GROWTH, LIPID PRODUCTION AND BIODIESEL POTENTIAL OF Chromulina freiburgensis Dofl., AN ACIDOPHILIC CHRYSOPHYTE ISOLATED FROM BERKELEY PIT LAKE

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GROWTH, LIPID PRODUCTION AND BIODIESEL POTENTIAL OF Chromulina freiburgensis Dofl., AN ACIDOPHILIC CHRYSOPHYTE ISOLATED FROM BERKELEY PIT LAKE, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research.

Abstract

Microalgae remain a promising, but underdeveloped source of lipids for sustainable biodiesel. Some of the obstacles to cost-effective commercial-scale production have been culture contamination and expensive harvest methods. A chrysophyte isolated from Berkeley Pit Lake and identified as Chromulina freiburgensis, was found to grow rapidly in a pH 2.5 liquid medium and to amass numerous intracellular lipid bodies. This research addresses the scarcity of published knowledge on the topic of chrysophyte species as potential lipid sources for biodiesel. It investigates how growth phase, culture conditions, and harvest timing influence the quantity and composition of lipids produced by this alga. This research serves as a foundation for optimizing production of lipids that contain the most desirable fatty acid composition for biodiesel. Six experimental treatments, representing six different combinations of nutrient concentrations, were monitored and sampled during a 52-day growth period, while cellular lipid content was tracked by Nile Red fluorescence measurements. Lowering medium nitrogen concentration resulted in increased lipid production, which was further increased by lowering phosphorus concentration and supplementing with CO2. The combination of lowered nitrogen
and phosphorus concentrations resulted in the highest proportion of C18:1 (50.1%) in the composition of fatty acid methyl esters from algal lipids, after approximately 22 days of stationary growth. The alga maintained its growth and favorable fatty acid composition with a
modest increase in CO2. Although C. freiburgensis from Berkeley Pit Lake did not clearly demonstrate a high lipid content, its fatty acid composition is favorable for biodiesel production, and it has additional traits which may prove advantageous. Its acidic medium provides
protection from culture contamination, and could potentially utilize acid mine drainage water. Fungal-assisted bioflocculation could then provide an economical means of harvest. This unique microalga is well suited for both cost-saving methods, and it has the potential to serve secondary
roles in bioremediation or in CO2 removal from flue gases.

Table of Contents

ABSTRACT …………………………………………………………………………………………………………………………… II
DEDICATION ………………………………………………………………………………………………………………………. III
ACKNOWLEDGEMENTS ………………………………………………………………………………………………………… IV
LIST OF TABLES …………………………………………………………………………………………………………………… XV
LIST OF FIGURES …………………………………………………………………………………………………………………. XX
LIST OF EQUATIONS ………………………………………………………………………………………………………….. XLII
1. INTRODUCTION ………………………………………………………………………………………………………………… 1
1.1. Berkeley Pit Lake and Mining in Butte, Montana ……………………………………………………… 1
1.1.1. Berkeley Pit Lake Chemistry, Past and Current ……………………………………………………………………….. 2
1.1.2. Extremophilic Organisms of Berkeley Pit Lake ………………………………………………………………………… 6
1.2. Biofuels and Biodiesel ………………………………………………………………………………………….. 7
1.2.1. Advantages of Biofuels and Biodiesel …………………………………………………………………………………….. 7
1.2.2. History and Definition of Biodiesel ………………………………………………………………………………………. 10
1.2.3. Four Generations of Biofuels ………………………………………………………………………………………………. 11
1.2.3.1. First Generation Biofuels …………………………………………………………………………………………….. 12
1.2.3.1. Second Generation Biofuels ………………………………………………………………………………………… 13
1.2.3.2. Third Generation Biofuels …………………………………………………………………………………………… 13
1.2.3.3. Fourth Generation Biofuels …………………………………………………………………………………………. 14
1.2.4. Manufacture of Biodiesel from Biological Fats and Oils (Lipids) ………………………………………………. 14
1.2.5. Quality of Biodiesel …………………………………………………………………………………………………………… 18
1.3. Algae ……………………………………………………………………………………………………………….. 19
1.3.1. Definition of Algae …………………………………………………………………………………………………………….. 19
1.3.2. Chromulina freiburgensis from Berkeley Pit Lake …………………………………………………………………… 20
1.3.3. Chrysophytes, Chromulinales and Stomatocysts……………………………………………………………………. 21

1.3.4. Biofuels from Algae …………………………………………………………………………………………………………… 24
1.3.5. Advantages of Algal Products as Biofuel Feedstocks ………………………………………………………………. 25
1.3.6. Challenges of Producing Algal Lipids for Biodiesel …………………………………………………………………. 26
1.3.7. Potential Solutions to Problems with Algal Biofuels ……………………………………………………………….. 27
1.3.8. Lowering cost and energy expenditure ………………………………………………………………………………… 28
1.3.8.1. Co-culture as a Method to Reduce Costs Associated with Harvest and Nutrient Supply ………. 28
1.3.8.2. Algal Side Products …………………………………………………………………………………………………….. 30
1.3.8.3. Algal Services…………………………………………………………………………………………………………….. 32
1.3.9. Large-Scale Algal Cultivation ………………………………………………………………………………………………. 37
1.3.9.1. Open Systems ……………………………………………………………………………………………………………. 38
1.3.9.2. Closed Systems ………………………………………………………………………………………………………….. 39
1.3.9.1. Batch vs. Continuous Cultures …………………………………………………………………………………….. 41
1.3.10. Algal Responses to Changes in Nutrient Concentrations ………………………………………………………. 42
1.3.11. Light Requirements …………………………………………………………………………………………………………. 43
1.3.12. Balancing Carbon Dioxide and Oxygen ……………………………………………………………………………….. 43
1.3.13. Stability of pH …………………………………………………………………………………………………………………. 44
1.3.14. Micronutrients and Concentrations of Metal Ions ……………………………………………………………….. 45
1.3.15. Microalgal Growth Patterns ……………………………………………………………………………………………… 46
1.4. Potential Advantages of C. freiburgensis from Berkeley Pit Lake ……………………………… 47
1.4.1. Potential High Lipid Content ………………………………………………………………………………………………. 47
1.4.2. Potential for Reduced Costs Associated with Contamination ………………………………………………….. 51
1.4.3. Potential for Use and Treatment of Acid Mine Drainage Water ………………………………………………. 52
1.4.4. Potential for CO2 Capture …………………………………………………………………………………………………… 52
1.5. Experiment to Investigate Biomass Productivity, Lipid Content and FAME Composition, in
Response to Changes in Nutrient and CO2 Concentrations ……………………………………………………….. 53
1.5.1. Medium Nitrogen Concentration ………………………………………………………………………………………… 54
1.5.2. Medium Phosphorus Concentration ……………………………………………………………………………………. 55
1.5.3. Supplemental CO2 vs. Ambient Air Only ……………………………………………………………………………….. 55
1.5.4. Timing of Harvest Relative to Growth Stage …………………………………………………………………………. 56

1.6. Suitability of C. freiburgensis as a Source of Lipids for Biodiesel ……………………………….. 56
1.6.1. Lipid Content ……………………………………………………………………………………………………………………. 56
1.6.2. Fatty Acid Composition of Algal Lipids for FAME Composition of Biodiesel Product …………………… 57
2. METHODS …………………………………………………………………………………………………………………….. 59
2.1. Preparation of Materials and Culture Methods ……………………………………………………… 59
2.1.1. Preparation of Glassware, Materials and Equipment ……………………………………………………………… 59
2.1.2. Observation Methods ………………………………………………………………………………………………………… 60
2.1.3. Culture System and Laboratory Growing Conditions ……………………………………………………………… 60
2.1.4. Modified Acid Medium (MAM) …………………………………………………………………………………………… 61
2.1.5. Re-isolation of C. freiburgensis from a Dormant Berkeley Pit Lake Algal Culture………………………… 62
2.1.6. Trial Run Cultures for Stock and Selection of Growing Conditions ……………………………………………. 63
2.1.7. Preliminary Test to Observe Responses to Lower Nutrient Concentrations ………………………………. 64
2.1.8. Monitoring Growth …………………………………………………………………………………………………………… 65
2.1.8.1. Cell Counting Method ………………………………………………………………………………………………… 65
2.1.8.2. Methods of Determining Growth Rates and Phase of Growth Cycle …………………………………. 68
2.1.9. Batch Culture Method, Simple Bioreactor …………………………………………………………………………….. 75
2.1.10. Source of Starter Cells for Inoculation ………………………………………………………………………………… 77
2.1.11. Six Experimental Treatments …………………………………………………………………………………………….. 77
2.1.11.1. Treatment 1, Standard MAM, Nutrient-Replete Control ……………………………………………….. 81
2.1.11.2. Treatment 2, Low Nitrogen Medium ………………………………………………………………………….. 81
2.1.11.3. Treatment 3, Low Phosphorus Medium ………………………………………………………………………. 81
2.1.11.4. Treatment 4, Low Nitrogen and Low Phosphorus Medium ……………………………………………. 82
2.1.11.5. Treatment 5, Low Nitrogen and Low Phosphorus Medium with Supplemental CO2 ………….. 82
2.1.11.6. Treatment 6, Intermittent Nitrogen Feeding ……………………………………………………………….. 84
2.1.12. Sampling and Cell Counting for Experimental Treatments …………………………………………………….. 84
2.1.13. Monitoring Nitrogen and Phosphorus Concentrations …………………………………………………………. 85
2.1.14. Monitoring pH ………………………………………………………………………………………………………………… 86
2.2. Nile Red Fluorescence Method to Monitor Lipid Content ………………………………………… 86
2.2.1. Equipment ……………………………………………………………………………………………………………………….. 88

2.2.2. Standard Preparation and Calibration ………………………………………………………………………………….. 89
2.2.3. Sample Preparation and Data Collection ………………………………………………………………………………. 90
2.2.4. Data Analysis ……………………………………………………………………………………………………………………. 90
2.3. Dry Weight Biomass Determination and Freeze-drying Method ………………………………. 93
2.4. GC/MS Method for Determining FAME Composition ………………………………………………. 94
2.4.1. Sample Preparation …………………………………………………………………………………………………………… 95
2.4.1.1. Lipid Extraction and Transesterification ………………………………………………………………………… 95
2.4.1.2. 2.4.2.2 Filtering to Remove Algal Solids ………………………………………………………………………… 95
2.4.1.3. FAME Extraction ………………………………………………………………………………………………………… 95
2.4.1.4. Concentration by Evaporation …………………………………………………………………………………….. 96
2.4.2. Naphthalene Internal Standard …………………………………………………………………………………………… 97
2.4.3. GC/MS Equipment, Data Collection and Analysis …………………………………………………………………… 97
2.4.3.1. GC/MS Electron Ionization Method ……………………………………………………………………………… 97
2.4.3.2. GC/MS Chemical Ionization Method …………………………………………………………………………… 101
2.4.4. Relating EI Chromatography Peak Areas (Signal Intensities) to Quantities of FAMEs in Each Sample ..
……………………………………………………………………………………………………………………………………… 110
2.4.4.1. Response Factors from FAME Standards …………………………………………………………………….. 111
2.5. Additional FAME Analysis by Algal Genomics and Synthetic Biology Laboratory, National Research
Council Canada …………………………………………………………………………………………………………………. 113
3. RESULTS AND DISCUSSION ………………………………………………………………………………………………… 114
3.1. Initial Observations of Culture Re-Isolated from a Dormant Culture ……………………….. 114
3.2. Cell Types and Behavior ……………………………………………………………………………………. 114
3.3. Observation of External Features of Cell Wall and Siliceous Cyst ……………………………. 116
3.4. Growth and Cell Density ……………………………………………………………………………………. 118
3.5. Slight Decrease in Medium pH of Three Treatments ……………………………………………… 119
3.6. Growth Changes in Responses to Experimental Nutrient Concentrations ………………… 120
3.6.1. Rapid Transition to Stationary Phase in Low Nitrogen Treatments …………………………………………. 122
3.6.2. Extended Phase of Linear Growth in High Nitrogen Treatments…………………………………………….. 123

3.6.1. Changes in Growth Rates as a Response to Nitrogen Depletion …………………………………………….. 124
3.6.2. Physiological Changes Associated with Lipid Accumulation …………………………………………………… 126
3.6.3. Aggregation, Settling and Algal-Fungal Flocculation …………………………………………………………….. 128
3.6.4. Swimming Behavior …………………………………………………………………………………………………………. 131
3.6.5. Extra-Large Cells ……………………………………………………………………………………………………………… 135
3.7. Nitrogen and Phosphorus Concentrations …………………………………………………………… 136
3.7.1. Nitrogen ………………………………………………………………………………………………………………………… 136
3.7.2. Phosphorus …………………………………………………………………………………………………………………….. 138
3.8. Biomass (Dry Weight) Productivity Responses to Treatments ………………………………… 140
3.9. Relationship Between Nutrient Concentrations, Biomass and Lipid Content …………….. 145
3.10. Total Lipid Content, Nile Red Fluorescence and CNRC Results ………………………………… 147
3.11. Triglyceride (Triacylglycerol) Portion of Total Algal Lipids ……………………………………… 153
3.12. GC/MS Results for FAME Composition ………………………………………………………………… 154
3.12.1. FAMEs detected in Transesterified Algal Lipids ………………………………………………………………….. 154
3.12.1. Changes in FAME Concentrations with Time ……………………………………………………………………… 156
3.12.2. Interpreting GC/MS Results, with Comparison to FAME Composition Results from CNRC GC and
NMR Methods. ……………………………………………………………………………………………………………………………………. 158
3.12.3. Highest Total Detected FAME Content and Highest Proportion of C18:1 ………………………………. 160
3.12.3.1. Accumulation of Total Detected FAME Yield Over Time ………………………………………………. 161
3.12.3.2. Highest Detected Total FAME on One Sample Day ……………………………………………………… 162
3.12.3.3. Treatments Resulting in the Highest Proportions of C18:1 …………………………………………… 163
3.12.4. Disparity in Detected Total FAME Between GC/MS Methods and CNRC Methods ………………….. 165
3.12.5. Consistent Results for Percentages of individual FAME Types Between GC/MS Methods and CNRC
Methods ……………………………………………………………………………………………………………………………………. 167
3.12.6. Changes in Fatty Acid Composition and Proportions FAMEs with Time …………………………………. 168
3.12.7. Common Patterns of Change in FAME Compositions with Time …………………………………………… 168
3.12.8. Contrasting Patterns of Change in FAME Compositions with Time ……………………………………….. 175
3.12.8.1. Treatment 1, Nutrient Replete, Standard MAM …………………………………………………………. 175
3.12.8.2. Treatment 2, Low Nitrogen MAM …………………………………………………………………………….. 179

3.12.8.3. Treatment 3, Low Phosphorus MAM ………………………………………………………………………… 184
3.12.8.4. Treatment 4, Low Nitrogen and Low Phosphorus MAM ………………………………………………. 188
3.12.8.6. Treatment 5, Low Nitrogen, Low Phosphorus MAM with Supplemental CO2 ………………….. 193
3.12.8.7. Treatment 6 ………………………………………………………………………………………………………….. 198
4. SUMMARY …………………………………………………………………………………………………………………… 204
4.1. The Effects of Nutrients on Growth, Biomass Production, and Lipid Content ……………. 204
4.1.1. Starting Nitrogen Concentration ……………………………………………………………………………………….. 204
4.1.2. Intermittent Nitrogen Feeding (Treatment No. 6) ……………………………………………………………….. 205
4.1.3. Phosphorus …………………………………………………………………………………………………………………….. 206
4.2. Lipid Content and FAME Yields for the Final Product …………………………………………….. 206
4.3. FAME Composition …………………………………………………………………………………………… 209
4.4. Harvest Timing ………………………………………………………………………………………………… 210
4.5. Fatty Acid and FAME Composition ……………………………………………………………………… 212
4.6. CO2 Supplementation ……………………………………………………………………………………….. 219
4.6.1. Biomass Productivity with Supplemental CO2 ……………………………………………………………………… 219
4.6.2. Lipid Productivity and FAME Composition with Supplemental CO2 ………………………………………… 219
5. CONCLUSIONS ………………………………………………………………………………………………………………. 220
5.1. Changes in Biomass Production in Response to Nutrient Concentrations and CO2 Supplementation
……………………………………………………………………………………………………………………… 220
5.2. Changes in Lipid Production in Response to Nutrient Concentrations and CO2 Supplementation
……………………………………………………………………………………………………………………… 221
5.3. Changes in FAME Composition in Response to Nutrient Concentrations and CO2 Supplementation
……………………………………………………………………………………………………………………… 221
5.4. Change in FAME Composition with Harvest Timing ………………………………………………. 222
5.5. Differences Between the Total Lipid and Total FAME Content Reported by CNRC and Those
Detected by the Nile Red and GC/MS Methods……………………………………………………………………… 223
5.5.1. Total Lipid Content Detection by Nile Red Fluorescence Method Compared to CNRC Solvent
Extraction Method …………………………………………………………………………………………………………………………………….. 223

5.5.2. Nile Red Detection of Lipid Increase and Differences in Total Lipid Content ……………………………. 224
5.5.3. Total FAME Content Detection by GC/MS Method Compared to CNRC Methods …………………….. 224
5.5.4. FAME Composition Detection by GC/MS Method Compared to CNRC Methods ………………………. 225
6. SUGGESTIONS FOR FUTURE WORK ………………………………………………………………………………………. 226
6.1. Replication of Experimental Treatments ……………………………………………………………… 226
6.2. Suggestions for Future Experiments Investigating Nutrient Concentrations …………….. 226
6.3. Supplemental CO2 and the Potential for Carbon Capture from Flu Gas Emissions …….. 227
6.4. Potential for Bioremediation, Acid Mine Drainage Use and Treatment …………………… 228
6.5. Potential for Fungus-assisted Flocculation and Co-culture to Improve Productivity …… 228
6.6. Remaining Unanswered Questions …………………………………………………………………….. 229
6.7. A Unique Alga that Deserves Further Study …………………………………………………………. 231
7. REFERENCES CITED …………………………………………………………………………………………………………. 233
8. APPENDIX A: TECHNICAL NOTE – ERROR IN CELL COUNTING, CELEROMICS, 2015 ……………………………….. 255
9. APPENDIX B: TABLE OF DRY WEIGHT BIOMASS YIELDS ………………………………………………………………. 259
10. APPENDIX C: MONITORING PH IN EXPERIMENTAL TREATMENTS ……………………………………………………. 262
11. APPENDIX D: NILE RED FLUORESCENCE LIPID CALCULATIONS ……………………………………………………….. 263
11.1. Example Excel Charts: Fluorescence Results for Triolein Standards and Algal Samples 263
11.2. Example: Nile Red Fluorescence Data from March 3rd, 2018 ………………………………….. 264
11.3. Nile Red Fluorescence Calibration Curves ……………………………………………………………. 265
11.4. Line Equations from Linear Calibration Curves …………………………………………………….. 272
11.1. Line Equations from Polynomial Calibration Curves ……………………………………………… 273
11.1. Nile Red Fluorescence Data and Calculations Summary Table ……………………………….. 274
12. APPENDIX E: GC/MS INSTRUMENT METHOD SETTINGS …………………………………………………………….. 280
12.1. Electron Ionization (EI) Settings …………………………………………………………………………. 280
12.1. Chemical Ionization (CI) Settings ………………………………………………………………………… 283
12.2. Appendix F: GC/MS File Name List …………………………………………………………………….. 286
12.3. Appendix G: GC/MS Electron Ionization Chromatograms ……………………………………… 288

12.3.1. Treatment 1, Standard MAM (Nutrient Replete) ……………………………………………………………….. 288
12.3.1.1. Treatment 1, Standard MAM, Day 6 …………………………………………………………………………. 288
12.3.1.2. Treatment 1, Standard MAM, Day 9 …………………………………………………………………………. 289
12.3.1.3. Treatment 1, Standard MAM, Day 52 ……………………………………………………………………….. 292
12.3.2. Treatment 2, Low Nitrogen …………………………………………………………………………………………….. 293
12.3.2.1. Treatment 2, Low Nitrogen, Day 6 ……………………………………………………………………………. 293
12.3.2.2. Treatment 2, Low Nitrogen, Day 9 ……………………………………………………………………………. 294
12.3.2.3. Treatment 2, Low Nitrogen, Day 19 ………………………………………………………………………….. 295
12.3.2.4. Treatment 2, Low Nitrogen, Day 27 ………………………………………………………………………….. 296
12.3.2.5. Treatment 2, Low Nitrogen, Day 52 ………………………………………………………………………….. 297
12.3.3. Treatment 3, Low Phosphorus ………………………………………………………………………………………… 298
12.3.3.1. Treatment 3, Low Phosphorus, Day 6 ……………………………………………………………………….. 298
12.3.3.2. Treatment 3, Low Phosphorus, Day 9 ……………………………………………………………………….. 299
12.3.3.3. Treatment 3, Low Phosphorus, Day 19 ……………………………………………………………………… 300
12.3.3.4. Treatment 3, Low Phosphorus, Day 27 ……………………………………………………………………… 301
12.3.3.5. Treatment 3, Low Phosphorus, Day 52 ……………………………………………………………………… 302
12.3.4. Treatment 4, Low Nitrogen and Low Phosphorus ………………………………………………………………. 303
12.3.4.1. Treatment 4, Low Nitrogen and Low Phosphorus, Day 6 ……………………………………………… 303
12.3.4.2. Treatment 4, Low Nitrogen and Low Phosphorus, Day 9 ……………………………………………… 304
12.3.4.3. Treatment 4, Low Nitrogen and Low Phosphorus, Day 19 ……………………………………………. 305
12.3.4.4. Treatment 4, Low Nitrogen and Low Phosphorus, Day 27 A …………………………………………. 306
12.3.4.5. Treatment 4, Low Nitrogen and Low Phosphorus, Day 27 B …………………………………………. 307
12.3.4.6. Treatment 4, Low Nitrogen and Low Phosphorus, Day 27 C …………………………………………. 308
12.3.4.7. Treatment 4, Low Nitrogen and Low Phosphorus, Day 52 ……………………………………………. 309
12.3.5. Treatment 5, Low Nitrogen, Low Phosphorus + CO2 …………………………………………………………… 310
12.3.5.1. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 6 ………………………………………….. 310
12.3.5.2. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 9 ………………………………………….. 311
12.3.5.3. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 15 A ……………………………………… 312
12.3.5.4. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 15 B ……………………………………… 313

12.3.5.5. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 15 C ……………………………………… 314
12.3.5.6. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 19 A ……………………………………… 315
12.3.5.7. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 19 B ……………………………………… 316
12.3.5.8. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 19 C ……………………………………… 317
12.3.5.9. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 19 D ……………………………………… 318
12.3.5.10. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 27 ………………………………………. 319
12.3.5.11. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 52 ………………………………………. 320
12.3.6. Treatment 6, Intermittent Nitrogen Feeding …………………………………………………………………….. 321
12.3.6.1. Treatment 6, Intermittent Nitrogen Feeding, Day 6 ……………………………………………………. 321
12.3.6.2. Treatment 6, Intermittent Nitrogen Feeding, Day 9 ……………………………………………………. 322
12.3.6.3. Treatment 6, Intermittent Nitrogen Feeding, Day 19 ………………………………………………….. 323
12.3.6.4. Treatment 6, Intermittent Nitrogen Feeding, Day 27 ………………………………………………….. 324
12.3.6.5. Treatment 6, Intermittent Nitrogen Feeding, Day 52 ………………………………………………….. 325
12.4. Appendix H: GC/MS Chemical Ionization Chromatograms …………………………………….. 326
12.4.1. Treatment 1, Standard MAM (Nutrient Replete) ……………………………………………………………….. 326
12.4.1.1. Treatment 1, Standard MAM, Day 6 …………………………………………………………………………. 326
12.4.1.2. Treatment 1, Standard MAM, Day 27 ……………………………………………………………………….. 327
12.4.2. Treatment 2, Low Nitrogen …………………………………………………………………………………………….. 328
12.4.2.1. Treatment 2, Low Nitrogen, Day 6 ……………………………………………………………………………. 328
12.4.2.2. Treatment 2, Low Nitrogen, Day 27 ………………………………………………………………………….. 329
12.4.3. Treatment 3, Low Phosphorus ………………………………………………………………………………………… 330
12.4.3.1. Treatment 3, Low Phosphorus, Day 6 ……………………………………………………………………….. 330
12.4.3.2. Treatment 3, Low Phosphorus, Day 27 ……………………………………………………………………… 331
12.4.4. Treatment 4, Low Nitrogen and Low Phosphorus ………………………………………………………………. 332
12.4.4.1. Treatment 4, Low Nitrogen and Low Phosphorus, Day 6 ……………………………………………… 332
12.4.5. Treatment 5, Low Nitrogen, Low Phosphorus + CO2 …………………………………………………………… 333
12.4.5.1. Treatment 5, Low Nitrogen, Low Phosphorus + CO2, Day 6 ………………………………………….. 333
12.4.6. Treatment 6, Intermittent Nitrogen Feeding …………………………………………………………………….. 334
12.4.6.1. Treatment 6, Intermittent Nitrogen Feeding, Day 6 ……………………………………………………. 334

13. APPENDIX I: CALCULATIONS OF FAME CONCENTRATIONS SUMMARY ……………………………………………… 335
13.1. Treatment 1. MAM (Nutrient Replete Control) …………………………………………………….. 335
13.1. Treatment 2. Low Nitrogen ……………………………………………………………………………….. 339
13.1. Treatment 3. Low Phosphorus …………………………………………………………………………… 343
13.1. Treatment 4. Low Nitrogen & Low Phosphorus ……………………………………………………. 347
13.1. Treatment 5. Low Nitrogen & Low Phosphorus + CO2 ……………………………………………. 352
13.1. Treatment 6. Low Nitrogen and Phosphorus with Intermittent Nitrogen Feeding …….. 360
14. APPENDIX J: DESCRIPTION OF AN UNUSUAL OPTICAL PHENOMENON CAUSED BY A FEW SPECIES OF GOLDEN ALGAE
……………………………………………………………………………………………………………………………….. 364
15. APPENDIX K: CONTACT INFORMATION, PRODUCTS AND SERVICES …………………………………………………. 366
15.1. Algaebase ……………………………………………………………………………………………………….. 366
15.2. Canadian Phycological Culture Centre ………………………………………………………………… 366
15.1. EMtrix, University of Montana …………………………………………………………………………… 366
15.2. Ground Water Information Center, MBMG Data Center ……………………………………….. 367
15.3. Hausser Scientific …………………………………………………………………………………………….. 367
15.4. Laboratory for Environmental Analysis ……………………………………………………………….. 367
15.5. National Research Council Canada, Conseil national de recherches Canada …………….. 367

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Author

June E. Mohler Mitman

No of Chapters

15

No of Pages

411

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