Effects of Light Quality and Light Quantity on the Growth Kinetics of a Louisiana Native Microalgal

157 Pages·2017·4.4 MB·English

Louisiana State University
LSU Digital Commons
LSU Master's Teses Graduate School
2015
Efects of Light Quality and Light Quantity on the
Growth Kinetics of a Louisiana Native Microalgal/
Cyanobacterial Co-culture
Jonathan Zell Barnet
Louisiana State University and Agricultural and Mechanical College,
EFFECTS OF LIGHT QUALITY AND LIGHT QUANTITY ON THE
GROWTH KINETICS OF A LOUISIANA NATIVE
MICROALGAL/CYANOBACTERIAL CO-CULTURE

A Thesis

Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Master of Science

in

The Department of Civil and Environmental Engineering

by:
Jonathan Z. Barnett
B.S., Texas A&M University, 2013
December 2015
Acknowledgements

First and for most I would like to thank my father (Jonathan Barnett) and mother (Cathie
Barnett) for their unfailing love and support of me throughout not only my time as a graduate
student but through my life. Without their unyielding support it would have been very difficult to
accomplish my master’s degree. I would also like to thank the love and support by my three
sisters (Emily, Allyson and Madison), my brother (Michael) and my brother in law (Charlie
Roberts).
Special thanks to my graduate advisory co-chairs Dr. Maria Teresa Gutierrez-Wing and
Dr. Ronald Malone for their guidance throughout my time at LSU. They constantly challenged
me to be the best researcher I could be and gave me a vast array of knowledge I can take with me
for the rest of my life. I would also like to thank my other committee members, Dr. Kelly Rusch
and Dr. Jin-Woo Choi who helped guide me through my thesis with thought provoking questions
and helpful critiquing.
Additional gratitude is extended to the faculty and staff of the LSU Civil &
Environmental Department, especially Mrs. Janet Labatut, Dave Roberston and Ms. Julie
Mueller for their help whenever I requested it. Lastly, I would like to thank my fellow graduate
students, Jacob Foy, Charles Davis Lofton and Glenn Shin. Their companionship throughout my
time at LSU will stick with me for the rest of my life. A thank you goes to all the student workers
that assisted me in lab, Mary Beth Dixon, Anteneh “Fasil” Lisan, Kristen Courville, Lisa Weaver
and Rhine Perrin. Their moral support and engaging conservations, provided me with a fun
atmosphere to conduct my research. I would like to give a special thank you to Mary Beth for her
constant support and love.

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Table of Contents

Acknowledgements ......................................................................................................................... ii

List of Tables ................................................................................................................................. vi

List of Figures .............................................................................................................................. viii

Abstract ......................................................................................................................................... xii

1. Global Introduction.................................................................................................................. 1
1.1 Background ...................................................................................................................... 1
1.2 Microalgae ........................................................................................................................ 3
1.2.1 Microalgae/Cyanobacteria Photosynthesis ............................................................... 4
1.2.2 Light Dependent Reaction ........................................................................................ 5
1.2.3 Light Independent Reaction (Calvin Cycle) ............................................................. 8
1.2.4 Cyanobacteria Photosynthesis .................................................................................. 9
1.3 Light ............................................................................................................................... 10
1.3.1 Light Quality ........................................................................................................... 10
1.3.2 Light Quantity ......................................................................................................... 14
1.4 Culture Systems.............................................................................................................. 17
1.4.1 Open Systems.......................................................................................................... 17
1.4.2 Closed Systems ....................................................................................................... 18
1.4.3 Hybrid Systems ....................................................................................................... 19
1.5 Research Objectives ....................................................................................................... 21
1.6 Thesis Organization........................................................................................................ 21

2. Impact of Light Quality on a Native Louisiana Chlorella vulgaris/Leptolyngbya sp. Co-
culture ........................................................................................................................................... 22
2.1 Introduction ......................................................................................................................... 22
2.2 Materials and Methods ........................................................................................................ 24
2.2.1 Co-culture Species ........................................................................................................ 24
2.2.2 Experimental Design .................................................................................................... 25
2.2.3 Distinguishing between Microalgae and Cyanobacteria .............................................. 29
2.2.4 Statistics ........................................................................................................................ 30
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2.3 Results and Discussion ........................................................................................................ 31
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2.3.1 Growth under Irradiance Level 80 and 400 μmol m s ............................................. 31
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2.3.2 Growth under Irradiance Level 400 μmol m s ......................................................... 36
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2.3.3 Light Attenuation at 400 μmol m s .......................................................................... 40
2.4 Conclusions ......................................................................................................................... 42

3. Impact of Irradiance Levels for Red and White Light on the Growth of a Native Louisiana
Chlorella vulgaris/Leptolyngbya sp. Co-culture........................................................................... 44
3.1 Introduction ......................................................................................................................... 44
3.2 Materials and Methods ........................................................................................................ 46
3.2.1 Co-culture Species ........................................................................................................ 47
3.2.2 Experimental Design .................................................................................................... 47
3.2.3 Distinguishing between Microalgae and Cyanobacteria .............................................. 51
3.2.4 Biomass Yield on Light energy and Photosynthetic Efficiency Equations .................. 52
3.2.5 Statistics ........................................................................................................................ 54
3.3 Results and Discussion ........................................................................................................ 54
3.3.1 Growth rates under Red and White Light ..................................................................... 54
3.3.2 Biomass and Biomass Productivity under Red and White Light ................................. 57
3.3.3 Biomass Yield on Light Energy and Photosynthetic Efficiency under Red and White
Light....................................................................................................................................... 62
3.3.4 Chlorophyll-a Content under Red and White Light ..................................................... 64
3.3.5 Lipid Content under Red and White Light ................................................................... 66
3.3.6 Light Attenuation under Red and White Light ............................................................. 68
3.3.7 Species Composition under Red and White Light........................................................ 71
3.4 Conclusions ......................................................................................................................... 72

4. Culturing a Louisiana Native Chlorella vulgaris/Leptolyngbya sp. Co-culture in a
Hydraulically Integrated Serial Turbidostat Algal Reactor .......................................................... 74
4.1 Introduction ......................................................................................................................... 74
4.2 Materials and Methods ........................................................................................................ 76
4.2.1 Co-culture Species ........................................................................................................ 76
4.2.2 HISTAR Description .................................................................................................... 77
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4.2.3 HISTAR Experiment with Qf = 1 L min .................................................................... 79
4.2.4 Light Attenuation Measurements ................................................................................. 80
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4.3 Results and Discussion ........................................................................................................ 82
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4.3.1 Results from Qf = 1 L min .......................................................................................... 82
4.3.2 Light Attenuation in CFSTR Tanks.............................................................................. 86
4.4 Conclusions ......................................................................................................................... 88

5. Global Discussion ..................................................................................................................... 90

6. Global Conclusions and Recommendations ............................................................................. 93
6.1 Global Conclusions ............................................................................................................. 93
6.2 Recommendations ............................................................................................................... 94

References ..................................................................................................................................... 95

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Appendix A: Growth Experiments at 80 & 400 mol m s ..................................................... 106

Appendix B: Growth Experiments under Red and White Light at Various Irradiance Levels .. 119

Appendix C: HISTAR Experiment ............................................................................................. 137

Vita .............................................................................................................................................. 144

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List of Tables

Table 1.1 Comparison of oil yields for various oil crops in terms of
a
liters/hectares. Microalgae has 70% oil by dry biomass weight and
b
m icroalgae has 30% oil by dry biomass weight. Adapted from ( Chisti,
2007 )……………………………………………………………………. 3

Table 1.2 Different models used to describe the relationship between irradiance
level and growth rate. Adapted from Acién Fernández et al. (1999) …… 16

Table 2.1 Average ± standard d eviations for growth rates (based on the cell
counts), biomass and cell concentrations for samples grown at 80 and
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400 μmol m s . μ micro is the microalgae growth rate, μ cyano is the
cyanobacteria growth rate, X final is the final biomass concentration, C i
is the cell concentration at initial biomass, C f is the cell concentration
at final biomass. For C i and C f the number on top represents C. vulgaris

and the number on bottom represents Leptolyngbya sp. Excluded from
34
this table are the cyanobacteria filamen t growth……………………….

Table 2.2 Average and standard deviations of growth rates based on biomass
(μmax), final biomass (X final), biomass productivity (X prod.) lipid content,
chlorophyll - a content (Chl - a) and energy per mole of photon for co -
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culture samples grown at 400 μmol m s . Xmax was calculated from
eq 2.4 . The energy listed for white light are calculated from the two
peaks in its wavelength distribution ……………………………………. 38

Table 2.3 Light attenuation coefficients (kb) for the different wavelength
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distributions at 400 μmol m s ………………………………………... 41

Table 3.1 Growth rates (average ± standard deviations) for the co -culture grown
under red and white light at various irradiance levels ………………….. 55

Table 3.2 Final biomass concentrations (average ± standard deviations) of the co -
culture under red and white light at various irradiance levels ………….. 58

Table 3.3 Biomass productivity (average ± standard deviations) of the co -culture
under red and white light at various irradiance levels………………….. 60

Table 3.4 Chlorophyll-a content (average ± standard deviation) of the co -culture
under red and white light at various irradiance levels ………………….. 65

Table 3.5 Light attenuation coefficients of the co -culture under red and white
light at various irradiance levels using eq. 3.3………………………….. 70

Table 4.1 Average ± standard deviations for growth rates, volumetric
productivity, areal productivity and wet biomass from the centrifuge …. 86

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Table 4.2 ka,k aw k w and k values for the equations 4.6, 4.7 and 4.8 ……………….. 86

Table A.1 Excitation laser and peak fluorescence of FL2, FL3 and FL4. The ‘LP’
under FL3 stands for long pass…………………………………………. 117

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List of Figures

Figure 1.1 Schematic of the light dependent reaction in the chloroplast of
microalgae. Taken from http://en.wikipedia.org/wiki/Photosynthesis......... 7

Figure 1.2 Electron transport chain (‘Z’ scheme) showing the transfer of electrons
from one pigment to another and the associated energy. Adapted from
Richmond (2004)………………………………………………………….. 8

Figure 1.3 Comparison of photosynthesis light harvesting complexes in
cyanobacteria/red algae (left image) and microalgae (right image). Image
adapted from Kanehisa Laboratories 2010………………………………... 10

Figure 1.4 Schematic of the P vs I curve. The region from Ic to I s is the light
independent region, from I s to I h is the light saturation region and after I h
this is the photoinhibition region. Image adapted from Carvalho et al.
(2011 )……………………………………………………………………… 15

Figure 2.1 Wavelength distributions of the four colors used in this experiment…....... 25

Figure 2.2 LEDs coiled around the plastic mesh (left image). Experimental setup
showing the cylidrical chambers made from a steel sheet 0.5 mm thick
(right image)……………………………………………………………...... 27

Figure 2.3 Flow cytometer graph showing the distinction between microalgae
stained with Nile Red dye (box P1) and microalgae not stained (box P5)... 28

Figure 2.4 Flow cytometer graphs showing a shift from microalgae dominant (left
image) to cyanobacteria dominant (right image). The y-axis labeled FL3-
A was used to detect chlorophyll-a fluorescence and the x-axis labeled
FL4-A was used to detect phycocyanin fluorescence. The left image
shows a strong microalgae population enclosed by P3. The right image
shows a higher population of cyanobacteria (P2) as compared to
microalgae (P3)……………………………………………………………. 30

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Figure 2.5 Growth curves of C. vulgaris and Leptolyngbya sp. at 80 μmol m s
under the different wavelength distributions, 1a) blue light, 1b) green
light, 1c) red light, 1d) white light. Microalgae growth curves were fitted
with a Gaussian curve and cyanobacteria growth curves were fitted with a
sigmoidal curve ……………………………………………………………. 32

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Figure 2.6 Growth curves of C. vulgaris and Leptolyngbya sp. at 400 μmol m s
under the different wavelength distributions, 1a) blue light, 1b) green
light, 1c) red light, 1d) white light. Microalgae growth curves were fitted
with sigmoidal curves and cyanobacteria curves were fitted with
exponential growth curves ………………………………………………… 33
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Figure 2.7 Biomass growth curves for the co-culture at 400 μmol m s under four
wavelength distributions fitted with a logistic growth curve. The dashed
line represents the growth curve for the green light that was not found
using the logistic model …………………………………………………… 37

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Figure 2.8 Light attenuation for the four wavelength distributions at 400 μmol m s 41

Figure 2.9 Absorba nce spectrum of the co - culture shows more blue light is absorbed
as compared to other colors…………………………………...................... 42

Figure 3.1 Wavelength distributions of the red and white LEDs. The figure shows
the peak at 640 nm for the red light and a narrow peak at 450 nm and a
wide peak centered on 550 nm for the white light………………………… 48

Figure 3.2 Experimental set up showing the cylindrical chambers made from the
steel sheet 0.5 mm thick (right image) and the plastic mesh with the LED
lights coiled around it (left image)………………………………………… 49

Figure 3.3 Flow cytometer graph showing the distinction between microalgae
stained with Nile Red dye (box P1) and microalgae not stained (box P5)... 50

Figure 3.4 Flow cytometer graphs showing a shift from microalgae dominant (left
image) to cyanobacteria dominant (right image). The y-axis labeled FL3-
A was used to detect chlorophyll-a fluorescence and the x-axis labeled
FL4-A was used to detect phycocyanin fluorescence. The left image
shows a strong microalgae population enclosed by P3. The right image
shows a higher population of cyanobacteria (P2) as compared to
microalgae (P3)……………………………………………………………. 52

Figure 3.5 Growth rate versus irradiance level curves for co-cultures grown under
red and white light fitted with Steele’s kinetics model……………………. 57

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Figure 3.6 Cell sizes taken of co-culture grown at 1400 µmol m s showing larger
cells under white light (right image) compared to smaller cells under red
® ®
light (left image). Images taken using CFlow Sampler from Accuri …… 59

Figure 3.7 Growth curves for the co-culture under red (black dots) and white (white
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dots) at a) 180 b) 400 c) 600 d) 800 e) 1000 f) 1200 g) 1400 µmol m s .. 61

Figure 3.8 Biomass yield on light energy of the co - culture under red and white light.
2 2
Red and white light fitted with a logistic curve (red r = 0.982, white r =
0.985)……………………………………………………………………… 63

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