.00
6.95E+05
7.74E+05
6.78E+05
7.16E+05
5.12E+04
4.00
2.89E+06
3.28E+06
2.69E+06
2.95E+06
3.00E+05
6.00
4.45E+06
4.39E+06
4.28E+06
4.37E+06
8.62E+04
8.00
6.75E+06
7.48E+06
7.03E+06
7.09E+06
3.68E+05
10.00
7.13E+06
7.95E+06
7.43E+06
7.50E+06
4.15E+05
12.00
8.23E+06
9.48E+06
9.38E+06
9.03E+06
6.95E+05
14.00
7.17E+06
1.10E+07
1.09E+07
9.69E+06
2.18E+06
16.00
7.58E+06
8.73E+06
7.08E+06
7.80E+06
8.46E+05
17.00
6.15E+06
5.20E+06
4.90E+06
5.42E+06
6.53E+05
17.88
5.18E+06
4.00E+06
3.87E+06
4.35E+06
7.22E+05
St Dev
total population did Not change, cells were only counted if alive based on healthy chlorophyll
Table B.79. pH for Strain 92 on NH4+, Air and No Buffer.
0.00
Tube
1
7.41
Tube
2
7.46
Tube
3
7.47
Average
pH
7.45
2.00
7.35
7.41
7.41
7.39
0.03
4.00
7.13
7.16
7.16
7.15
0.02
6.00
6.74
6.83
6.84
6.80
0.06
8.00
6.41
6.50
6.54
6.48
0.07
10.00
5.75
5.91
6.04
5.90
0.15
12.00
4.98
5.02
5.12
5.04
0.07
14.00
4.82
4.73
4.73
4.76
0.05
16.00
4.45
4.55
4.52
4.51
0.05
17.00
4.42
4.47
4.48
4.46
0.03
17.88
4.52
4.54
4.54
4.53
0.01
Time (days)
St Dev
0.03
117
Table B.80. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and No Buffer.
Time (days)
Tube 1
Tube 2
Tube 3
0.00
-9
92
-40
Average Nile Red
Fluorescence
14
2.00
125
79
155
120
38
4.00
85
180
226
164
72
6.00
400
287
180
289
110
8.00
397
293
296
329
59
10.00
638
528
647
604
66
12.00
1271
712
662
882
338
14.00
1540
1925
1510
1658
231
16.00
2045
1880
2250
2058
185
17.00
1800
1835
2080
1905
153
17.88
1830
1865
1800
1832
33
St Dev
69
Table B.81. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and No Buffer.
Time
(days)
0.00
-0.61
Tube
2
6.52
-2.82
Average Specific
Fluorescence
1.03
2.00
1.80
1.02
2.29
1.70
0.64
4.00
0.29
0.55
0.84
0.56
0.27
6.00
0.90
0.65
0.42
0.66
0.24
8.00
0.59
0.39
0.42
0.47
0.11
10.00
0.89
0.66
0.87
0.81
0.13
12.00
1.54
0.75
0.71
1.00
0.47
14.00
2.15*
1.75
1.39
1.76*
0.38
16.00
2.70*
2.15*
3.18*
2.68*
0.51
17.00
2.93*
3.53*
4.24*
3.57*
0.66
17.88
3.53*
4.66*
*False increase due to decrease in live cells
4.65*
4.28*
0.65
Tube 1
Tube 3
St Dev
4.88
118
Table B.82. Ammonium Concentration for Strain 92 on NH4+, Air and No Buffer (mg/L).
Time
(days)
0.00
Tube
1
Tube
2
Tube
3
Average NH4+
Concentration
St Dev
54
54
55
54.31
0.80
2.00
51
53
53
52.36
1.20
4.00
43
46
46
45.02
1.41
6.00
35
44
38
39.12
4.41
8.00
28
29
31
29.34
1.71
10.00
22
24
25
23.36
1.55
12.00
19
19
20
19.34
0.99
14.00
18
19
19
18.68
0.62
16.00
18
17
19
17.74
0.70
17.00
17
17
18
17.48
0.82
17.88
17
17
18
17.53
0.75
Table B.83. Dry Cell Weight for Strain 92 on NH4+, Air and No Buffer (g/L).
Time
(days)
17.88
Tube 1
Tube 2
Tube 3
0.35
0.39
0.36
Average Dry
Weight
0.37
St Dev
0.02
Table B.84. % Biofuel Potential for Strain 92 on NH4+, Air and No Buffer.
Time
(days)
17.88
Tube 1
Tube 2
Tube 3
14.45
13.86
14.10
Average Biofuel
Potential
14.14
St Dev
0.29
119
B.7 Growth on Ammonium and CO2 and PIPES buffer
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the medium. The reactors were grown
on 5% CO2 and 8 mM PIPES.
Scenedesmus sp. 131
Table B.85. Cell Concentration for Strain 131 on NH4+, 5% CO2 and PIPES Buffer (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
5.06E+04
5.06E+04
4.30E+04
4.81E+04
4.39E+03
2.00
1.11E+05
7.74E+04
9.50E+04
9.45E+04
1.68E+04
3.00
2.14E+05
1.70E+05
2.21E+05
2.02E+05
2.76E+04
4.00
6.85E+05
6.75E+05
4.93E+05
6.18E+05
1.08E+05
5.00
2.08E+06
2.47E+06
2.01E+06
2.19E+06
2.48E+05
6.00
6.19E+06
6.00E+06
5.61E+06
5.93E+06
2.96E+05
7.00
8.80E+06
7.94E+06
8.25E+06
8.33E+06
4.36E+05
8.00
1.73E+07
1.37E+07
1.60E+07
1.57E+07
1.82E+06
9.00
1.56E+07
1.52E+07
1.38E+07
1.49E+07
9.45E+05
10.00
1.79E+07
1.65E+07
1.80E+07
1.75E+07
8.39E+05
Table B.86. pH for Strain 131 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
6.64
6.65
6.63
6.64
0.01
2.00
6.70
6.70
6.63
6.68
0.04
3.00
6.68
6.63
6.59
6.63
0.05
4.00
6.47
6.49
6.48
6.48
0.01
5.00
6.26
6.24
6.29
6.26
0.03
6.00
6.09
6.10
6.08
6.13
0.03
7.00
6.11
6.12
6.17
6.05
0.02
8.00
6.07
6.05
6.04
6.05
0.02
9.00
6.07
6.09
6.10
6.09
0.02
10.00
6.09
6.10
6.10
6.10
0.01
120
Table B.87. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
-9
101
-15
26
65
2.00
122
49
80
84
37
3.00
122
125
257
168
77
4.00
41
237
119
132
99
5.00
670
60
550
427
323
6.00
515
520
385
473
77
7.00
1235
1130
460
942
420
8.00
2745
2260
2945
2650
352
9.00
2560
3070
2445
2692
333
10.00
2715
3325
2500
2847
428
Table B.88. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
-1.78
19.96
-3.49
4.90
13.07
2.00
10.99
6.33
8.42
8.58
2.33
3.00
5.70
7.35
11.63
8.23
3.06
4.00
0.60
3.51
2.41
2.17
1.47
5.00
3.22
0.24
2.74
2.07
1.60
6.00
0.83
0.87
0.69
0.79
0.10
7.00
1.40
1.42
0.56
1.13
0.49
8.00
1.59
1.65
1.84
1.69
0.13
9.00
1.64
2.02
1.77
1.81
0.19
10.00
1.52
2.02
1.39
1.64
0.33
Table B.89. Ammonium Concentration for Strain 131 on NH4+, 5% CO2 and PIPES Buffer (mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
6.00
Tube
1
49
47
45
38
0
0
Tube
2
49
47
44
36
15
0
Tube
3
50
48
45
40
22
0
Average NH4+
Concentration
49.29
47.69
44.98
37.97
12.14
0.00
St Dev
0.40
0.67
0.47
1.59
11.11
0.00
121
Table B.90. Dry Cell Weight for Strain 131 on NH4+, 5% CO2 and PIPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
10.00
1.62
1.65
1.65
1.64
0.01
Table B.91. % Biofuel Potential for Strain 131 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
10.00
18.92
19.54
18.58
Average Biofuel
Potential
19.01
St Dev
0.48
122
Kirchneriella sp. 92
Table B.92. Cell Concentration for Strain 92 on NH4+, 5% CO2 and PIPES Buffer (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
2.19E+05
2.62E+05
2.51E+05
2.44E+05
2.23E+04
2.00
9.18E+05
9.50E+05
1.03E+06
9.66E+05
5.77E+04
3.00
4.58E+06
4.94E+06
4.57E+06
4.70E+06
2.11E+05
4.00
1.24E+07
1.44E+07
1.28E+07
1.32E+07
1.06E+06
5.00
1.78E+07
1.88E+07
1.78E+07
1.81E+07
5.92E+05
6.00
2.69E+07
2.53E+07
2.45E+07
2.56E+07
1.22E+06
7.00
2.39E+07
2.45E+07
2.72E+07
2.52E+07
1.76E+06
8.00
2.45E+07
2.43E+07
2.53E+07
2.47E+07
5.29E+05
9.00
2.80E+07
2.71E+07
2.93E+07
2.81E+07
1.11E+06
Table B.93. pH for Strain 92 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
6.63
6.62
6.62
6.62
0.01
2.00
6.64
6.64
6.63
6.64
0.01
3.00
6.55
6.54
6.57
6.55
0.02
4.00
6.16
6.16
6.17
6.16
0.01
5.00
6.17
6.13
6.11
6.14
0.03
6.00
6.12
6.12
6.15
6.13
0.02
7.00
6.17
6.17
6.18
6.17
0.01
8.00
6.12
6.11
6.13
6.12
0.01
9.00
6.18
6.19
6.18
6.18
0.01
Table B.94. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
43
85
73
67
22
2.00
329
314
379
341
34
3.00
1034
1224
1279
1179
129
4.00
2110
2490
2685
2428
292
5.00
15430
14605
14830
14955
426
6.00
25150
26125
30215
27163
2687
7.00
37520
47025
43230
42592
4785
8.00
33110
30180
35830
33040
2826
9.00
32840
27040
28780
29553
2976
123
Table B.95. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
1.96
3.24
2.91
2.71
0.66
2.00
3.58
3.31
3.68
3.52
0.19
3.00
2.26
2.48
2.80
2.51
0.27
4.00
1.70
1.73
2.10
1.84
0.22
5.00
8.67
7.77
8.35
8.26
0.46
6.00
9.35
10.33
12.33
10.67
1.52
7.00
15.70
19.19
15.89
16.93
1.96
8.00
13.51
12.42
14.16
13.37
0.88
9.00
11.73
9.98
9.82
10.51
1.06
Table B.96. Ammonium Concentration for Strain 92 on NH4+, 5% CO2 and PIPES Buffer (mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
Tube
1
49
47
37
4
0
Tube
2
56
51
38
4
0
Average NH4+
Concentration
51.78
48.53
37.84
4.04
0.00
Tube
3
50
48
38
5
0
St Dev
3.99
1.94
0.47
0.72
0.00
Table B.97. Dry Cell Weight for Strain 92 on NH4+, 5% CO2 and PIPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
9.00
1.73
1.69
1.67
1.70
0.03
Table B.98. % Biofuel Potential for Strain 92 on NH4+, 5% CO2 and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
9.00
32.35
38.40
32.38
Average Biofuel
Potential
34.37
St Dev
3.48
124
B.8 Growth on Ammonium and CO2 and PIPES Buffer with KOH spike
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the Medium. The reactors were grown
on 5% CO2 and 8 mM PIPES in addition, a spike of 2 mM KOH (final solution concentration) was added to
increase the pH to increase the DIC of the medium.
Scenedesmus sp. 131
Table B.99. Cell Concentration for Strain 131 on NH4+, 5% CO2, PIPES Buffer and 2 mM KOH (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
4.00E+04
4.44E+04
3.89E+04
4.11E+04
2.91E+03
2.00
6.28E+04
7.00E+04
6.39E+04
6.56E+04
3.88E+03
3.00
2.30E+05
2.18E+05
2.19E+05
2.22E+05
6.66E+03
4.00
8.02E+05
8.22E+05
6.73E+05
7.66E+05
8.09E+04
5.00
3.24E+06
2.90E+06
2.82E+06
2.99E+06
2.23E+05
6.00
7.30E+06
7.28E+06
7.35E+06
7.31E+06
3.61E+04
7.00
1.22E+07
1.19E+07
1.27E+07
1.23E+07
4.04E+05
8.00
1.23E+07
1.10E+07
1.10E+07
1.14E+07
7.51E+05
9.00
1.46E+07
1.62E+07
1.35E+07
1.48E+07
1.36E+06
10.00
1.31E+07
1.58E+07
1.38E+07
1.42E+07
1.40E+06
11.00
1.35E+07
1.64E+07
1.40E+07
1.46E+07
1.55E+06
12.00
1.37E+07
1.50E+07
1.57E+07
1.48E+07
1.01E+06
Table B.100. pH for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
6.74
6.75
6.74
6.74
0.01
2.00
6.58
6.58
6.59
6.58
0.01
3.00
6.58
6.58
6.61
6.59
0.02
4.00
6.62
6.59
6.6
6.60
0.02
4.71*
6.29
6.30
6.31
6.30
0.01
4.73
6.82
6.82
6.82
6.82
0.00
5.00
6.77
6.77
6.77
6.77
0.00
6.00
6.78
6.78
6.8
6.79
0.01
7.00
6.79
6.77
6.79
6.78
0.01
8.00
6.77
6.79
6.80
6.79
0.02
9.00
6.74
6.74
6.75
6.74
0.01
10.00
6.83
6.81
6.78
6.81
0.03
11.00
6.85
6.86
6.87
6.86
0.01
6.77
6.74
6.75
6.75
0.02
12.00
*Injection occurred at 4.71 days
125
Table B.101. Total Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
21
89
25
45
38
2.00
79
45
43
56
20
3.00
92
83
73
83
10
4.00
171
140
321
211
97
5.00
266
339
409
338
72
6.00
1085
1665
960
1237
376
7.00
3345
3310
2870
3175
265
8.00
2365
2900
2730
2665
273
9.00
3675
2975
3845
3498
461
10.00
3955
4685
4595
4412
398
11.00
5020
5665
5600
5428
355
12.00
4940
5760
5700
5467
457
Table B.102. Specific Nile Red Fluorescence for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH.
Time
(days)
Tube 1
Tube 2
Tube
3
Average Specific
Fluorescence
St Dev
0.00
5.25
20.05
6.43
10.57
8.22
2.00
12.58
6.43
6.73
8.58
3.47
3.00
4.00
3.81
3.33
3.71
0.34
4.00
2.13
1.70
4.77
2.87
1.66
5.00
0.82
1.17
1.45
1.15
0.32
6.00
1.49
2.29
1.31
1.69
0.52
7.00
2.74
2.78
2.26
2.59
0.29
8.00
1.92
2.64
2.48
2.35
0.38
9.00
2.52
1.84
2.85
2.40
0.52
10.00
3.02
2.97
3.33
3.10
0.20
11.00
3.72
3.45
4.00
3.72
0.27
12.00
3.61
3.84
3.63
3.69
0.13
126
Table B.103. Ammonium Concentration for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH
(mg/L).
Time
(days)
Tube
1
Tube
2
Tube
3
Average NH4+
Concentration
St Dev
0.00
63
58
57
59.20
3.31
2.00
56
55
55
55.40
0.41
3.00
51
53
51
51.57
0.87
4.00
37
40
39
38.39
1.59
5.00
0
0
0
0.00
0.00
Table B.104. Dry Cell Weight for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
12.00
2.17
2.08
2.00
2.09
0.09
Table B.105. % Biofuel Potential for Strain 131 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH.
Time
(days)
Tube 1
Tube 2
Tube 3
12.00
19.17
18.80
18.99
Average Biofuel
Potential
18.99
St Dev
0.26
127
Kirchneriella sp. 92
Table B.106. Cell Concentration for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH
(cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
9.50E+04
7.22E+04
9.72E+04
8.81E+04
1.38E+04
2.00
4.79E+05
4.18E+05
4.44E+05
4.47E+05
3.06E+04
3.00
3.12E+06
2.15E+06
2.50E+06
2.59E+06
4.91E+05
4.00
1.09E+07
1.06E+07
1.01E+07
1.05E+07
4.04E+05
5.00
1.41E+07
1.41E+07
1.24E+07
1.35E+07
9.81E+05
6.00
3.07E+07
1.38E+07
1.62E+07
2.02E+07
9.14E+06
7.00
2.86E+07
1.60E+07
1.57E+07
2.01E+07
7.36E+06
8.00
2.81E+07
1.76E+07
1.62E+07
2.06E+07
6.50E+06
9.00
2.90E+07
1.38E+07
1.64E+07
1.97E+07
8.13E+06
10.00
3.18E+07
1.51E+07
1.76E+07
2.15E+07
9.01E+06
11.00
3.12E+07
1.50E+07
1.57E+07
2.06E+07
9.16E+06
1.43E+07
1.94E+07
9.21E+06
12.00
3.00E+07
1.38E+07
Tube 1 had smaller cells but over density was similar
Table B.107. pH for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
6.76
6.74
6.74
6.75
0.01
2.00
6.63
6.63
6.63
6.63
0.00
3.00
6.61
6.58
6.58
6.59
0.02
4.00
6.49
6.48
6.46
6.48
0.02
4.71*
6.29
6.30
6.31
6.30
0.01
4.73
6.82
6.82
6.82
6.82
0.00
5.00
6.82
6.82
6.83
6.82
0.01
6.00
6.87
6.87
6.87
6.87
0.00
7.00
6.88
6.85
6.83
6.85
0.03
8.00
6.86
6.91
6.89
6.89
0.03
9.00
6.83
6.81
6.81
6.82
0.01
10.00
6.83
6.81
6.78
6.81
0.03
11.00
6.88
6.88
6.88
6.88
0.00
12.00
*Spike occurred at 4.71 days
6.82
6.82
6.81
6.82
0.01
128
Table B.108. Total Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
-49
16
52
6
51
2.00
217
107
226
183
66
3.00
849
669
684
734
100
4.00
2530
2215
1620
2122
462
5.00
12300
15380
15380
14353
1778
6.00
21580
32350
27650
27193
5400
7.00
33670
35530
31410
33537
2063
8.00
31010
33330
35240
33193
2118
9.00
38180
32310
35230
35240
2935
10.00
34910
34850
39250
36337
2523
11.00
41440
40440
41410
41097
569
12.00
40010
35860
37510
37793
2089
Table B.109. Specific Nile Red Fluorescence for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
-5.16
2.22
5.35
0.80
5.39
2.00
4.53
2.56
5.09
4.06
1.33
3.00
2.72
3.11
2.74
2.86
0.22
4.00
2.32
2.09
1.60
2.00
0.37
5.00
8.72
10.91
12.40
10.68
1.85
6.00
7.03
23.44
17.07
15.85
8.27
7.00
11.77
22.21
20.01
18.00
5.50
8.00
11.04
18.94
21.75
17.24
5.56
9.00
13.17
23.41
21.48
19.35
5.45
10.00
10.98
23.08
22.30
18.79
6.77
11.00
13.28
26.96
26.38
22.21
7.73
12.00
13.34
25.99
26.23
21.85
7.37
129
Table B.110. Ammonium Concentration for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM
KOH(mg/L).
Time
(days)
Tube
1
Tube
2
Tube
3
Average NH4+
Concentration
St Dev
0.00
58
55
55
55.82
1.80
2.00
56
53
52
53.61
2.02
3.00
48
45
45
45.88
2.10
4.00
17
12
12
13.72
2.49
5.00
0
0
0
0.00
0.00
Table B.111. Dry Cell Weight for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
12.00
1.52
1.18
1.08
1.26
0.23
Table B.112. % Biofuel Potential for Strain 92 on NH4+, 5% CO2, PIPES Buffer, and 2 mM KOH.
Time
(days)
Tube 1
Tube 2
Tube 3
12.00
31.83
35.48
34.19
Average Biofuel
Potential
33.83
St Dev
1.85
130
B.9 Growth on Ammonium and air with HEPES buffer
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the medium. The reactors were grown
on compressed air with 8mM HEPES.
Scenedesmus sp. 131
Table B.113. Cell Concentration for Strain 131 on NH4+, Air and HEPES Buffer (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
6.11E+04
6.83E+04
5.22E+04
6.05E+04
8.06E+03
2.00
1.05E+05
1.02E+05
1.02E+05
1.03E+05
1.73E+03
4.00
4.75E+05
6.45E+05
7.81E+05
6.34E+05
1.53E+05
6.00
1.00E+06
1.07E+06
1.19E+06
1.09E+06
9.61E+04
8.00
1.24E+06
1.44E+06
2.05E+06
1.58E+06
4.22E+05
10.00
1.92E+06
2.49E+06
2.73E+06
2.38E+06
4.16E+05
12.00
2.24E+06
2.61E+06
3.04E+06
2.63E+06
4.00E+05
14.04
2.33E+06
3.56E+06
4.09E+06
3.33E+06
9.03E+05
16.00
3.32E+06
3.61E+06
5.22E+06
4.05E+06
1.02E+06
18.00
3.67E+06
4.49E+06
5.13E+06
4.43E+06
7.32E+05
20.00
3.81E+06
5.70E+06
6.25E+06
5.25E+06
1.28E+06
22.00
5.53E+06
5.55E+06
7.50E+06
6.19E+06
1.13E+06
24.00
5.42E+06
6.48E+06
6.35E+06
6.08E+06
5.78E+05
26.00
6.58E+06
6.81E+06
7.12E+06
6.84E+06
2.71E+05
Table B.114. pH for Strain 131 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
7.69
7.69
7.68
7.69
0.01
2.00
7.48
7.52
7.54
7.51
0.03
4.00
7.64
7.62
7.61
7.62
0.02
6.00
7.57
7.54
7.54
7.55
0.02
8.00
7.56
7.54
7.50
7.53
0.03
10.00
7.48
7.44
7.37
7.43
0.06
12.00
7.35
7.31
7.24
7.30
0.06
14.04
7.36
7.36
7.29
7.34
0.04
16.00
7.23
7.19
7.18
7.20
0.03
18.00
7.24
7.23
7.26
7.24
0.02
20.00
7.18
7.19
7.22
7.20
0.02
22.00
7.14
7.17
7.19
7.17
0.03
24.00
7.14
7.12
7.12
7.13
0.01
26.00
7.20
7.20
7.22
7.21
0.01
131
Table B.115. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube
1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
21
-25
25
7
28
2.00
33
77
55
55
22
4.00
171
162
223
185
33
6.00
198
220
290
236
48
8.00
131
198
244
191
57
10.00
238
228
296
254
37
12.00
505
385
455
448
60
14.04
260
580
260
367
185
16.00
320
305
640
422
189
18.00
15
410
765
397
375
20.00
825
675
900
800
115
22.00
90
710
520
440
318
24.00
580
915
1340
945
381
26.00
370
1125
1620
1038
629
Table B.116. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
3.44
-3.66
4.79
1.52
4.54
2.00
3.14
7.55
5.39
5.36
2.20
4.00
3.60
2.51
2.86
2.99
0.56
6.00
1.98
2.06
2.44
2.16
0.24
8.00
1.06
1.38
1.19
1.21
0.16
10.00
1.24
0.92
1.08
1.08
0.16
12.00
2.25
1.48
1.50
1.74
0.44
14.04
1.12
1.63
0.64
1.13
0.50
16.00
0.96
0.84
1.23
1.01
0.20
18.00
0.04
0.91
1.49
0.82
0.73
20.00
2.17
1.18
1.44
1.60
0.51
22.00
0.16
1.28
0.69
0.71
0.56
24.00
1.07
1.41
2.11
1.53
0.53
26.00
0.56
1.65
2.28
1.50
0.87
132
Table B.117. Ammonium Concentration for Strain 131 on NH4+, Air and HEPES Buffer (mg/L).
Time
(days)
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.04
16.00
18.00
20.00
22.00
24.00
26.00
Tube
1
53
47
37
32
27
22
16
11
5
0.6
0.8
1.6
0.9
1.0
Tube
2
53
44
40
35
23
20
11
8
2.4
0.5
0.7
1.3
1.2
1.0
Tube
3
52
48
35
29
27
22
11
1.0
0.8
0.8
0.7
1.4
1.1
1.0
Average NH4+
Concentration
52.64
46.41
37.31
31.92
25.54
21.39
12.51
6.75
2.77
0.60
0.72
1.43
1.08
1.00
St Dev
0.55
1.96
2.82
3.05
2.53
1.24
3.05
5.16
2.13
0.16
0.07
0.17
0.13
0.00
Concentrations are approximate due to HEPES interaction on the cation column used for ammonium
analysis. Using Nessler's Assay low levels of ammonium (below 1 mg/L) were detected throughout the
entire experiment, which were below the limit of detection of the IC in the presence of HEPES Buffer
Table B.118. Dry Cell Weight for Strain 131 on NH4+, Air and HEPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
26.00
0.56
0.70
0.82
0.70
0.13
Table B.119. % Biofuel Potential for Strain 131 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
26.00
18.40
19.10
17.70
Average Biofuel
Potential
18.40
St Dev
0.99
133
Kirchneriella sp. 92
Table B.120. Cell Concentration for Strain 92 on NH4+, Air and HEPES Buffer (cells/mL).
Time (days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
1.47E+05
1.52E+05
1.59E+05
1.53E+05
6.03E+03
2.00
6.70E+05
6.77E+05
6.40E+05
6.62E+05
1.97E+04
4.00
3.45E+06
3.82E+06
3.59E+06
3.62E+06
1.87E+05
6.00
5.33E+06
4.75E+06
5.67E+06
5.25E+06
4.65E+05
8.00
1.12E+07
8.95E+06
8.40E+06
9.52E+06
1.48E+06
10.00
1.48E+07
1.40E+07
1.27E+07
1.38E+07
1.06E+06
12.00
1.62E+07
1.35E+07
1.55E+07
1.51E+07
1.40E+06
14.04
1.64E+07
1.60E+07
1.68E+07
1.64E+07
4.00E+05
16.00
2.12E+07
2.01E+07
2.13E+07
2.09E+07
6.66E+05
18.00
2.24E+07
2.03E+07
2.00E+07
2.09E+07
1.31E+06
20.00
2.04E+07
2.06E+07
2.07E+07
2.06E+07
1.53E+05
22.00
2.04E+07
2.07E+07
2.12E+07
2.08E+07
4.04E+05
24.00
1.99E+07
2.06E+07
2.11E+07
2.05E+07
6.03E+05
26.00
2.23E+07
2.25E+07
2.05E+07
2.18E+07
1.10E+06
Table B.121. pH for Strain 92 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
7.71
7.71
7.70
7.71
0.01
2.00
7.57
7.56
7.56
7.56
0.01
4.00
7.67
7.66
7.65
7.66
0.01
6.00
7.56
7.59
7.56
7.57
0.02
8.00
7.53
7.56
7.55
7.55
0.02
10.00
7.43
7.46
7.44
7.44
0.02
12.00
7.35
7.34
7.34
7.34
0.01
14.04
7.43
7.40
7.40
7.41
0.02
16.00
7.36
7.32
7.34
7.34
0.02
18.00
7.47
7.42
7.42
7.44
0.03
20.00
7.42
7.42
7.42
7.42
0.00
22.00
7.40
7.41
7.42
7.41
0.01
24.00
7.35
7.36
7.36
7.36
0.01
26.00
7.41
7.44
7.47
7.44
0.03
134
Table B.122. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
-36
-61
61
-12
64
2.00
189
177
73
146
64
4.00
67
119
125
104
32
6.00
220
247
205
224
21
8.00
345
272
250
289
50
10.00
1100
165
715
660
470
12.00
395
365
535
432
91
14.04
1525
430
1100
1018
552
16.00
2105
1190
870
1388
641
18.00
4425
1880
2605
2970
1311
20.00
4365
2535
4000
3633
969
22.00
6745
3970
4180
4965
1545
24.00
8565
5070
6840
6825
1748
26.00
12055
7155
8605
9272
2517
Table B.123. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube
3
Average Specific
Fluorescence
St Dev
0.00
-2.45
-4.01
3.84
-0.88
4.15
2.00
2.82
2.61
1.14
2.19
0.92
4.00
0.19
0.31
0.35
0.28
0.08
6.00
0.41
0.52
0.36
0.43
0.08
8.00
0.31
0.30
0.30
0.30
0.01
10.00
0.74
0.12
0.56
0.47
0.32
12.00
0.24
0.27
0.35
0.29
0.05
14.04
0.93
0.27
0.66
0.62
0.33
16.00
0.99
0.59
0.41
0.66
0.30
18.00
1.98
0.93
1.30
1.40
0.53
20.00
2.14
1.23
1.93
1.77
0.48
22.00
3.31
1.92
1.97
2.40
0.79
24.00
4.30
2.46
3.24
3.34
0.93
26.00
5.41
3.18
4.20
4.26
1.11
135
Table B.124. Ammonium Concentration for Strain 92 on NH4+, Air and HEPES Buffer (mg/L).
Time
(days)
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.04
16.00
Tube
1
51
50
39
28
20
8
1
0
0
Tube
2
55
53
44
35
26
17
8
4
0
Average NH4+
Concentration
53.45
51.10
41.89
31.94
23.20
13.11
4.67
2.33
0.00
Tube
3
54
51
43
32
23
13
5
3
0
St Dev
2.16
1.63
2.66
3.27
2.84
4.51
3.51
2.08
0.00
Concentrations are approximate due to HEPES interaction on the cation column used for ammonium
analysis.
Table B.125. Dry Cell Weight for Strain 92 on NH4+, Air and HEPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
26.00
0.96
0.90
0.94
0.93
0.03
Table B.126. % Biofuel Potential for Strain 92 on NH4+, Air and HEPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
26.00
24.64
21.87
19.23
Average Biofuel
Potential
21.91
St Dev
2.71
136
B.10 Growth on Ammonium and air with PIPES buffer
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the medium. The reactors were grown
on compressed air with 8mM PIPES.
Scenedesmus sp. 131
Table B.127. Cell Concentration for Strain 131 on NH4+, Air and PIPES Buffer (cells/mL).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Cell
Count
St Dev
0.00
3.11E+04
2.89E+04
3.61E+04
3.20E+04
3.69E+03
2.00
1.02E+05
1.13E+05
1.02E+05
1.06E+05
6.35E+03
4.00
3.03E+05
3.23E+05
3.49E+05
3.25E+05
2.31E+04
6.00
6.78E+05
7.40E+05
1.07E+06
8.29E+05
2.11E+05
8.00
9.25E+05
1.10E+06
1.39E+06
1.14E+06
2.35E+05
10.00
1.10E+06
1.42E+06
1.55E+06
1.36E+06
2.32E+05
12.00
1.63E+06
1.90E+06
2.45E+06
1.99E+06
4.18E+05
14.00
1.96E+06
2.65E+06
2.60E+06
2.40E+06
3.85E+05
16.00
2.18E+06
2.60E+06
3.82E+06
2.87E+06
8.52E+05
18.00
2.74E+06
3.65E+06
3.56E+06
3.32E+06
5.01E+05
20.00
2.40E+06
3.07E+06
4.11E+06
3.19E+06
8.62E+05
22.00
2.71E+06
3.55E+06
5.50E+06
3.92E+06
1.43E+06
24.00
2.91E+06
4.08E+06
4.60E+06
3.86E+06
8.66E+05
26.00
2.86E+06
4.64E+06
5.01E+06
4.17E+06
1.15E+06
Table B.128. pH for Strain 131 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
7.02
7.00
7.00
7.01
0.01
2.00
7.02
7.01
7.01
7.01
0.01
4.00
6.98
6.95
6.92
6.95
0.03
6.00
6.92
6.88
6.85
6.88
0.04
8.00
6.88
6.83
6.78
6.83
0.05
10.00
6.78
6.71
6.71
6.73
0.04
12.00
6.72
6.65
6.59
6.65
0.07
14.00
6.59
6.59
6.54
6.57
0.03
16.00
6.65
6.55
6.47
6.56
0.09
18.00
6.57
6.45
6.45
6.49
0.07
20.00
6.55
6.45
6.44
6.48
0.06
22.00
6.54
6.46
6.46
6.49
0.05
24.00
6.45
6.41
6.43
6.43
0.02
26.00
6.44
6.43
6.43
6.43
0.01
137
Table B.129. Total Nile Red Fluorescence for Strain 131 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
-37
122
73
53
81
2.00
31
34
45
37
7
4.00
70
83
95
83
13
6.00
122
232
171
175
55
8.00
195
277
263
245
44
10.00
198
161
140
166
29
12.00
187
205
217
203
15
14.00
205
186
238
210
26
16.00
238
152
214
201
44
18.00
205
195
220
207
13
20.00
214
266
608
363
214
22.00
275
445
730
483
230
24.00
488
666
1036
730
280
26.00
470
824
1098
797
315
Table B.130. Specific Nile Red Fluorescence for Strain 131 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
-11.90
42.21
20.22
16.85
27.21
2.00
3.04
3.01
4.41
3.49
0.80
4.00
2.31
2.57
2.72
2.53
0.21
6.00
1.80
3.14
1.60
2.18
0.84
8.00
2.11
2.52
1.89
2.17
0.32
10.00
1.80
1.13
0.90
1.28
0.47
12.00
1.15
1.08
0.89
1.04
0.14
14.00
1.05
0.70
0.92
0.89
0.17
16.00
1.09
0.58
0.56
0.75
0.30
18.00
0.75
0.53
0.62
0.63
0.11
20.00
0.89
0.87
1.48
1.08
0.35
22.00
1.01
1.25
1.33
1.20
0.16
24.00
1.68
1.63
2.25
1.85
0.35
26.00
1.64
1.78
2.19
1.87
0.29
138
Table B.131. Ammonium Concentration for Strain 131 on NH4+, Air and PIPES Buffer (mg/L).
Time
Tube Tube Tube
Average NH4+
St Dev
(days)
1
2
3
Concentration
0.00
54
56
52
53.89
2.10
2.00
51
51
49
50.25
0.90
4.00
47
45
42
44.60
2.67
6.00
43
39
34
38.63
4.48
8.00
36
32
27
31.92
4.71
10.00
32
27
21
26.65
5.68
12.00
28
22
14
21.29
6.72
14.00
23
16
8
15.66
7.73
16.00
19
9
2
9.89
8.57
18.00
14
3
1
6.34
7.09
20.00
11
2
1
4.82
5.52
22.00
7
2
2
3.37
3.17
24.00
2
1
2
1.85
0.52
26.00
1
2
1
1.27
0.36
The final data points are below the limit of quantification and therefore are approximated based on the
standard curve
Table B.132. Dry Cell Weight for Strain 131 on NH4+, Air and PIPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
26.00
0.47
0.64
0.65
0.58
0.10
Table B.133. % Biofuel Potential for Strain 131 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
26.00
9.09
11.17
17.06
Average Biofuel
Potential
12.44
St Dev
4.13
139
Kirchneriella sp. 92
Table B.134. Cell Concentration for Strain 92 on NH4+, Air and PIPES Buffer (cells/mL).
Time
(days)
Tube 1
Average Cell
Count
St Dev
0.00
1.00E+05
9.83E+04
4.84E+03
2.00
3.80E+05
3.41E+05
4.00
2.11E+06
1.98E+06
4.41E+05
3.87E+05
5.04E+04
2.19E+06
2.09E+06
1.06E+05
6.00
4.11E+06
3.96E+06
4.51E+06
4.19E+06
2.84E+05
8.00
8.10E+06
7.55E+06
7.80E+06
7.82E+06
2.75E+05
10.00
1.13E+07
8.70E+06
1.03E+07
1.01E+07
1.31E+06
12.00
1.20E+07
1.22E+07
1.32E+07
1.25E+07
6.43E+05
14.00
1.44E+07
1.44E+07
1.56E+07
1.48E+07
6.93E+05
16.00
1.90E+07
1.58E+07
1.60E+07
1.69E+07
1.78E+06
18.00
2.19E+07
1.67E+07
1.95E+07
1.94E+07
2.60E+06
20.00
2.65E+07
2.04E+07
2.09E+07
2.26E+07
3.39E+06
22.00
2.66E+07
2.09E+07
2.46E+07
2.40E+07
2.89E+06
24.00
3.02E+07
2.46E+07
2.89E+07
2.79E+07
2.93E+06
26.00
2.98E+07
2.49E+07
2.98E+07
2.82E+07
2.83E+06
Tube 2
Tube 3
1.02E+05
9.28E+04
Table B.135. pH for Strain 92 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube
1
Tube
2
Tube
3
Average
pH
St Dev
0.00
7.02
7.02
7.02
7.02
0.00
2.00
7.02
7.02
7.02
7.02
0.00
4.00
6.98
6.98
6.96
6.97
0.01
6.00
6.91
6.91
6.89
6.90
0.01
8.00
6.84
6.86
6.84
6.85
0.01
10.00
6.72
6.74
6.72
6.73
0.01
12.00
6.63
6.66
6.63
6.64
0.02
14.00
6.55
6.61
6.59
6.58
0.03
16.00
6.52
6.58
6.52
6.54
0.03
18.00
6.48
6.51
6.48
6.49
0.02
20.00
6.49
6.45
6.48
6.47
0.02
22.00
6.52
6.50
6.51
6.51
0.01
24.00
6.53
6.50
6.51
6.51
0.02
26.00
6.53
6.52
6.52
6.52
0.01
140
Table B.136. Total Nile Red Fluorescence for Strain 92 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Nile Red
Fluorescence
St Dev
0.00
113
-51
-43
6
92
2.00
55
125
98
93
35
4.00
101
101
71
91
17
6.00
196
153
248
199
48
8.00
345
314
363
341
25
10.00
369
424
412
402
29
12.00
537
500
576
538
38
14.00
1035
945
1070
1017
64
16.00
930
1115
960
1002
99
18.00
1130
1295
1175
1200
85
20.00
1540
1570
2390
1833
482
22.00
2705
1710
3085
2500
710
24.00
3450
2250
3450
3050
693
26.00
4740
4820
5950
5170
677
Table B.137. Specific Nile Red Fluorescence for Strain 92 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
Average Specific
Fluorescence
St Dev
0.00
11.30
-5.00
-4.63
0.56
9.31
2.00
1.45
3.67
2.22
2.45
1.13
4.00
0.48
0.51
0.32
0.44
0.10
6.00
0.48
0.39
0.55
0.47
0.08
8.00
0.43
0.42
0.47
0.44
0.03
10.00
0.33
0.49
0.40
0.40
0.08
12.00
0.45
0.41
0.44
0.43
0.02
14.00
0.72
0.66
0.69
0.69
0.03
16.00
0.49
0.71
0.60
0.60
0.11
18.00
0.52
0.78
0.60
0.63
0.13
20.00
0.58
0.77
1.14
0.83
0.29
22.00
1.02
0.82
1.25
1.03
0.22
24.00
1.14
0.91
1.19
1.08
0.15
26.00
1.59
1.94
2.00
1.84
0.22
141
Table B.138. Ammonium Concentration for Strain 92 on NH4+, Air and PIPES Buffer (mg/L).
Time
(days)
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
Tube
1
61
58
55
37
36
29
22
14
7
1
2
1
2
2
Tube
2
62
59
54
46
39
32
26
19
12
5
1
1
1
1
Average NH4+
Concentration
61.13
59.12
54.25
43.07
37.70
30.21
23.00
15.74
8.83
2.82
1.56
1.31
1.50
1.68
Tube
3
60
60
53
45
37
29
22
14
7
2
2
1
1
2
St Dev
1.12
0.82
0.99
4.86
1.58
1.95
2.43
2.80
3.08
2.29
0.18
0.13
0.04
0.20
Table B.139. Dry Cell Weight for Strain 92 on NH4+, Air and PIPES Buffer (g/L).
Time
(days)
Tube 1
Tube 2
Tube 3
Average Dry
Weight
St Dev
26.00
0.90
0.82
0.93
0.88
0.06
Table B.140. % Biofuel Potential for Strain 92 on NH4+, Air and PIPES Buffer.
Time
(days)
Tube 1
Tube 2
Tube 3
26.00
13.57
13.28
14.64
Average Biofuel
Potential
13.83
St Dev
0.72
142
B.11 Growth on Ammonium and CO2 using pH controllers
The experiment was run using Bold's Basal Medium with ammonium substituted for nitrate at 2.94
mM to ensure molar concentration of nitrogen remained constant in the medium. The reactors were grown
using 0.1 M KOH solution attached to a pH controller and dosed in when the pH dropped below the set
point. The pH of the medium was allowed to be lower the first two days, and then the pH was set at 6.5.
Scenedesmus sp. 131
Run 1
Table B.141. Cell Concentration for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1
(cells/mL).
4.83E+04
Average Cell
Count
4.72E+04
1.56E+03
7.33E+04
6.62E+04
1.01E+04
5.90E+04
1.98E+05
1.29E+05
9.83E+04
4.00
1.84E+05
8.65E+05
5.25E+05
4.82E+05
5.00
7.83E+05
3.33E+06
2.06E+06
1.80E+06
6.00
2.56E+06
7.05E+06
4.81E+06
3.17E+06
7.00
6.52E+06
1.22E+07
9.36E+06
4.02E+06
8.00
1.10E+07
1.11E+07
1.11E+07
7.07E+04
Time (days)
Tube 1
Tube 2
0.00
4.61E+04
2.00
5.90E+04
3.00
St Dev
9.00
1.07E+07
1.45E+07
1.26E+07
2.69E+06
10.00
1.11E+07
1.45E+07
1.28E+07
2.40E+06
11.00
1.11E+07
1.43E+07
1.27E+07
2.26E+06
12.00
1.03E+07
1.42E+07
1.23E+07
2.76E+06
13.00
1.08E+07
1.38E+07
1.23E+07
2.09E+06
143
Table B.142. pH for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
Tube 1
Tube 2
Average
pH
St Dev
0.00
6.35
6.31
6.33
0.03
2.00
6.56
6.55
6.56
0.01
3.00
6.55
6.56
6.56
0.01
4.00
6.50
6.46
6.48
0.03
5.00
6.58
6.50
6.54
0.06
6.00
6.72
6.72
6.72
0.00
7.00
6.63
6.61
6.62
0.01
8.00
6.69
6.64
6.67
0.04
9.00
6.60
6.64
6.62
0.03
10.00
6.65
6.65
6.65
0.00
11.00
6.56
6.49
6.53
0.05
12.00
6.56
6.55
6.56
0.01
13.00
6.49
6.53
6.51
0.03
With the slight increase in growth, and mostly in the size of the organisms, the pH started to increase. The
first day was increased with KOH addition to a pH of 6.45the next day, the increase in carbon uptake by the
organisms and KOH addition, the pH was increased to 6.55. This occurs prior to enough ammonium
utilization that the amount of protons released become the dominant pH force. pH is at the point of 6.5
where to 6.55 is affected by slight changes in the flow of CO 2 and can prevent the medium from staying at
6.55. The set point will be changed to 6.5. furthermore, the pH at night increases above 8 due to the extra
KOH added attempting to keep the pH at 6.55 small variations in pH are attributed to fluctuation in CO2
concentration
Table B.143. Total Nile Red Fluorescence for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
Tube 1
Tube 2
Average Nile Red
Fluorescence
St Dev
0.00
-37
122
43
112
2.00
55
128
92
52
3.00
-52
94
21
103
4.00
58
208
133
106
5.00
150
214
182
45
6.00
560
565
563
4
7.00
975
1510
1243
378
8.00
1785
1940
1863
110
9.00
1910
1785
1848
88
10.00
2060
1665
1863
279
11.00
2445
1635
2040
573
12.00
2380
1755
2068
442
13.00
3140
1960
2550
834
144
Table B.144. Specific Nile Red Fluorescence for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run
1.
Time
(days)
Tube 1
Tube 2
Average Specific
Fluorescence
St Dev
0.00
-8.03
25.26
6.16
17.18
2.00
9.32
17.46
9.40
8.02
3.00
-8.81
4.75
-2.03
9.59
4.00
3.15
2.40
2.78
0.53
5.00
1.92
0.64
1.28
0.90
6.00
2.19
0.80
1.49
0.98
7.00
1.50
1.24
1.37
0.18
8.00
1.62
1.75
1.69
0.09
9.00
1.79
1.23
1.51
0.39
10.00
1.86
1.15
1.50
0.50
11.00
2.20
1.14
1.67
0.75
12.00
2.31
1.24
1.77
0.76
13.00
2.91
1.43
2.17
1.05
Table B.145. Ammonium Concentration for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1
(mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
6.00
Tube
1
56
55
53
47
33
0
Tube
2
58
56
51
33
1
0
Average NH4+
Concentration
57.02
55.66
51.97
39.88
16.91
0.00
St Dev
1.88
1.10
1.78
10.30
22.50
0.00
Table B.146. Dry Cell Weight for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1 (g/L).
Time
(days)
Tube 1
Tube 2
Average Dry
Weight
St Dev
13.00
2.13
2.37
2.25
0.17
Table B.147. % Biofuel Potential for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
Tube 1
Tube 2
13.00
18.34
20.36
Average Biofuel
Potential
19.76
St Dev
1.24
145
Run 2
Table B.148. Cell Concentration for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2
(cells/mL).
3.17E+04
Average Cell
Count
3.56E+04
5.44E+03
1.36E+05
1.11E+05
1.24E+05
1.77E+04
3.00
2.89E+05
2.19E+05
2.54E+05
4.95E+04
4.00
9.88E+05
8.20E+05
9.04E+05
1.19E+05
5.00
3.43E+06
2.37E+06
2.90E+06
7.50E+05
6.00
4.20E+06
3.70E+06
3.95E+06
3.54E+05
7.00
7.20E+06
6.25E+06
6.73E+06
6.72E+05
8.00
6.82E+06
7.20E+06
7.01E+06
2.69E+05
9.00
8.12E+06
8.77E+06
8.45E+06
4.60E+05
10.00
8.08E+06
8.35E+06
8.22E+06
1.91E+05
10.96
8.40E+06
8.23E+06
8.32E+06
1.20E+05
Time (days)
Tube 1
Tube 2
0.00
3.94E+04
2.00
St Dev
Table B.149. pH for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
0.00
Tube 1
Tube 2
6.21
6.25
Average
pH
6.23
2.00
6.54
6.59
6.57
0.04
3.00
6.55
6.61
6.58
0.04
4.00
6.58
6.59
6.59
0.01
5.00
6.58
6.56
6.57
0.01
6.00
6.53
6.53
6.53
0.00
7.00
6.59
6.62
6.61
0.02
8.00
6.69
6.74
6.72
0.04
9.00
6.55
6.62
6.59
0.05
10.00
6.61
6.65
6.63
0.03
10.96
6.66
6.64
6.65
0.01
St Dev
0.03
146
Table B.150. Total Nile Red Fluorescence for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
0.00
Tube 1
Tube 2
70
-67
Average Nile Red
Fluorescence
2
2.00
6
125
66
84
3.00
161
110
136
36
4.00
174
266
220
65
5.00
674
415
545
183
6.00
582
644
613
44
7.00
2245
2365
2305
85
8.00
2490
2500
2495
7
9.00
2895
3370
3133
336
10.00
3665
3020
3343
456
10.96
3495
3460
3478
25
St Dev
97
Table B.151. Specific Nile Red Fluorescence for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run
2.
Time
(days)
0.00
Tube 1
Tube 2
17.77
-21.14
Average Specific
Fluorescence
-1.68
2.00
0.44
11.26
5.85
7.65
3.00
5.57
5.02
5.30
0.39
4.00
1.76
3.24
2.50
1.05
5.00
1.97
1.75
1.86
0.15
6.00
1.39
1.74
1.56
0.25
7.00
3.12
3.78
3.45
0.47
8.00
3.65
3.47
3.56
0.13
9.00
3.57
3.84
3.70
0.20
10.00
4.54
3.62
4.08
0.65
10.96
4.16
4.20
4.18
0.03
St Dev
27.51
147
Table B.152. Ammonium Concentration for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2
(mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
Tube
1
50
46
39
20
0
Tube
2
50
45
40
26
0
Average NH4+
Concentration
50.12
45.05
39.22
23.16
0.00
St Dev
0.46
0.65
0.78
4.09
0.00
Table B.153. Dry Cell Weight for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2 (g/L).
Time
(days)
10.96
Tube 1
Tube 2
1.89
1.84
Average Dry
Weight
1.868
St Dev
0.03
Table B.154. % Biofuel Potential for Strain 131 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
Tube 1
Tube 2
10.96
18.56
19.45
Average Biofuel
Potential
19.01
St Dev
0.63
148
Kirchneriella sp. 92
Run 1
Table B.155. Cell Concentration for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1 (cells/mL).
Time
(days)
0.00
Tube 1
Tube 2
2.88E+05
Average Cell
Count
2.69E+05
2.49E+05
2.76E+04
2.00
3.15E+05
3.04E+05
3.10E+05
7.78E+03
3.00
4.52E+05
4.11E+05
4.32E+05
2.90E+04
4.00
2.76E+06
1.66E+06
2.21E+06
7.78E+05
5.00
1.47E+07
8.13E+06
1.14E+07
4.65E+06
6.00
3.19E+07
2.97E+07
3.08E+07
1.56E+06
7.00
4.43E+07
3.98E+07
4.21E+07
3.18E+06
8.00
4.16E+07
4.34E+07
4.25E+07
1.27E+06
9.00
5.07E+07
4.70E+07
4.89E+07
2.62E+06
10.00
4.73E+07
4.35E+07
4.54E+07
2.69E+06
11.00
4.55E+07
4.35E+07
4.45E+07
1.41E+06
12.00
4.24E+07
4.67E+07
4.46E+07
3.04E+06
13.00
4.40E+07
4.16E+07
4.28E+07
1.70E+06
14.00
4.37E+07
4.92E+07
4.65E+07
3.89E+06
15.00
4.59E+07
4.69E+07
4.64E+07
7.07E+05
Table B.156. pH for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
0.00
Tube
1
6.35
Tube
2
6.30
Average
pH
6.33
2.00
6.56
6.42
6.49
0.10
3.00
6.50
6.50
6.50
0.00
4.00
6.65
6.56
6.61
0.06
5.00
6.72
6.56
6.64
0.11
6.00
6.65
6.50
6.58
0.11
7.00
6.65
6.48
6.57
0.12
8.00
6.59
6.55
6.57
0.03
9.00
6.70
6.55
6.63
0.11
10.00
6.74
6.58
6.66
0.11
11.00
6.74
6.73
6.74
0.01
12.00
6.62
6.49
6.56
0.09
13.00
6.66
6.55
6.61
0.08
14.00
6.61
6.47
6.54
0.10
15.00
6.58
6.48
6.53
0.07
St Dev
0.04
St Dev
149
Table B.157. Total Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
Tube 1
Tube 2
Average Nile Red
Fluorescence
St Dev
0.00
58
21
40
26
2.00
153
116
135
26
3.00
323
159
241
116
4.00
614
360
487
180
5.00
1540
1325
1433
152
6.00
7555
3355
5455
2970
7.00
18495
13385
15940
3613
8.00
27070
22010
24540
3578
9.00
26950
24720
25835
1577
10.00
25580
26090
25835
361
11.00
29810
22490
26150
5176
12.00
28530
22860
25695
4009
13.00
32300
35720
34010
2418
14.00
37040
34480
35760
1810
15.00
23680
18980
21330
3323
Table B.158. Specific Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run
1.
Time
(days)
Tube
1
Tube
2
Average Specific
Fluorescence
St Dev
0.00
2.33
0.73
2.37
1.66
2.00
4.86
3.82
4.34
0.74
3.00
7.15
3.87
5.51
2.32
4.00
2.22
2.17
2.20
0.04
5.00
1.05
1.63
1.34
0.41
6.00
2.37
1.13
1.75
0.88
7.00
4.17
3.36
3.77
0.57
8.00
6.51
5.07
5.79
1.02
9.00
5.32
5.26
5.29
0.04
10.00
5.41
6.00
5.70
0.42
11.00
6.55
5.17
5.86
0.98
12.00
6.73
4.90
5.81
1.30
13.00
7.34
8.59
7.96
0.88
14.00
8.48
7.01
7.74
1.04
15.00
5.16
4.05
4.60
0.79
150
Table B.159. Ammonium Concentration for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1
(mg/L).
Time
(days)
0.00
2.00
3.00
4.00
5.00
6.00
Tube
1
51
48
50
37
5
0
Average NH4+
Concentration
51.82
49.77
48.18
41.31
14.32
0.00
Tube
2
53
51
46
45
24
0
St Dev
1.21
2.27
2.59
5.46
13.58
0.00
Table B.160. Dry Cell Weight for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1 (g/L).
Time
(days)
Tube 1
Tube 2
Average Dry
Weight
St Dev
15.00
1.52
1.41
1.46
0.08
Table B.161. % Biofuel Potential for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 1.
Time
(days)
Tube 1
Tube 2
15.00
30.92
26.01
Average Biofuel
Potential
28.55
St Dev
2.46
151
Run 2
Table B.162. Cell Concentration for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2 (cells/mL).
Time
(days)
Tube 1
Tube 2
Average Cell
Count
St Dev
0.00
9.83E+04
1.00E+05
9.92E+04
1.20E+03
2.00
2.54E+05
3.20E+05
2.87E+05
4.67E+04
3.00
4.68E+05
1.45E+06
9.59E+05
6.94E+05
4.00
2.92E+06
6.48E+06
4.70E+06
2.52E+06
5.00
1.42E+07
2.60E+07
2.01E+07
8.34E+06
6.00
2.43E+07
3.37E+07
2.90E+07
6.65E+06
7.00
3.57E+07
4.05E+07
3.81E+07
3.39E+06
8.00
4.00E+07
4.88E+07
4.44E+07
6.22E+06
9.00
4.64E+07
4.38E+07
4.51E+07
1.84E+06
10.00
4.60E+07
4.84E+07
4.72E+07
1.70E+06
11.00
4.66E+07
4.04E+07
4.35E+07
4.38E+06
12.00
4.85E+07
4.30E+07
4.58E+07
3.89E+06
Table B.163. pH for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
Tube
1
Tube
2
Average
pH
St Dev
0.00
6.17
6.20
6.19
0.02
2.00
6.54
6.64
6.59
0.07
3.00
6.57
6.66
6.62
0.06
4.00
6.60
6.67
6.64
0.05
5.00
6.86
6.77
6.82
0.06
6.00
6.66
6.53
6.60
0.09
7.00
6.63
6.55
6.59
0.06
8.00
6.58
6.54
6.56
0.03
9.00
6.71
6.58
6.65
0.09
10.00
6.65
6.54
6.60
0.08
11.00
6.67
6.54
6.61
0.09
12.00
6.68
6.57
6.63
0.08
152
Table B.164. Total Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
Tube 1
Tube 2
Average Nile Red
Fluorescence
St Dev
0.00
19
101
60
58
2.00
76
156
116
57
3.00
299
492
396
136
4.00
916
1398
1157
341
5.00
1330
3205
2268
1326
6.00
8330
13610
10970
3734
7.00
20140
28870
24505
6173
8.00
24960
23020
23990
1372
9.00
32480
32840
32660
255
10.00
30100
38940
34520
6251
11.00
26980
27160
27070
127
12.00
21620
18420
20020
2263
Table B.165. Specific Nile Red Fluorescence for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run
2.
Time
(days)
Tube
1
Tube 2
Average Specific
Fluorescence
St Dev
0.00
1.93
10.10
6.02
5.78
2.00
2.99
4.88
3.93
1.33
3.00
6.39
3.39
4.89
2.12
4.00
3.14
2.16
2.65
0.69
5.00
0.94
1.23
1.08
0.21
6.00
3.43
4.04
3.73
0.43
7.00
5.64
7.13
6.38
1.05
8.00
6.24
4.72
5.48
1.08
9.00
7.00
7.50
7.25
0.35
10.00
6.54
8.05
7.29
1.06
11.00
5.79
6.72
6.26
0.66
12.00
4.46
4.28
4.37
0.12
153
Table B.166. Ammonium Concentration for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2
(mg/L).
Time (days)
0.00
2.00
3.00
4.00
5.00
6.00
Tube
1
53
49
47
38
3
0
Tube
2
53
48
43
29
0
0
Average NH4+
Concentration
53.17
48.62
44.81
33.67
1.37
0.00
St Dev
0.34
0.97
2.91
6.59
1.71
0.00
Table B.167. Dry Cell Weight for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2 (g/L).
Time
(days)
Tube 1
Tube 2
Average Dry
Weight
St Dev
12.00
1.66
1.76
1.71
0.07
Table B.168. % Biofuel Potential for Strain 92 on NH4+ and 5% CO2 Using pH Controller Run 2.
Time
(days)
Tube 1
Tube 2
12.00
34.09
29.54
Average Biofuel
Potential
31.81
St Dev
3.21
154
APPENDIX C
HEPES BUFFER INTERACTION ON IC
155
Analysis of ammonium samples in the presence of HEPES biological buffer.
During IC analysis of ammonium, it became apparent that HEPES had a very similar
retention time to ammonium. To assess the implications, ammonium standards were
made in an 8 mM HEPES only buffer to look at the interaction of these two elements
(Fig. C.1 and C.2). The other assessment that was made at this time was whether the two
peaks were additive or were convoluting each other and preventing an accurate analysis
of ammonium. The final determination was that the HEPES buffer increased the limit of
detection. To access the capability to determine ammonium concentrations the HEPES
with no ammonium (Fig. C.3) was subtracted from the total peak area. This was done
with varying levels of ammonium to determine whether the peaks were additive or if
HEPES was masking the peak creating a matrix effect. The ammonium concentrations
used were 5, 10, 50 , and 100 mg/L. For a value of 5 mg/L of NH4+, the value calculated
in HEPES buffer was 4.1 mg/L. Other columns were considered, but no other columns
had a significant increase in separation based on retention time and were therefore not
pursued. Nessler's assay was not deemed quantitative enough or accurate enough to have
a smaller deviation than 1 mg/L and was therefore only used for real time monitoring of
the system and to check the presence of ammonium at low concentrations where HEPES
completely masked the ammonium.
156
ADC1A, ADC1CHANNELA(CATIONS\110406_A2011-04-0612-44-50\RG000016.D)
mAu
210000
205000
200000
195000
190000
175000
2.5
20.045
7.754
5.380
180000
6.035
16.113
185000
5
7.5
10
12.5
15
+
17.5
20
min
Figure C.1 Bolds Basal Medium with 50 mg/L of NH4 , which shows good peak
separation. Ammonium peak is at approximately 6 minutes.
ADC1A, ADC1CHANNELA(CATIONS\110406_A2011-04-0612-44-50\RG000016.D)
mAu
210000
205000
200000
195000
190000
175000
1
2
3
4
5
6
7.754
5.380
180000
6.035
185000
7
8
min
Figure C. 2 This is the same chromatogram as Figure C.1 zoomed in for a better view of
the separation between the sodium peak (5.3 minutes) and the ammonium peak (6
minutes).
157
ADC1A, ADC1CHANNELA(CATIONS\110406_A2011-04-0612-44-50\RG000019.D)
5.335
mAu
180000
178000
176000
174000
172000
5.852
170000
168000
1
2
3
4
5
6
7
min
Figure C.3 Chromatogram of HEPES buffer in nanopure water showing a large peak that
comes out just before 6 minutes and has the potential to mask the ammonium peak.
ADC1A, ADC1CHANNELA(CATIONS\110406_A2011-04-0612-44-50\RG000022.D)
mAu
190000
5.341
185000
180000
175000
5.870
170000
1
2
3
4
5
+
6
Figure C.4 Chromatogram of HEPES buffer with 50 mg/L NH4 . It is evident by the
peak size and shape that the two peaks are combined.
min
158
ADC1A, ADC1CHANNELA(CATIONS\110406_A2011-04-0612-44-50\RG000023.D)
mAu
210000
205000
200000
195000
190000
5.355
185000
180000
5.877
6.173
175000
170000
1
2
3
4
5
+
7 min
6
Figure C.5 Chromatogram of 8 mM HEPES buffer with 100 mg/L NH4 .
Table C.1. Data from Day 4-12-2011 Analyzing HEPES and Ammonium Interaction.
Concentration
of NH4+
Run 1
Run 2
Run 3
0
180751.6
180951.5
5
186162.8
185565
10
197122
50
100
Average
St Dev
180868.5
180857.2
100.428
185877.3
185868.37
299
197353.6
197380.6
197285.4
142.151
266633.2
266141.1
266484.6
266419.63
252.401
352999.7
353177.1
353203.1
353126.63
110.694
Subtracted
0 from Avg
Nanopure
Comparison
error
5011.167
8856.1
43.42%
16428.2
17270.6
4.88%
85562.43
90068.9
5.00%
172269.4
177734.5
3.07%
Table C.2. Comparison of Nanopure to Calculated Value of Ammonium from 4-12-2011
Actual NH4+
Concentration (mg/L)
Nanopure NH4+
Concentration (mg/L)
Subtracted NH4+
Concentration from
HEPES (mg/L)
5
4.94
4.25
10
9.62
10.72
50
50.11
49.86
100
98.86
98.96
159
Table C.3. Data from Day 4-13-2011 Analyzing HEPES and Ammonium Interaction.
Concentration
of NH4+
Run 1
Run 2
Run 3
Average
St Dev
Subtracted
0 from Avg
Nanopure
Comparison
0
185028.5
183577.4
183404.8
184003.57
891.80
error
5
188857
188601
188596.9
188684.97
149.00
4681.40
8764.60
46.59%
10
200603.1
200407.4
200088.8
50
270557.4
270534.8
271216.8
200366.43
259.59
16362.87
17503.30
6.52%
270769.67
387.39
86766.10
91097.80
100
358682.8
356742.9
357338
4.75%
357587.90
993.80
173584.33
179222.50
3.15%
Table C.4. Comparison of Nanopure to Calculated Value of Ammonium from 4-13-2011.
5
4.88
Subtracted NH4+
Concentration from
HEPES (mg/L)
4.07
10
9.75
10.68
50
50.68
50.55
100
99.69
99.71
Actual NH4+
Concentration (mg/L)
Nanopure NH4+
Concentration (mg/L)
When analysis was completed on actual samples, the injection volume was
decreased from 10 μL to 5 μL due to additional salts in the Bold's media (Fig. C.6). This
prevented an overlap of the sodium peak with the HEPES buffer peak (Fig. C.7), which
would decrease the accuracy of the results. To ensure that this method had little effect on
the overall accuracy, the HEPES standards were run with the same samples at the 5 μL
injection volume.
160
ADC1A, ADC1CHANNELA(CATIONS\110324_A2011-03-2412-14-06\RG000017.D)
mAu
210000
200000
190000
1
2
3
4
5
7.864
170000
6.173
5.704
5.924
180000
6
7
min
8
Figure C.6 Sample with 10 μL injection had overlap of the sodium peak with the HEPES
peak making analysis impossible.
ADC1A, ADC1CHANNELA(CATIONS\110416_A2011-04-1613-03-25\RG000018.D)
mAu
195000
190000
185000
5.503
175000
1
2
3
4
5
5.932
7.789
180000
6
7
Figure C.7 When the samples were run with a 5 μL injection to essentially dilute the
sample by 2 allowed for peak separation between the sodium and HEPES.
8
min
161
APPENDIX D
BOLD'S BASAL MEDIUM
.
162
Component
For 1 L Media
KH2PO4
CaCl2*2H2O
MgSO4*7H2O
NaNO3
K2HPO4
NaCl
H3BO3
Trace Metal Solution
Solution 1
Solution 2
175 mg
25 mg
75 mg
250 mg
75 mg
25 mg
11.42 mg
1 mL
1 mL
1 mL
Component
ZnSO4*7H2O
MnCl2*4H2O
MoO3
CuSO4*5H2O
Co(NO3)2*6H2O
Final Medium
Concentration
1.29 x 10-3 M
1.70 x 10-4 M
3.04 x 10-4 M
2.94 x 10-3M
4.31 x 10-4 M
4.28 x 10-4 M
4.62 x 10-4 M
Trace Metal Solution
For 1 L stock Final medium concentration
8.82 g
7.67 x 10-5 M
1.44 g
1.82 x 10-5 M
0.71 g
1.23 x 10-5 M
1.57 g
1.57 x 10-5 M
0.49 g
4.21 x 10-6 M
Component
EDTA
KOH
Solution 1
For 1 L stock Final medium concentration
50.0 g
4.28 x 10-4 M
31.0 g
1.38 x 10-3 M
Component
FeSO4*7H2O
H2SO4
Solution 2
For 1 L stock Final medium concentration
4.89 g
4.48 x 10-5 M
1.0 mL
163
APPENDIX E
DETAILED METHODS
164
Organism Isolation and Culture
The two green algae Scenedesmus sp. 131 (131) and Kirchneriella sp. 92 (92)
(Figure E.1 and E.2, respectively) were isolated from wastewater settling ponds in Deer
Lodge, Montana, and identified through screening experiments as the two primary
candidates to continue with for further studies. Comparison of Strains during initial
testing can be found in Appendix A. The Strains were isolated using Bold's Basal
Medium (BBM) agar plates (2% w/w). Using a standard stereoscope single colonies
were picked, using glass pipettes that had been sterilized and flamed prior to use, and
transferred into 1 mL of liquid medium. This method reduces the amount of Strains that
will be isolated because 1) not all Strains are capable of growing on solid agar medium
and 2) BBM is a basic medium and does not have any vitamins that the Strain may
require. Once the colonies had grown in the 1 mL volumes, the 1mL was added to 5 mL
of BBM and bacteria check broth. The bacteria check broth was made by adding 0.5%
(w/v) dextrose to BBM prior to autoclaving. If the bacteria check broth was positive for
bacterial growth and/or the microscopy of the morphology showed signs of multiple
Strains, the culture was then streaked on a new plate for further isolation. The Strains
were streaked a total of five times to remove bacteria and provide a unialgal culture.
165
Figure E.1. Light micrograph of Scenedesmus sp. 131, along with a fluorescence
micrograph stained with Nile Red.
Figure E.2. Light micrograph of Kirchneriella sp. 92, along with a fluorescence
micrograph stained with Nile Red.
Culture Identification
The strains were first identified as unialgal by morphology and genus level
identification was made using past research papers, which provided key morphological
markers to identify Scenedesmus sp. 131 and Kirchneriella sp. 92 (Marvan et al. 1984;
166
Prescott 1978; Trainor et al. 1976). Furthermore, Scenedesmus sp. 131 was identified
based on sequencing of the SSU 18S RNA gene. Molecular sequencing of the SSU 18S
region of Scenedesmus sp. 131 shows > 99% alignment to Scenedesmus communis.
DNA extraction was completed using a modified CTAB
(hexadecyltrimethylammonium bromide) method. 1 to 2 mL of algal culture was
centrifuge in a conical screw top microcentrifuge tube at 14,500 rcf (relative centrifugal
force) for 1 minute to pellet the sample. The supernatant was then discarded and 200 μL
of extraction buffer (1M NaCl, 70mM Tris, 30mM NaEDTA, pH 8.6) was added before
centrifuging the sample for 1 minute at 14,500 rcf. The supernatant was discarded and
500 μL of new extraction buffer was added to the sample. In addition to the buffer, glass
beads were added to fill the bottom of the conical section followed by 125 μL of 2%
CTAB solution (hexadecyltrimethylammonium bromide. 200 μL of chloroform was also
added to the system to create an organic/aqueous separation. Samples were then vortexed
for several minutes to break open the cells, and were centrifuged at 12,000 rcf for 15
minutes to separate the organic and aqueous phases. The bead beating was considered
successful when the organic (bottom) layer was colored green due to chlorophyll. The
top aqueous layer was removed and placed into a new sterile microcentrifuge tube where
40 μL of 3 M sodium acetate and 480 μL of 95 % ethanol were added to the tube and
allowed to extract at -20°C overnight. The next day, the tubes were centrifuged at 12,000
rcf for 15 minutes to pellet the DNA, and the supernatant was decanted. The pellets were
washed with 200 μL of 80% ethanol and centrifuged for 15 minutes at 12,000 rcf. The
167
ethanol was removed and the samples were air dried to ensure ethanol removal. The
samples were then resuspended in 100 μL of 10 mM Tris-HCl.
The DNA was purified using the Promega Wizard Genomic DNA Purification to
eliminate any remaining RNA and proteins. 3 μL of RNase Solution were added to the
samples and incubated at 37°C for 15 minutes. The samples were cooled to room
temperature and 200 μL of Protein Precipitation Solution were added, inverting the tubes
several times. Samples were centrifuged for 3 minutes at 13,000 rcf. The supernatant
containing the DNA was trasnfered to clean tubes containing 600 μL of isopropanol at
room temperature. The tubes were inverted several times to allow the DNA to
precipitate. Samples were centrifuged for 1 minute at 13,000 rcf. The supernatant was
decanted, discarded, and 600 μL of 70% ethanol added. The samples were inverted
several times and centrifuged for 1 minute at 13,000 rcf. The ethanol was carefully
removed and the pellet allowed to air dry. The pellet was resuspended in 100 μL of DNA
Rehydration Solution and incubated in the fridge overnight.
Once the samples had been extracted and the DNA was purified, the next step was
to determine if DNA was successfully extracted. This was quickly accomplished using
Qubit analysis and NanoDrop detection to get an approximate concentration of DNA
extracted. PCR amplification was accomplished using 25 μL of 2x master mix (10 mM
dNTP, Taq, 50 mM MgCl2, and 10 mM PCR buffer) with 2.5 μL of NS1 (10 mM)
(forward primer for 18S) , 2.5 μL of NS8 (10mM) (reverse primer for 18S) and 20 μL of
sample. PCR amplification was accomplished using an Eppendorf Mastercycle Gradient
with a set protocol. The protocol was designed specifically for the NS1 and NS8 primers.
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The protocol started with an initialization step where the block heated the product to
95°C for 7 min to denature the DNA prior to starting the standard amplification cycles.
Once this step was completed, the temperature was dropped to 52°C for annealing of the
primers to the templates. The temperature was then increased to 72°C for elongation
using TAQ. This cycle was repeated 32 times before a final elongation step of 7 minutes
was completed. Once the reactions were completed, the system dropped the temperature
to 4°C to prevent degradation of the PCR product.
Once PCR was completed the product was run on a 1% agarose gel with 1x TBE
(Tris base, boric acid and EDTA). Along with the product, a 1kb step-ladder (promega)
was used to identify the proper band and quantify the PCR product in the gel based on
intensity of Ethidium Bromide fluorescence. Samples were also quantified using Qubit.
Once the PCR product concentration had been quantified, the next step was to use a PCR
clean up kit to remove any additional salts, primers, and junk DNA. The PCR product
was then diluted to the proper concentration of DNA was sent with internal primers for
sequencing at Idaho State University.
Experimental Conditions
Media
All strains were grown in Bold's Basal Medium with the pH adjusted to 7.8 with
KOH (Bischoff and Bold 1963). This medium was modified for different nitrogen source
experiments for strains Scenedesmus sp. 131 and Kirchneriella sp. 92, including
modification by replacing the sodium nitrate (2.94 mM) with either urea (1.47 mM) or
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ammonium chloride (2.94 mM), both of which were filter sterilized into the autoclaved
medium after it reached room temperature. The concentration of nitrogen in BBM is
with the concentration range found in wastewater. The range varies between 2.5 and 3.5
mM, but depending on the facility can vary outside of this range (Sedlak 1991; Woertz et
al. 2009a). Experiments with ammonium were run unbuffered or buffered with 8 mM
Piperazine-1,4-bis(2-ethanesulfonic Acid)] (PIPES, pKa 6.8) or N-(2hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES, pKa 7.5) depending on the
test parameters. The 8 mM concentration was chosen over 10 mM due to precipitant
after autoclaving and because it provided adequate buffering to minimize pH change.
Experimental System
The cultures were grown in a 14:10 light/dark cycle at room temperature (24 ±
1°C). Biological triplicate cultures were grown in vertical, tubular photobioreactors
(diameter = 70 mm, height = 500mm) filled with 1 L of BBM. Reactors were inserted
into a circulating water bath (aquarium) to increase temperature control, and were
illuminated by 12 T5 4 ft fluorescent lights for a total illumination of 350 µmoles m-2 s-1,
measured using a photosynthetically active radiation (PAR) meter (LI-COR). Each
reactor utilized a rubber stopper with three ports for sampling, aeration, and ventilation.
The reactors were also modified for pH control by the addition of an inlet port for base
and a slot for an autoclavable pH probe (Cole-Parmer). The probe was attached to a pH
controller (HANNA Instruments BL 931700-1) with a minimum pH set point, to activate
a dosing pump (HANNA Instrument BL 1.5-1) with 0.1 M KOH. The aeration and
ventilation ports were equipped with 0.2 μm filters (Millipore) to prevent contamination
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and release of algae. Incoming air was bubbled through water prior to entering the
reactors to saturate the incoming gas with water and reduce the rate of evaporation in the
reactors. The reactors were aerated with either 400 mL*min-1 of air or air with 5% CO2.
The CO2 tank was mixed with compressed house air, run through a water/oil trap, and
utilized an automated on/off controller set to turn the CO2 on with the lights.
Figure E.3. 3D schematic of tube reactor system, where the aquarium acts as a water
bath to increase temperature stability.
Analysis
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pH and Ion Chromatography
Medium pH was measured on samples using a pH meter (AB15pH, Accumet).
Concentrations of phosphate, sulfate, and nitrate were measured by ion chromatography
(IC) using an IonPac AS9-HC Anion-Exchange Column (Dionex) with a 9.0 mM sodium
carbonate buffer set at a flow rate of 1.0 mL*min-1. Detection was performed using a
CD20 conductivity detector (Dionex) at 21°C, and IC data were analyzed on Dionex
PeakNet 5.2 software. Phosphate and sulfate concentrations were measured by IC to
confirm they were in excess during experimentation.
Concentrations of ammonium were measured by IC using a Metrohm Metrosep
C4 150/4.0 mm IC column (Metrohm) controlled to 25 °C on an Agilent 1200 series
column compartment with dipicolinic acid solution at a flow rate of 0.9 mL*min-1.
Dipicolinic acid solution was made dissolving 0.1170 g of Fluka 2,6-pyridinedicarboxylic
acid >99.5% purity for ion chromatography (Sigma-Aldrich) and 0.1011 ml of 67-70%
trace metal grade nitric acid (Fisher) into 1 L of nanopure water. The detection was
performed using a Metrohm 732 IC detector. Data were analyzed using HP ChemStation
software.
Concentrations of urea were measured using an Agilent HPLC with a Zorbax
Eclipse XDB-C18 column (Agilent Technologies) controlled to 35 °C with 20 mM
sodium acetate ACS reagent (Sigma-Aldrich) adjusted to pH 7.20 and 10% acetonitrile
added (100% HPLC Grade acetonitrile, Burdick and Jackson). Eluent was delivered
using an Agilent 1100 series pump programmed to deliver gradient of 2 solutions (20
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mM sodium acetate with 10% ACN and 100% ACN) at 1.0 ml*min-1 as shown in Table
E.1.
Table E.1. Eluent Gradient for HPLC and Urea Analysis.
Time
(min)
0
12.6
13.6
22.6
20 mM Sodium Acetate with 10% ACN
(% of 1.0 mL/min)
88.9
55.5
1.1
88.9
100 % ACN
(% of 1.0 mL/min)
11.1
44.5
98.9
11.1
Detection was performed using an Agilent 1100 series UV-visisble diode-array detector
programmed to record results at 230 nm. Samples were run using an Agilent 1100 series
autosampler programmed to perform autosampler derivatization (xanthydrol
derivatization) (Clark et al. 2007). Data was analyzed using HP ChemStation software.
Nile Red Staining Protocol
Algae were stained with Nile Red (9-diethylamino-5H-benzo(α)phenoxazine-5one) (Kodak) at a concentration (4 µL of Nile Red for 1 mL sample from a 250 µg/mL in
acetone stock solution) to monitor TAG accumulation over time by using the method
developed by Cooksey et al. (1987). The correlation between Nile Red fluorescence and
TAG was recently reconfirmed by Gardner et al. (2010). Aliquots of 1 mL of sample
were removed from cultures and depending on the cell density was either stained without
dilution, diluting with 4 mL ultrapure H2O, or 0.5 mL of sample with 4.5 mL ultrapure
H2O before assaying for Nile Red fluorescence. Dilution was determined using two
methods. The first method was a standard dilution curve to determine the linear range of
the instrument, which is strain specific. This was accomplished by running a serial
dilution on a culture with high cell concentration and nitrogen depleted to increase the
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TAG content. The second method was done using real-time analysis of each experiment
by running two dilutions of each sample to verify the fluorescence values were
equivalent. This was necessary due to variations in cell size and lipid content at given
cell concentrations, which changed the linear range. Nile Red fluorescence was
quantified on a microplate reader (Bio-Tek instruments Inc.) utilizing 480/580 nm
excitation/emission filters. A baseline sensitivity setting of 75 was experimentally
determined to maximize the signal-to-noise ratio while ensuring a range that
accommodated fluorescent level changes over 10,000 units. To minimize fluorescence
spillover, black walled 96-well plates were loaded with 200 µL of sample. Unstained
samples were used for background medium and cellular autofluorescence correction. It
has been shown by Cooksey et al. that the Nile Red intensity shifts for different algal
strains over time (1987). This was recently reconfirmed (Elsey et al. 2007).
Measurement times of 60 min after staining were optimal for both strain 131 and strain
92.
Biofuel Potential Measured by Gas Chromatography
Biofuel Potential of the cell was analyzed using direct transesterification based on
the protocol designed by Griffiths (2010). For Griffiths' alkali-acid method, 10 mg of
lyophilized biomass was added to 5 mL glass serum vials along with 1.0 mL toluene.
Sodium methoxide (2 ml) (Pure, titrated, ACROS) was added to the mixture prior to
crimp sealing with Teflon lined septa and vortexing. Samples were incubated at 80 oC
for 30 min with intermittent vortexing and cooled for 10 min. 2 mL 14% boron
trifluoride methanol (BF3-methanol, Thermo Scientific) was added before repeating the
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incubation. The bottles were cooled to room temperature for 10 min before 0.8 mL H2O
and 0.8 mL hexane were added and vortexed. Samples were centrifuged at 3,000 x g for
5 min and the upper hexane/toluene layer, containing the fatty acid methyl esters (FAME)
extract, was transferred to vials for appropriate dilution with triple solvent (chloroform
(HPLC grade, EMD)/tetrahydrofuran (HPLC grade, Fisher Scientific)/hexanes (GC
grade, Fisher Scientific), 1:1:1 by volume) and analysis by GC.
In preparation for analysis by GC, extracts were diluted to the appropriate
concentration range using triple solvent. Prior to analysis, 10 L of 10 mg/mL octacosane
was added as an internal standard to 1 mL of diluted sample in a 1.5 mL GC autosampler
vial capped with a teflon lined septum. Samples (1 L) were injected into an Agilent
6890N GC and quantified with a flame ionization detector using a 15 m Restek
RTX65TG column (fused silica with a film thickness of 0.1 μm; Restek, Bellefonte, PA).
The oven temperature increased from 60-370 oC at a rate of 10 oC/min using helium as
the carrier gas at a pressure of 61.2 kPa and a flow rate of 1.3 mL/min. A split mode with
a ratio of 1:30.8 was used for analysis of FAMEs from direct transesterification reactions.
Palmitic acid methyl ester, nonadecanoic acid methyl ester, glyceryl tripalmitin and
glyceryl tristearin were used as calibration standards at a concentration range of 0.001
mg/mL to 2 mg/mL. Peak area for standards were calculated and using a linear
correlation allowed for calculation of sample concentrations. The concentration in the
samples run on GC were not the concentration of the initial samples. The concentration
analyzed on the GC was multiplied the initial volume of the organic phase, multiplied by
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the dilution factor, and divided by the biomass initially added. This provided a final
answer in % FAME/Biofuel Potential (w/w).
Cell Concentrations and Harvesting
Algal cells were counted directly using a hemocytometer with a minimum of 400
cells counted for statistical reliability (Andersen 2005). Micrographs of cell morphology
were taken using a transmitted light microscope (Nikon Eclipse E800) with an Infinity 2
color camera equipped with fluorescence capabilities to record Nile Red stained samples
for comparison between consecutive experiments. Cells were harvested at the end of the
experiment by centrifugation (4000x g for 10 min) of 750 mL of the reactor contents (3 x
250 mL bottles), washed once, and frozen for lyophilization at -80 °C. An additional 25
mL was collected from each reactor and filtered, using 0.7 µm glass fiber filters (Fischer
Scientific), to determine dry cell weight (DCW). The filters with algae were dried in an
oven at 60 °C until the weight of the biomass and filter remained constant between
weight measurements. Biomass yields were calculated by subtracting the dry weight of
the clean filter from the oven-dried weight of the filter with biomass, and cell density was
determined by dividing by the volume of sample filtered.
Kinetics
Growth Rate
The specific growth rate for each experiment was calculated using the exponential
growth equation (Equation E.1). Even though algae do not always follow the standard
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growth pattern of one parent cell producing two daughter cells (Trainor et al. 1976), the
basic equation for exponential growth given in Equation E.1 provides a method for
determining the differences between different strains and environmental conditions.
X 2 = X 1e μt ⇒ μ =
ln(
X2
X1 )
t
(Equation E.1)
The specific growth rate for each experiment was analyzed using data from the
exponential growth excluding the lag phase and the first data point of stationary phase.
The reason for excluding the first point of the stationary phase is the lack of knowledge
of the precise time the system reached stationary phase.
Nitrogen Yields
Nitrogen yields were analyzed for the different experiments. This was very
important information when analyzing the optimum conditions for bioremediation of
ammonium. Nitrogen yields were analyzed by looking at the final DCW and dividing by
the amount of nitrogen assimilated as shown in Equation E.2.
Yield XN = -
(final - inoculum) g Biomass
(final - initial) g Nitrogen
(Equation E.2)
Where the amount of nitrate, urea or ammonium was converted from the respective mass
of the molecule used to elemental nitrogen. The equation includes a negative sign to
make the yield positive. This methodology provided a consistent basis for comparing
biomass production with nitrogen uptake. The weight of the inoculum assumed to be
insignificant due to the large change in cell concentration.