Identifying, Monitoring, Quantifying and Converting Algae to Bio-Fuels in Bio-Reactors

113 Pages·2016·3 MB·English

by  Alice C. Jernigan

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Graduate Theses and Dissertations
5-2014
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Alice C. Jernigan
University of Arkansas, Fayetteville
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Identifying, Monitoring, Quantifying and Converting
Algae to Bio-Fuels in Bio-Reactors
Identifying, Monitoring, Quantifying and Converting Algae to Bio-Fuels in Bio-Reactors

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy in Chemical Engineering

by
Alice C. Jernigan
University of Missouri
Bachelor of Arts in Biology, 1982
University of Arkansas
Master of Science in Chemical Engineering, 2009

May 2014
University of Arkansas

This dissertation is approved for recommendation to the Graduate Council.

__________________________________
Dr. Christa Hestekin
Dissertation Director

__________________________________ ________________________________
Dr. Robert Beitle Dr. Jamie Hestekin
Committee Member Committee Member

__________________________________ ________________________________
Dr. Ed Clausen Dr. Michelle Evans-White
Committee Member Committee Member
ABSTRACT
Growing algae as a source for bio-fuels has become an area of interest due to concerns
about global warming and the reliability and ecology of the production of fossil fuels. Dried
algae harvested from a pilot water quality improvement technology at the Rockaway Wastewater
Treatment Facility in New York were examined as a source of carbohydrates and lipids for the
production of bio-fuels in bio-reactors. The length of storage time, storage conditions, sugar and
lipid extraction processes, and fuel production were studied. The results show that if the algae is
stored dry (<25% moisture), the algae stock can be used for up to a year with good conversion of
carbohydrates to sugars using a 10% w/v of dried algae, yielding an average of 0.11 (g butanol/g
sugar) from the bioreactors year-round from a wide range of diatoms and other microalgae used
to treat wastewater. Similarly, lipids could be obtained from the stored algal with value of >0.015
g/g algae even after a year in storage. The types of algae harvested has an effect on the amounts
of sugars and lipids extracted, so two different methods to identify, monitor and quantify algae
grown in both open and closed systems were evaluated. Capillary electrophoresis single strand
conformational polymorphism (CE-SSCP) was able to identify known algae samples in an
environmental system by “fingerprint” comparison, but may be most useful as a fast, accurate
method of monitoring changes in the species in closed systems. We also examined and found
capillary electrophoresis single base extension (CE-SBE) to be an extremely fast and accurate
method to quantify the algae DNA of Chlorella vulgaris and Spirulina platensis in a closed
system photo-bioreactor. A primer was designed that allowed the accurate correlation of the
algae DNA amounts with the area under the curve in an electropherogram. This primer also
distinguished between and quantified each species. CE-SBE demonstrated great potential for
quantification of algae with difficult morphologies, and algae grown in a co-culture photo-
bioreactor.
ACKNOWLEDGEMENTS
I would like to thank my research advisor, Dr. Christa Hestekin for her calm patience and
guidance during this long and arduous journey. Her love of teaching shines through with good
humor, no matter the crisis at hand, and she has inspired all of her graduate and undergraduate
students to face all the trials and tribulations of research with patience, logic and laughter.
I would also like to thank the members of my committee, Dr. Bob Beitle, Dr. Ed Clausen,
Dr. Jamie Hestekin and Dr. Michelle Evans-White who have all been supportive in different
ways. I would also like to thank Dr. Sonja Hausmann for all of her help with understanding algae
and interest in my research.
Thank you to the Faculty and Staff of the Ralph E. Martin Chemical Engineering
Department, who gave very good advice when asked, and had patience with my mistakes when
they were not consulted.
Thank you to all of my graduate student friends, Ellen Brune, Mckinzie Fructl, Michael
May, Melissa Hubert, and Alex Lopez, without whose support, both physical and mental, I could
not have withstood the demands of this project. A special thank you to Tom Potts, who made
sure I was not the oldest graduate student, and kept me sane amongst all the youngsters, and
especially, thank you to Elizabeth Pryor for being a friend and a colleague and the best younger
sister I never had.
DEDICATION
This dissertation is dedicated to my wonderful husband, James Scott Jernigan, without
whose support, physical, mental, emotional and especially computer, this long journey would
never have been completed. I am the luckiest woman in the world to have such a loving partner.
TABLE OF CONTENTS
INTRODUCTION .......................................................................................................................... 1
REFERENCES ............................................................................................................................. 11
CHAPTER 1: EFFECTS OF DRYING AND STORAGE ON YEAR-ROUND PRODUCTION
OF BUTANOL AND BIODIESEL FROM ALGAL CARBOHYDRATES AND LIPIDS USING
ALGAE FROM WATER REMEDIATION ................................................................................. 19
ABSTRACT .................................................................................................................................. 19
INTRODUCTION ........................................................................................................................ 20
MATERIALS AND METHODS .................................................................................................. 24
Algae Growth, Harvest, and Storage ......................................................................................... 24
Algae Pretreatment .................................................................................................................... 24
Reducing Sugar Analysis .......................................................................................................... 25
Algae Concentration Study ....................................................................................................... 26
Wet vs. Dry Storage of Algae and the Effect on Sugar for Butanol Production ....................... 26
Inoculum Preparation for Acetone-Butanol-Ethanol (ABE) Fermentation .............................. 27
ABE Fermentation from Algal Media ....................................................................................... 28
Butanol Analysis by Gas Chromatography ............................................................................... 28
Lipid Extraction and Fatty Acid Methyl Ester (FAME) Production ......................................... 29
RESULTS ..................................................................................................................................... 30
Drying of Algae for Extraction of Carbohydrates and Lipids ................................................... 30
Effect of Processing Conditions ................................................................................................ 34
Biofuel Production .................................................................................................................... 36
Case Study ................................................................................................................................. 38
CONCLUSIONS........................................................................................................................... 39
ACKNOWLEDGEMENTS .......................................................................................................... 40
REFERENCES ............................................................................................................................. 40
APPENDIX ................................................................................................................................... 45
CHAPTER 2: EVALUATION OF THE POTENTIAL FOR CAPILLARY
ELECTROPHORESIS-SINGLE STRAND CONFORMATIONAL POLYMORPHISMS (CE-
SSCP) AS A METHOD TO IDENTIFY AND MONITOR ALGAL SPECIES FROM PURE
CULTURES AND ENVIRONMENTAL SAMPLES.................................................................. 48
INTRODUCTION ........................................................................................................................ 48
Algae Speciation ....................................................................................................................... 48
16S gene vs. 18S gene ............................................................................................................... 50
Amplification by Polymerase Chain Reaction .......................................................................... 51
CE-SSCP ................................................................................................................................... 51
MATERIALS AND METHODS .................................................................................................. 54
Pure Cultures ............................................................................................................................. 54
Environmental Samples............................................................................................................. 54
DNA Extraction......................................................................................................................... 55
Reagents and Primers for PCR Amplifications ......................................................................... 55
PCR Amplified DNA Samples Purification, Quantification and Storage ................................. 57
CE-SSCP Sample Preparation ................................................................................................... 57
Preparation of Polymer.............................................................................................................. 58
Preparation of Samples for Sequencing .................................................................................... 59
RESULTS ..................................................................................................................................... 59
Pure Culture “Fingerprints” ...................................................................................................... 59
Comparison of CE-SSCP to Traditional Methods in Environmental Samples ......................... 67
CONCLUSIONS........................................................................................................................... 73
REFERENCES ............................................................................................................................. 73
CHAPTER 3: QUANTIFYING TWO SPECIES OF ALGAL DNA USING CE-SBE
(CAPILLARY ELECTROPHORESIS – SINGLE BASE EXTENSION) IN A PHOTO-
BIOREACTOR SYSTEM ............................................................................................................ 77
INTRODUCTION ........................................................................................................................ 77
Quantifying Algae in Culture Samples ..................................................................................... 77
MATERIALS AND METHODS .................................................................................................. 82
Photo Bioreactor Cultures ......................................................................................................... 82
Chlorella vulgaris ................................................................................................................. 82
Spirulina platensis ................................................................................................................ 83
Co-Cultures ........................................................................................................................... 83
DNA Extraction......................................................................................................................... 84
PCR Amplification .................................................................................................................... 84
CE-SBE ..................................................................................................................................... 86
SBE Primer Design ................................................................................................................... 87
RESULTS ..................................................................................................................................... 89
Calibration Studies .................................................................................................................... 89
DNA Calibration Curve ............................................................................................................ 91
Mixed Cultures of Chlorella and Spirulina............................................................................... 92
Verification of the Calibration Curve........................................................................................ 95
DISCUSSION ............................................................................................................................... 96
REFERENCES ............................................................................................................................. 97
CONCLUSIONS......................................................................................................................... 100
FUTURE WORK ........................................................................................................................ 102
LIST OF PAPERS

Chapter 1: Jernigan, A.; May, M.; Potts, T.; Rodgers, B.; Hestekin, J.; May, P. I,; McLaughlin,
J.; Beitle, R. R.; Hestekin, C. N. Environmental Progress & Sustainable Energy Effects of
Drying and Storage on Year-Round Production of Butanol and Biodiesel from Algal
Carbohydrates and Lipids using Algae from Water Remediation 2013, 32, 4, 1013-1022