USE OF BICARBONATE SALTS IN ALGAL GROWTH FOR ENHANCEMENT OF LIPID CONTENT ...

132 Pages·2016·1.89 MB·English

by  Pedersen, Todd

USE OF BICARBONATE SALTS IN ALGAL GROWTH FOR

ENHANCEMENT OF LIPID CONTENT

by

Todd Christian Pedersen

A thesis submitted in partial fulfillment
of the requirements for the degree

of

Master of Science

in

Chemical Engineering

MONTANA STATE UNIVERSITY
Bozeman, Montana

November 2016
©COPYRIGHT

by

Todd Christian Pedersen

2016

All Rights Reserved
ii

DEDICATION

I dedicate my thesis to Dick and Linda, whose everlasting inspiration will always
determine me; and to my parents Pam and Rob, who have always been unconditionally
supportive in my endeavors.
iii

ACKNOWLEDGEMENTS

A special thanks to my committee chair, Dr. Brent Peyton, who helped provide
me with the opportunity to complete a M.S. in chemical engineering. He was a patient
mentor and an attentive advisor. I would also thank the other members of the committee,
Dr. Robin Gerlach and Dr. Robert Gardner, who were also instrumental in providing me
with an opportunity to complete a graduate degree. Dr. Gerlach was influential in adeptly
educating me regarding the use analytical instruments and their maintenance. Dr. Gardner
is an admired mentor, a cherished friend, and largely responsible for ingraining
fundamental scientific discipline into my research through his continued close
mentorship.
Thanks to Dr. Egan Lohman, who was a pleasure to work with in the early days of
my graduate studies and was sincerely missed for the remainder. Graduate students Karen
Moll, Muneeb Rathore, and Matthew Jackson, and undergraduate students Ashley
Berninghaus and James Crawford all deserve distinguished thanks. Their accommodating
help in the lab was refreshing and are all admirable people to work with. Final thanks to
the MSU Algal Biofuels Group for setting a high standard of research in our growing
interdisciplinary collaborations, and to all the wonderful faculty and staff at the Center
for Biofilm Engineering.
iv

TABLE OF CONTENTS

1. INTRODUCTION ...........................................................................................................1

Project Rationale ..............................................................................................................1
Background ......................................................................................................................2
Algae as a Biomass Alternative .......................................................................................4
Cultivation of Algae for Biomass ............................................................................6
Harvesting of Algal Biomass ...................................................................................7
Extraction and Conversion of Algal Biomass ..........................................................8
High pH and Alkaline Systems for Growth of Microalgae ...........................................10

2. ASSESMENT OF NANNOCHLOROPSIS GADITANA GROWTH
AND LIPID ACCUMULATION WITH ENHANCED INORGANIC
CARBON DELIVERY ..................................................................................................13

Contribution of Authors and Co-Authors ......................................................................13
Manuscript Information Page ........................................................................................14
Abstract ..........................................................................................................................15
Introduction ....................................................................................................................16
Materials and Methods ...................................................................................................20
Strain, Experimental, and Culturing Conditions ....................................................20
Analysis of Culture and Media Components .........................................................22
Chlorophyll Measurements ....................................................................................23
Harvesting ..............................................................................................................23
Transesterification for Fatty Acid
Methyl Ester (FAME) Analysis .............................................................................23
GC-MS Analysis ....................................................................................................24
Calculations............................................................................................................25
Statistics .................................................................................................................26
Results and Discussion ..................................................................................................26
Cell Growth and Culture Density for
Different Carbon Supplementation Strategies .......................................................26
Total Chlorophyll Accumulation and
Degradation with Varying Carbon Supplementation .............................................34
Fatty Acid Methyl Ester (FAME) Analysis ...........................................................36
Conclusion .....................................................................................................................42
Competing Interests .......................................................................................................44
Acknowledgments..........................................................................................................45
References ......................................................................................................................46
v

TABLE OF CONTENTS – CONTINUED

3. MONITORING CHLORELLA VULGARIS LIPIDS DURING
BICARBONATE INDUCED LIPID ACCUMULATION
USING 1H HIGH-RESOULUTION MAGIC ANGLE SPINNING
(HR-MAS) NMR SPECTROSCOPY ............................................................................51

Contribution of Authors and Co-Authors ......................................................................51
Manuscript Information Page ........................................................................................52
Abstract ..........................................................................................................................53
Introduction ....................................................................................................................54
Materials and Methods ...................................................................................................57
Experimental Conditions .......................................................................................57
Strain and Culturing Conditions ............................................................................58
Analysis of Culture and Media Components .........................................................59
Dissolved Inorganic Carbon Analysis ...................................................................59
Chlorophyll Measurements ....................................................................................60
Harvesting and Centrifugation ...............................................................................60
1H HR-MAS NMR Spectroscopy ..........................................................................61
Extraction of Lipids from Dry Biomass Using Bead Beating ...............................61
Transesterification for GC-MS Analysis ...............................................................62
GC-MS Analysis ....................................................................................................63
Results and discussion ...................................................................................................64
Growth Characteristics of C. vulgaris
on Different Inorganic Carbon Sources .................................................................64
1H HR-MAS NMR Monitoring of In Vivo Metabolites ........................................71
13C FAME Isotopomer Distribution.......................................................................78
Conclusion .....................................................................................................................82
Competing Interests .......................................................................................................83
Acknowledgments..........................................................................................................84
References ......................................................................................................................85

4. CONCLUSIONS............................................................................................................88

Project Outcomes ...........................................................................................................88
Nannochloropsis gaditana Bicarbonate Studies ....................................................89
Chlorella vulgaris NaH13CO Labeling Studies ....................................................90
3
Concluding Remarks ......................................................................................................91
Funding Acknowledgments ...........................................................................................92
vi
TABLE OF CONTENTS – CONTINUED
5. FUTURE WORK ...........................................................................................................93
Integration of Bicarbonate Induced Lipid
Production and Continuous Growth...............................................................................93
REFERENCES CITED ......................................................................................................98
APPENDICES .................................................................................................................106
APPENDIX A: Tabulated Data from N. Gaditana Studies .................................107
APPENDIX B: Supplemental Material from C. Vulgaris Studies .......................116
vii

LIST OF TABLES

Table Page

2.1. Experimental design for assessment of N. gaditana growth
and lipid accumulation under various carbon application strategies. ....................21

2.2. N. gaditana growth and culturing characteristics when grown
utilizing various carbon delivery methods .............................................................29

2.3. N. gaditana total chlorophyll content [mg L-1] and cell
dry weight (CDW) [g L-1] at both nitrogen depletion and harvest
(72 hours post depletion) when grown utilizing various
carbon delivery methods ........................................................................................30

2.4. N. gaditana FAME profiles for cultures grown under
various carbon application strategies at nitrogen depletion ...................................38

2.5. N. gaditana FAME profiles for cultures grown under
various carbon application strategies at harvest (72 hours
post nitrogen depletion) .........................................................................................39

3.1. Growth [cells mL-1], pH, total chlorophyll [mg L-1],
dissolved inorganic carbon (DIC) [mmol C L-1], and cell
dry weight [g DW L-1] of cultures of C. vulgaris at
light-dark transition times over two successive diurnal cycles. .............................69

A.1. Air-1 average values ± 95% confidence interval for
cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................108
3

A.2. Air-2 average values ± 95% confidence interval for
cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................109
3

A.3. Air-3 average values ± 95% confidence interval for
cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................110
3

A.4. CO -1 average values ± 95% confidence interval for
2
cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................111
3
viii

LIST OF TABLES – CONTINUED

Table Page

A.5. CO -2 average values ± 95% confidence interval for
2
cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................112
3

A.6. CO /HCO -1 average values ± 95% confidence interval
2 3
for cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................113
3

A.7. CO /HCO -2 average values ± 95% confidence interval
2 3
for cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................113
3

A.8. CO /HCO -3 average values ± 95% confidence interval
2 3
for cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................114
3

A.9. CO /HCO -4 average values ± 95% confidence interval
2 3
for cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................114
3

A.10. CO /HCO -5 average values ± 95% confidence interval
2 3
for cell density [cells mL-1], pH, nitrate concentration
[mg N (NO -) L-1], and total chlorophyll [mg L-1] ...............................................115
3
ix

LIST OF FIGURES

Figure Page

2.1. Growth [cells mL-1] (a), pH (b), nitrate
concentration [mg N (NO -) L-1] (c), and total
3
chlorophyll [mg L-1] (d) of cultures of N. gaditana
grown using atmospheric air aprage ......................................................................28

2.2. Growth [cells mL-1] (a), pH (b), nitrate
concentration [mg N (NO -) L-1] (c), and total
3
chlorophyll [mg L-1] (d) of cultures of N. gaditana
cultured with CO ..................................................................................................32
2

2.3. Growth [cells mL-1] (a), pH (b), nitrate
concentration [mg N (NO -) L-1] (c), and total
3
chlorophyll [mg L-1] (d) of cultures of N. gaditana
cultured with CO and bicarbonate ........................................................................33
2

3.1. Growth [cells mL-1] (a), pH (b), nitrate
concentration [mg N (NO -) L-1] (c), and dissolved
3
inorganic carbon (DIC) [mmol C L-1] (d) of cultures
of C. vulgaris grown using either CO or NaHCO
2 3
under nitrogen replete conditions. ..........................................................................66

3.2. Saturated fatty acid chain accumulation monitored
in C. vulgaris using 1H HR-MAS NMR and H13CO - labeling .............................73
3

3.3. Polyunsaturated fatty acid (PUFA) chain accumulation
monitored in C. vulgaris using 1H HR-MAS NMR and
H13CO - labeling ....................................................................................................75
3

3.4. Sucrose accumulation monitored in C. vulgaris
using 1H HR-MAS NMR and H13CO - labeling ....................................................76
3

3.5. Fractional abundance of 13C labeled fatty acid
chains as a function of number of added 13C labels for
saturated (C16:0) fatty acid chains ........................................................................80