Determination of Freshwater Algal Biomass and Sulfolipid Content as Functions of Inorganic

149 Pages·2017·2.58 MB·English

Clemson University
TigerPrints
All Theses Theses
8-2011
Determination of Freshwater Algal Biomass and
Sulfolipid Content as Functions of Inorganic
Carbon Treatment
Melissa Morella
Clemson University, [email protected]
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Recommended Citation
Morella, Melissa, "Determination of Freshwater Algal Biomass and Sulfolipid Content as Functions of Inorganic Carbon Treatment"
(2011).All Theses. 1165.
https://tigerprints.clemson.edu/all_theses/1165
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DETERMINATION OF FRESHWATER ALGAL BIOMASS AND SULFOLIPID CONTENT AS FUNCTIONS
OF INORGANIC CARBON TREATMENT
___________________________________________________
A Thesis
Presented to
the Graduate School of
Clemson University
___________________________________________________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Biosystems Engineering
___________________________________________________
by
Melissa McKinney Morella
August 2011
___________________________________________________
Accepted by:
Dr. Caye Drapcho, Committee Chair
Dr. Melisssa Riley
Dr. Terry Walker
ABSTRACT

Cultures of mixed freshwater algae were grown in open batch reactors made up of
modified BG-11 media that contained 0, 25, 50 or 100% of the suggested inorganic carbon
content (0, 0.5, 0.1 and 0.2 g/L Na CO ) to assess the subsequent production of the high-value
2 3
product, sulfoquinovosyl diacylglycerol (SQDG). After 25 days of growth, the reactors were
composed of primarily Oscillatoria growth. All four reactors exhibited a trend of increasing
biomass and alkalinity with time, and an initial increase then decrease in pH. The total inorganic
carbon (TIC) in all reactors exhibited a mirrored, opposite trend as pH, with an initial decrease
then increase over time. A positive correlation was found between specific growth rate and
amount of initial total inorganic carbon in the reactors. The Monod constants µ and K were
max S
estimated to be 0.025 hr-1 and 0.00215 mol/L C, respectively. At 95% confidence, the amount of
initial TIC significantly affected the total lipid concentration, which increased with time in all
reactors, and showed a trend of increasing total lipids with increasing initial TIC. Additionally,
the initial TIC did not significantly affect the concentration of SQDG per mL culture or per mg dry
biomass, or the percent of SQDG within the total lipids. SQDG content per mg biomass
decreased with time for all TIC treatments.

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DEDICATION

I would like to dedicate my graduate thesis and associated research to my husband,
Daniel Morella. His unwavering support and encouragement were welcomed blessings
throughout the years-long and occasionally daunting course of this accomplishment. He is my
anchor, and I am inspired by him every day.
I would also like to dedicate this work to my mother, who is the smartest person I know.
She continues to teach me the importance of the constant pursuit of knowledge and has
instilled in me the same love of learning that she shares; for these gifts, I am truly thankful.

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ACKNOWLEDGEMENTS

I would like to thank Dr. Caye Drapcho, Dr. Melissa Riley, and Dr. Terry Walker for their
willingness to serve on my committee and to provide technical and practical expertise in order
to bring this project to successful completion.
I would also like to thank Dr. Melissa Riley, additionally, and Fran Harper of the Clemson
University Multi-User Analytical Lab for their assistance with the rigorous chromatography
section of this project, and Mr. Scott Davis of the Clemson University Aquaculture Facility for his
assistance in identifying algal species.

iv
TABLE OF CONTENTS

Page
TITLE PAGE ..................................................................................................................................... i
ABSTRACT ..................................................................................................................................... ii
DEDICATION ................................................................................................................................ iii
ACKNOWLEDGEMENTS ............................................................................................................... iv
LIST OF TABLES ........................................................................................................................... vii
LIST OF FIGURES .......................................................................................................................... ix
CHAPTER
1. INTRODUCTION AND LITERATURE REVIEW ................................................................ 1
Algal Biology and its Role in Carbon Capture ......................................................... 2
Products from Algal Biomass .................................................................................. 6
Effects of Culture Conditions on Algal Growth..................................................... 11
References ............................................................................................................ 19

2. EXPERIMENTAL METHODS ....................................................................................... 24
Overview .............................................................................................................. 24
Batch Reactor Algal Growth ................................................................................. 24
Algal Species Identification .................................................................................. 26
Algal Sample Collection Schedule ........................................................................ 26
Optical Density ..................................................................................................... 28
Total Suspended Solids ......................................................................................... 28
Alkalinity ............................................................................................................... 29
Total Inorganic Carbon ......................................................................................... 29
Extraction of Lipids from Frozen Samples ............................................................ 31
Solid Phase Extraction of Lipid Samples ............................................................... 32
Thin Layer Chromatography of Lipid Samples ...................................................... 33
HPLC Separation and Quantification of Lipid Samples ......................................... 35
Total Lipid Weight Measurement ......................................................................... 36
Growth Parameter Determination ....................................................................... 37
References ............................................................................................................ 38

v
Table of Contents (Continued)

Page

3. DETERMINATION OF FRESHWATER ALGAL BIOMASS AND SULFOLIPID
CONTENT AS FUNCTIONS OF INORGANIC CARBON TREATMENT ........................ 40
Abstract ................................................................................................................ 40
Introduction .......................................................................................................... 41
Literature Review ................................................................................................. 41
Experimental Methods ......................................................................................... 48
Results and Discussion ......................................................................................... 54
Conclusion ............................................................................................................ 70
References ............................................................................................................ 71

4. SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK .................................... 75
Experimental Summary ........................................................................................ 75
Recommendations for Future Work .................................................................... 77
References ............................................................................................................ 78

APPENDICES ................................................................................................................................ 80
A. Experimental Sampling Schedule ............................................................................. 81
B. Raw Data .................................................................................................................. 82
C. Chromatograms ........................................................................................................ 99
D. Graphical Representations of Data ........................................................................ 134

vi
LIST OF TABLES

Table Page
1.1 Algal Conversion Processes ....................................................................................... 8

1.2 Combined SQDG/PG Concentrations with Time ..................................................... 17

2.1 Modified 616 Medium BG-11 ................................................................................. 25

2.2 HPLC Gradient Time Table ...................................................................................... 35

3.1 Modified 616 Medium BG-11 ................................................................................. 48

3.2 HPLC Gradient Time Table ...................................................................................... 52

3.3 Specific Growth Rates ............................................................................................. 59

3.4 ANOVA-Effect of Initial TIC and Time on Total Lipid Concentration ...................... 62

3.5 ANOVA-Effect of Initial TIC and Time on SQDG per mL of Culture ......................... 65

3.6 ANOVA-Effect of Initial TIC and Time on SQDG per mg Dry Biomass ..................... 66

3.7 ANOVA-Effect of Initial TIC and Time on % SQDG in Total Lipids ........................... 67

3.8 Post Investigation Specific Growth Rates ................................................................ 69

A.1 Sampling Schedule Employed for Reactors 0, 25, 50 and 100 ............................... 81

B.1 Dry Weight and O.D. Measurements ..................................................................... 82

B.2 pH Measurements .................................................................................................. 83

B.3 Total Inorganic Carbon Measurements .................................................................. 84

B.4 Alkalinity Measurements ........................................................................................ 85

B.5 Total Inorganic Carbon Measurements in First Observed Growth Phase .............. 86

B.6 Total Lipid Concentration per mL Culture .............................................................. 87

B.7 Concentration of SQDG per mL Culture ................................................................. 90
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List of Tables (Continued)

Table Page

B.8 Concentration of SQDG per mg Algal Dry Weight .................................................. 92

B.9 Concentration of SQDG per mL Total Lipids ........................................................... 94

B.10 Percent of SQDG in Total Lipids............................................................................ 96

B.11 Post Experiment 5-Day Growth Data ................................................................... 97

B.12 Post Experiment 5-Day Concentration of SQDG per mg Dry Biomass ................. 98

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LIST OF FIGURES

Figure Page
1.1 SQDG Molecule ....................................................................................................... 10

2.1 Procedural Flow Chart for Sampling ....................................................................... 27

2.2 Procedural Flow Chart for Extraction and Analysis ................................................ 28

3.1 SQDG Molecule ....................................................................................................... 44

3.2 Procedural Flow Chart for Extraction and Analysis ................................................ 51

3.3 Oscillatoria Grown in All Four Reactors .................................................................. 54

3.4 Alkalinity versus Time ............................................................................................. 55

3.5 pH versus Time ....................................................................................................... 56

3.6 Total Inorganic Carbon versus Time ....................................................................... 57

3.7 Optical Density versus Time ................................................................................... 57

3.8 Biomass versus Time (n=2) ..................................................................................... 58

3.9 Natural Log of Biomass versus Time ....................................................................... 59

3.10 Lineweaver-Burk Plot for Determination of Growth Parameters ........................ 60

3.11 TLC Separation of Sample and Standards ............................................................. 61

3.12 Total Lipid Concentrations versus Time for All Reactors (n=4) ............................ 62

3.13 SQDG Standard, 0.05 mg/mL Chromatogram ...................................................... 63

3.14 SQDG Standard Curve ........................................................................................... 63

3.15 PG Standard, 0.05 mg/mL Chromatogram ........................................................... 64

3.16 Phospholipid Fraction from 0 hr Sample, Reactor 100 ........................................ 64

3.17 Concentration of SQDG per mL Culture (n=4) ...................................................... 64
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