UNIVERSITY OF THE PHILIPPINES

Bachelor of Science in Biology

Renarose Angela J. Cadiz

Induction of Lipid Accumulation in Chlorella sp. Using Cyanobacteria in Co-

culture for Biofuel Production

Thesis Adviser:
Pierangeli G. Vital, Ph.D.
Institute of Biology
University of the Philippines Diliman

Date of Submission
June 2016

Thesis Classification:
P

This thesis is not available to the public. Please ask the library for assistance.

i
 Institute of Biology

College of Science
University of the Philippines
Diliman, Quezon City

ENDORSEMENT

This is to certify that this undergraduate thesis entitled Induction of Lipid

Accumulation in Chlorella sp. Using Cyanobacteria in Co-culture for Biofuel
Production prepared and submitted by Renarose Angela Jonson Cadiz in partial
fulfillment of the requirements for the degree of Bachelor of Science in Biology, is
hereby accepted.

PIERANGELI G. VITAL, Ph.D.

Thesis Adviser

The Institute of Biology endorses acceptance of this undergraduate thesis as partial

fulfillment of the requirements for the degree of Bachelor of Science in Biology.

ERNELEA P. CAO, Ph.D.

Director
Institute of Biology

ii
 Institute of Biology
University of the Philippines
Microbiology and Cell Biology Academic Group

Undergraduate Thesis Defense Approval Form

Name of Student: Renarose Angela J. Cadiz

Student Number: 2012-59781

Date of Defense: May 7, 2016

Name of Adviser: Pierangeli G. Vital, Ph.D.

Thesis Title: Induction of Lipid Accumulation in Chlorella sp. Using Cyanobacteria in Co-
culture for Biofuel Production

We, the undersigned members of the Microbiology and Cell Biology Academic Group, have
read and examined the attached manuscript. We certify that it is adequate in scope and
quality as an undergraduate thesis and indicate our approval of its contents.

_________________________ _________________________
Windell L. Rivera, Ph.D. Joyce A. Ibana, Ph.D.
Professor Associate Professor
Academic Group Head

_________________________ _________________________
Gil M. Penuliar, Ph.D. Pierangeli G. Vital, Ph.D.
Assistant Professor Assistant Professor

_________________________
Ivan Christian V. J. Imperial, M.D.
Instructor

iii
 BIOGRAPHICAL DATA

Name: Renarose Angela Jonson Cadiz

Address: Blk 1 Lot 3 Amorsolo St., Phase 12-C, Bahayang Pag-Asa Subdivision,
Imus, Cavite
Birthday: November 5, 1995
Father: Noel B. Cadiz
Occupation: Airline Pilot
Mother: Jennifer J. Cadiz
Occupation: Teacher
Sibling: Fernel Anne J. Cadiz

Educational Background
Primary: Divine Light Academy-Molino
Intermediate: Divine Light Academy-Molino
High School: Divine Light Academy-Molino
College: University of the Philippines, Diliman

Affiliations:
UP Pre-Medical Honor Society, Academics Committee Member (2012-2016)
UP Zoological Society, Publicity Committee Member (2015-2016)

iv
 ACKNOWLEDGMENT

First of all, I would like to thank my thesis adviser, Dr. Pierangeli G. Vital for

accepting me as her thesis advisee and for sharing her vast knowledge in the field of

microbiology and experience in microalgal culture. I am also very much grateful for

the support I felt especially when I really needed the plate reader and the BG-11

medium for my experiment. No part of this thesis would have been possible without

her guidance.

I would also like to thank her research associates for their suggestions to further

improve my thesis methodology. To Mr. Jude Francisco, for supervising my

experimentation in the laboratory most of the time, and for reviewing my thesis

manuscript and suggesting points for improvement, to Ms. Joseth Abello, for

especially helping me to follow up for the BG-11 medium, and to Ms. Cielo Paraoan

for supervising my experimentation in the EML-NSRI.

To Ms. Kuselah Tayaban for entertaining me whenever I had questions, for also

guiding me during my experiments, and for thoroughly checking my thesis draft.

To Ms. Katherine Pintor for her guidance all throughout since the writing of my

thesis proposal draft during the midyear, up until the writing of my thesis manuscript

this day, and for picking up the Spirulina sp. culture from the University of the

Philippines, Los Baños.

To my family, for visiting me every now and then and bringing me lunch during

my heaviest experiment days, and for giving me motivation and undying support,

v
especially to my father for driving back and forth from Quezon to Cavite to deliver

the 6-L PET bottles for my set-ups, my mother for the improvised 50-mL conical tube

Styrofoam rack, my sister for helping me round off some numbers from my data, and

to my grandmother for providing me food after some tiring paperwork.

I would like to express my deepest gratitude to the following professors—Dr. Ian

Kendrich C. Fontanilla, Dr. Ernelea P. Cao, Dr. Elena S. Catap, and Dr. Windell

L. Rivera, for accommodating me in their laboratories and allowing me to use their

equipment for my experimentation. I would also like to mention Dr. Gil M. Penuliar,

and Ms. Brenda M. Hernandez, for helping me obtain the solvents for my lipid

extraction, and Dr. Ivan Christian V.J. Imperial as well, for giving me permission

to use the 15-mL centrifuge in the cell biology laboratory and for briefing me on how

to properly manipulate it.

I would also like to thank the PhD Incentive Award from the Office of the Vice

Chancellor for Research and Development in the University of the Philippines

Diliman, for funding this thesis.

And finally, I would like to thank the Almighty God for this miracle and the many

other blessings he showered upon me for the accomplishment of this thesis.

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 ABSTRACT

Biofuel production from microalgae is one of the most promising alternatives

being studied and developed for a more sustainable source of renewable energy. One

of the novel methods in maximizing the lipid content of microalgae for biofuel

production is through co-cultivation. In this study, two types of co-cultures of green

microalgae-cyanobacteria, namely Chlorella sp.-Microcystis sp., and Chlorella sp.-

Spirulina sp., were set in varying ratios of 1:1 (50% Chlorella sp.), 3:2 (60%

Chlorella sp.), and 3:1 (75% Chlorella sp.). Biomass (g/L) and lipid content (% lipid

w/w) of each set-up was quantified on the sixth, eighth, and tenth day of cultivation

and was compared with control set-ups and mono-culture. The results showed that co-

cultivation has a positive effect on biomass production, while for the lipid content the

effect varied depending on the type of co-culture. The set-up to yield the highest

biomass is the 3:1 (75%) Chlorella sp.-Spirulina sp. while the highest lipid yield is

from the 3:1 (75%) Chlorella sp.-Microcystis sp. set-up. The most probable rationale

behind the greater increase of lipids in co-cultivation using cyanobacteria is that,

cyanobacteria are capable of transforming organic compounds to a more usable form,

hastening cell reproducibility, and cyanobacteria initiate nitrogen starvation, thereby

increasing lipid accumulation. Thus, co-cultivation using cyanobacteria is more

effective in maximizing lipids in a culture and hence, a better source of biofuel

compared with mono-cultivation.

vii
 TABLE OF CONTENTS

Title Page i
Endorsement ii
Microbiology and Cell Biology Academic Group Approval Sheet iii
Biographical Data iv
Acknowledgment v
Abstract vii
Table of Contents viii
List of Tables x
List of Figures xi
List of Appendices xiii
Introduction 1
Review of Related Literature 4
Microalgae 4
Chlorella sp. 5
Cyanobacteria 6
Biofuel 7
Biodiesel Production 7
Generations of Biodiesel 9
Microalgae as Feedstock for Biodiesel Production 10
Materials and Methods 12
Cultures and Cultivation Conditions 12
Axenization of Cultures 12
Growth Curve Determination 12
Co-cultivation 12
Extraction and Quantification of Biomass and Lipid 13
Calculations and Statistical Analysis 14
Results 15
Growth Curve 15
Biomass Production 15
Lipid Content 17

viii
Discussion 20
Growth Curve 20
Biomass Production 20
Lipid Content 22
Conclusion 23
Recommendations 24
Literature Cited 25
Tables 30
Figures 34
Appendices 56

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 LIST OF TABLES

Table Title Page

1 Components for culture axenization of the three microalgae species: 30

Chlorella sp., Microcystis sp., and Spirulina sp.

2 Green algae-cyanobacteria co-culture and control set-ups for co- 31

cultivation experiment

3 Algal biomass (g/L) of monocultures of Chlorella sp., Microcystis sp., 32

and Spirulina sp. and co-cultures of Chlorella sp.-Microcystis sp. and
Chlorella sp.-Spirulina sp. in ratios of 1:1 (50% Chlorella sp.), 3:2 (60%
Chlorella sp.), and 3:1 (75% Chlorella sp.)

4 Lipid content (w/w%) of monocultures of Chlorella sp., Microcystis sp., 33

and Spirulina sp. and co-cultures of Chlorella sp.-Microcystis sp. and
Chlorella sp.-Spirulina sp. in ratios of 1:1 (50% Chlorella sp.), 3:2 (60%
Chlorella sp.), and 3:1 (75% Chlorella sp.)

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 LIST OF FIGURES

Figure Title Page

1 Chlorella sp. at x40 magnification inoculated from pure culture seed stock 34
of Chlorella sp.

2 Microcystis sp. at x40 magnification inoculated from pure culture seed 35

stock of Microcystis sp.

3 Spirulina sp. at x40 magnification inoculated from pure culture seed stock 36
of Spirulina sp.

4 Phase separation of three layers: methanol layer, algal layer, chloroform 37

layer. Methanol and algae layer (15-mL conical tube) separated from
chloroform layer (50-mL conical tube)

5 Bottom part of a 50-mL conical tube containing dried lipid residue 38

6 Growth curve of the three microalgae species expressed as absorbance vs. 39

time (h)

7 Graph showing comparison of biomass (g/L) between two controls— 40

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, six days after
cultivation

8 Graph showing comparison of biomass (g/L) between two controls— 41

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, eight days after
cultivation

9 Graph showing comparison of biomass (g/L) between two controls— 42

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, ten days after
cultivation

10 Graph showing the biomass (g/L) produced by the mono-culture vs. co- 43
culture of varying ratios of Chlorella sp.-Microcystis sp. and Chlorella
sp.-Spirulina sp. six days after cultivation.

11 Graph showing the biomass (g/L) produced by the mono-culture vs. co- 44
culture of varying ratios of Chlorella sp.-Microcystis sp. and Chlorella
sp.-Spirulina sp. eight days after cultivation.

12 Graph showing the biomass (g/L) produced by the mono-culture vs. co- 45
culture of varying ratios of Chlorella sp.-Microcystis sp. and Chlorella
sp.-Spirulina sp. ten days after cultivation.

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13 Biomass (g/L) vs. time (h) graph of the three different ratios of the 46
Chlorella sp.-Microcystis sp. co-culture

14 Biomass (g/L) vs. time (h) graph of the three different ratios of the 47
Chlorella sp.-Spirulinasp. co-culture

15 Graph showing comparison of % lipid (w/w) between two controls— 48

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, six days after
cultivation

16 Graph showing comparison of % lipid (w/w) between two controls— 49

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, eight days after
cultivation

17 Graph showing comparison of % lipid (w/w) between two controls— 50

Chlorella sp. and Microcystis sp./Spirulina sp., and the actual biomass
(g/L) harvested from each different co-culture set-up, ten days after
cultivation

18 Graph showing the calculated % lipid (w/w) in mono-culture vs. % lipid 51

(w/w) in co-culture of varying ratios of Chlorella sp.-Microcystis sp. co-
culture and Chlorella sp.-Spirulina sp. co-culture, six days after
cultivation

19 Graph showing the calculated % lipid (w/w) in mono-culture vs. % lipid 52

(w/w) in co-culture of varying ratios of Chlorella sp.-Microcystis sp. co-
culture and Chlorella sp.-Spirulina sp. co-culture, eight days after
cultivation

20 Graph showing the calculated % lipid (w/w) in mono-culture vs. % lipid 53

(w/w) in co-culture of varying ratios of Chlorella sp.-Microcystis sp. co-
culture and Chlorella sp.-Spirulina sp. co-culture, ten days after
cultivation

21 Lipid content (% lipid w/w) vs. time (h) graph of the three different ratios 54
of the Chlorella sp.-Microcystis sp. co-culture

22 Lipid content (% lipid w/w) vs. time (h) graph of the three different ratios 55
of the Chlorella sp.-Spirulina sp. co-culture

xii
 LIST OF APPENDICES

Appendix Title Page

A Raw Data for Biomass Extraction -Day 6 56

B Raw Data for Biomass Extraction -Day 8 57

C Raw Data for Biomass Extraction -Day 10 58

D Raw Data for Lipid Extraction -Day 6 59

E Raw Data for Lipid Extraction -Day 8 60

F Raw Data for Lipid Extraction -Day 10 61

xiii
 INTRODUCTION

Oil production is one of the most important economic aspects of a country. The

Philippines consists of many areas for exploration for potential oil reserves, however,

exploration of these potential oil sites are not that easy to conduct. Discovery of oil

and natural gas reserves are very risky since there is no guaranteed success and the

whole process is capital intensive (Gault, 2014). Exploration companies have to invest

at about 85% of the cost which ranges from USD 25 million to USD 50 million

according to the Department of Energy. Hence, some exploration companies pulled

out and abandoned the exploration projects. Also, these exploration projects prove to

be bothersome to some fishermen as the drilling may affect catches and destroy

marine life.

The Philippines consumes an average of 300,000 barrels of oil daily (Department

of Energy, 2014). However, from the data gathered by the United States Energy

Information Administration, the daily production of crude oil was nowhere near the

average daily consumption of oil. The daily production rate did not even exceed

80,000 barrels according to the Department of Energy.

Although it was found out by the United States Energy Information

Administration, that the Spratly Islands has been estimated to contain 5.4 billion

barrels of oil and 55.1 trillion cubic feet (tcf) of natural gas, it cannot be explored and

consumed by the country due to the ongoing territorial dispute between four other

countries including China, Brunei, Taiwan, and Vietnam. Hence, there has to be an

alternative solution to obtain oil for a sufficient production.

Microalgae are microorganisms which are very diverse and strive in various

habitats (Tomaselli, 2004). These microorganisms are very much abundant and have

1
different uses and benefits to the environment such as bioremediation and biofuel

production which aids in the reduction of greenhouse gas emissions. They also have

commercial uses, medical uses, and economical uses since they contain a lot of

different essential proteins, carbohydrates, lipids, vitamins, and many other chemical

compounds and molecules that have important uses such as the β-1,3-glucan that can

be extracted from a certain species of microalgae, and omega-3 fatty acids (Singh &

Saxena, 2015). Proteins, carbohydrates, and lipids found in these microorganisms are

not only utilized for health purposes but also, are required for a potential source of

biofuel (Ma & Hanna, 1999; Brenan & Owende, 2010; Owolabi et al., 2012). In the

recent times, biofuel production are the focus of many researches since there is an

increasing demand for fuel and that, fuel from fossils are non-renewable or takes a

long time to reoccur. Because biodiesel, a type of biofuel, share almost similar

physical and chemical properties as gasoline, biodiesel became an alternative fuel for

gasoline (Hafizan & Zainura, 2013). Moreover, biodiesel have economical and

environmental benefits such as minimal to no modifications required in diesel or

gasoline-run engines (Choo et al., 2007), and reduced green house gas emissions

(Christi, 2007). There were a number of studies reporting that microalgae can actually

be utilized as a source of lipid for the synthesis of biodiesel, and there were already

many procedures to maximize lipid production such as nutrient starvation, particularly

nitrogen (Dianursati & Santoso, 2015). However, although manual nitrogen starvation

increases lipid accumulation, biomass production is compromised thus, limiting the

amount of lipid that can be extracted in the culture (Zhao et al., 2014). Fortunately,

there was a novel procedure, known as co-cultivation, which can counter this trade-off

between lipid accumulation and biomass production. Hence, this study aims to

determine the effect of co-cultivation on biomass production and lipid content, and to

2
determine which of the two co-cultures, Chlorella sp.-Microcystis sp. and Chlorella

sp.-Spirulina sp., will induce a greater increase in biomass production and lipid

content.

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 REVIEW OF RELATED LITERATURE

Microalgae

The term microalgae simply refers to algae that are microscopic in nature. This

term also encompass a group of prokaryotic microorganisms, the cyanobacteria,

which apparently is the only group of oxygenic photosynthetic bacteria to exist (Starr

et al., 2014). According to Tomaselli in 2004, microalgal cells exhibit different types

of organization. They can exist as unicellular, colonial, or filamentous

microorganisms. Some species form coenobic colonies, or the type of colony that

contains a fixed number of cells (e.g. Scenedesmus), while other species form non-

coenobic colonies, with varied cell numbers (e.g. Pediastrum). Their movement can

vary from non-motile, to an occurrence of a swimming and gliding motility.

Microalgal colonies also exist in different shapes—they can be flat, spherical, cubic,

dendroid, palmelloid, flagellate, and non-flagellate.

Given these attributes, it can be said that microalgae are indeed very diverse in

nature. In fact, there are 200 million different species of microalgae estimated to exist

(Singh & Saxena, 2015). This diversity in the morphology and movement of

microalgae can be attributed to the wide range of habitat they inhabit.

Like macroscopic algae, microalgae are also found in freshwater and in marine

water environments. These microscopic algae however, are more extreme than

macroscopic algae since they were reportedly found in highly saline environments

such as the Great Salt Lake in the United States, and even in the Dead Sea in Israel,

which is roughly eight times more saline than the average ocean (Singh & Saxena,

2015). Microalgae can also be found in different depths of an aquatic environment.

Some inhabits the surface of the water layer, some remains beneath as a pelagic or a

4
benthic species, while a few live at about 200-300 meters below the water surface

(Tomaselli, 2004). Microalgae thrives in terrestrial environments as well, such as in

humus soil, sand, and rocks, and interestingly, in furs of animals like sloths and polar

bears. Microalgae can also survive in biotic crusts in hot deserts, snowfields, and even

in air at about 2000 meters high (Singh & Saxena, 2015).

Another thing that makes microalgae distinct with macroscopic algae is that they

possess several morphological adaptations which perform specialized functions

unique to them. These specialized cells are in form of akinetes, or spore-like cells,

which is prominent during unfavorable conditions, heterocysts, which are the sites of

nitrogen-fixation, hormogonia, which refers to the developmental stage of a

cyanobacteria usually consisting of gas vacuoles necessary for light shielding and

buoyancy, and lastly, pili or fimbriae, which are protein-rich protrusions from the cell

wall which functions for adherence or for reproduction (Tomaselli, 2004).

Microalgae, due to their availability and abundance in nature, have been exploited

and have been discovered to have plenty of benefits in many different aspects of the

human living such as livelihood, health, research, industry, economy, and the

environment, but one of the most important and novel benefit is that, microalgae can

also be a source of renewable energy in the form of biofuel.

Chlorella sp.

Chlorella is a genus of unicellular green-algae or Phylum Chlorophyta, which is

spherical in shape and about 10μm or less in size. Chlorella has many uses and has

been included in many dietary supplements. And according to an article written by

Scheffler in 2007, it’s photosynthetic ability can also be used for power generation,

and construction of underwater habitats as its can be a source of oxygen and food.

5
Chlorella can also be a source for feed stock in biofuel production since it contains

high protein and high lipid content (Mahmoud et. al., 2014). Among the different and

various microalgae species, Chlorella sp., based on literature, usually has the highest

lipid yield compared with the other microalgae species studied for lipid quantification

such as Scenedesmus sp., Microcystis sp., Dunaliella sp., and etc. (Liu et al., 2011;

Mahmoud et al., 2014; Taher et al., 2014; Duong et al., 2015). Also, due to this

property of Chlorella sp., the species became the usual subject for biofuel production

studies in microalgae.

Cyanobacteria

Cyanobacteria, also called blue-green algae, is a phylum of unicellular and

photosynthetic bacteria that usually thrives in aquatic environments but can also be

found on various environments such as damp soil and rocks. Some of the remarkable

features of cyanobacteria include possession of gas vacuoles, undergoing

morphological changes such as the appearance of heterocysts during nitrogen

starvation, and hair formation in the filaments during phosphate deficiency (Carr and

Whitton, 1982). These attributes however are not exhibited in all cyanobacteria

species. Cyanobacteria are also the main cause of algal blooms in water bodies which

can be harmful as cyanobacteria can release several toxins including neurotoxins,

endotoxins, cytotoxins, and hepatotoxins, which can result to mass mortalities of

marine or freshwater species. Cyanobacteria cause algal blooms in the presence of too

much nutrients such as phosphate and nitrogen. Nitrogen fixation is another one of the

special attributes of cyanobacteria. In some species, it takes place in a specialized cell

called heterocyst. When nitrogen enters the heterocyst, it is converted by nitrogenase

enzyme to ammonia. Nitrogenase is driven by energy produced from the

6
transformation of a carbohydrate, such as maltose from the neighboring vegetative

cell, to glucose-6-phosphate, then to 6-phospho-gluconate, and finally to ribulose-5-

phosphate (Sah, 2008).

Since cyanobacteria intake nitrogen, they limit the nitrogen from the surrounding

environment, and nitrogen limitation is known to cause accumulation of lipid bodies

in green algae (Dianursanti & Santoso, 2015). Moreover, a study by Dianursanti and

Santoso in 2015 has shown that lipid accumulation in Chlorella vulgaris was

increased by adding Spirulina platensis in culture.

Biofuel

Biofuel is the fuel produced from biological means that utilizes organic materials

as its constituents such as plant biomass and animal wastes (Ma & Hanna, 1999;

Hafizan & Zainura, 2013). Since plants, which are the main feedstock for biofuel

production, derive its energy from the sun, biofuels are hence renewable and

definitely more sustainable than fossil fuels. Few examples of bio-sourced fuel

include biomethanol, bioethanol, biobutanol, biomethane, biohydrogen, and biodiesel

(Owolabi et al., 2012). Among them, biodiesel and bioethanol are the most useful and

the most widely studied.

Biodiesel Production

Biodiesel is made from lipids extracted through the process of transesterification

(Wang et al., 2014). Transesterification is the breakdown of lipid molecules into fatty

acids and transforming them into its usable form—the fatty acid methyl ester (Knothe

et al., 2015). Lipids are first extracted from cells through the lysing of the cell

membrane using different techniques such as heating, ultrasonication, or mechanical

7
disruption, and then purified by separating it from other cell components such as

proteins, carbohydrates, and other cellular debris (Wang et al., 2014).

Transesterification starts upon addition of an alcohol, usually methanol, to the lipid

molecule and using an acid or a base as catalyst, the reaction yields glycerol and

biodiesel in the form of fatty acid methyl esters (Ma & Hanna, 1999).

Besides the sustainability of biodiesel, it was also discovered that biodiesel can

help lessen greenhouse gas emissions. In a project sponsored by Harvard University,

known as The Alternative Fuel Vehicle Program in 2001, the greenhouse gas

emissions per mile in a travelling vehicle using different fuel types was calculated and

ranked in accordance to decreasing emission of greenhouse gases. Among diesel,

gasoline, and biodiesel, gasoline has the greatest emission (~500 g CO 2

equivalent/mile), followed by diesel (~380 g CO2 equivalent/mile), followed by 20%

biodiesel blend (~320 g CO2 equivalent/ mile), and finally, full biodiesel (~120 g CO2

equivalent/mile). To put it briefly, biodiesel can reduce as much as five times the

carbon dioxide emission of gasoline and three times as much of diesel. Hence,

biodiesel can benefit the environment by aiding the alleviation of the effects of global

warming through reduction of greenhouse gas emissions.This is a very fortunate

discovery since the proclamation of the Kyoto Protocol in 1997 calling for a 5.2%

reduction in greenhouse gas emission worldwide (Brenan & Owende, 2010).

Biodiesel is also very economical to use because the engines designed for diesel

does not require complex modifications when using biodiesel as a substitute fuel

(Hafizan & Zainura, 2013). In fact, a test run done using thirty passenger buses

mounted with Mercedes Benz OM352 engines fueled by biodiesel successfully ran for

about 300,000 km with promising engine performance and fuel consumption without

even modifying the engine (Choo et al., 2007).

8
 In addition to the reasons stated, use and production of biodiesel is now

progressing globally. Countries in South America, namely Brazil, Ecuador, Peru,

Guatemala, Colombia, and the like, as well as neighboring countries in Asia such as

China, Malaysia, Indonesia, and Japan, had already platformed the production of

biodiesel through establishing national policies promoting biodiesel use, conducting

extensive research on biodiesel, and supporting plantations for biodiesel production

(USAID, 2009; Knothe et al., 2015). Moreover, Southeast Asian countries are the

major exporters of fats and oils. This further potentiates biodiesel production, making

it a good investment in Southeast Asian countries.

In the Philippines, efforts to promote biodiesel production have already been

initiated through the implementation of certain laws and policies such as the Republic

Act No. 9367, known as Biofuels Act of 2006, which mandates the use of 2%

minimum blend of biodiesel in all diesel fuels sold within the country, the Omnibus

Investment Code (EO 226) and PEZA Law (RA 7196), which give incentives to

economical projects including biodiesel (BOI Philippines, 2011), and through

supporting 13.7 million hectares of coconut plantations, and nine biodiesel refineries

which produces 393 million liters of biodiesel annually (USDA-FAS, 2013).

Generations of Biodiesel

There are different generations of biodiesel depending on the feedstock used in its

production. Aro (2015), identified first generation biodiesel as fuel extracted from

food and oil crops such as rapeseed oil, sugar beet, sugarcane, and coconut, and

second generation biodiesel as fuel extracted from lignocellulosic, non-food materials

including straw, bagasse, and forest residues. However, these generations of biodiesel

have great impacts on economy, environment, and biodiversity conservation in a

9
negative way as it can compete with land use for food and fibre production, require

enormous amounts of fertilizer and water for maintenance, and promote the

exploitation of forests.

Unfortunately, the Philippines rely heavily on first generation biodiesel using

coconut oil from coconut plantations. More unfortunately, another disadvantage of

using biodiesel from food crops and plantations is the vulnerability of these

plantations to natural calamities, and the Philippines is in fact, the second most

disaster-prone country in Asia, and third in the world in terms of weather related

disasters (UNISDR, 2014).

In 2013, the Food and Agriculture Organization of the United Nations, and the

Philippine Coconut Authority reported that typhoon Haiyan had destroyed 33 million

coconut trees in Tacloban, Leyte. The damage caused by the typhoon resulted to a

decline in biodiesel production in the Philippines the following year (Corpuz, 2014).

Hence, it is very timely for a third generation biodiesel to emerge—the microalgae.

Microalgae as Feedstock for Biodiesel Production

Due to the threat to food security and the controversial sustainability issues of the

first two generations of biodiesel, a third generation of biodiesel was discovered: the

microalgae. Firstly, the use of microalgae does not pose a threat to food security.

Second, the use of microalgae is more cost effective and economical than the first and

second generation of biodiesel because cultivation of microalgae requires minimum

water and no additional land use. Microalgae can also grow in artificial light unlike

plantations that require sunlight to grow. Microalgae provide better air quality

maintenance as biomass production effect biofixation of waste CO 2 (Christi, 2007).

Pesticides or any other chemicals are not necessary for the maintenance of microalgae

10
(Rodolfi et al., 2008). Microalgal cultivation can also have a dual purpose of waste

water treatment as the nutrients necessary for its growth can be obtained from waste

water (Cantrell et al., 2008), and their residual biomass can be used as fertilizer

(Spolaore et al., 2006), or be converted to other forms of biofuel such as bioethanol

(Hirano et al., 1997) and biohydrogen (Ghirardi et al., 2000). Most of all, microalgae

have the capability of an all year round production thus, exceeding the yield of the

best oil seed crops (Brennan & Owende, 2010). In fact, Schenk et al. (2008)

discovered that microalgae cultures produce ten times as much as biodiesel than

rapeseed.

11
 MATERIALS AND METHODS

Cultures and Cultivation Conditions

Chlorella sp. and Microcystis sp. were isolated from fresh water samples collected

in Central Luzon. Spirulina sp. culture was obtained from the University of the

Philippines, Los Banos. All isolates were cultivated using BG-11 medium at 25oC and

was subjected under continuous illumination and aeration. Figures 1-3 show the

morphology of the three microalgal species under the microscope.

Axenization of Cultures

Cultures were axenized using the antifungal drug, Nystatin, at three varying

concentrations expressed as units (refer to Table 1).

Growth Curve Determination

Growth curve of each isolates were determined in a 96-well plate through optical

density (OD) measurement at 620 nm using a plate reader for a period of 8 days at 48

hours interval per reading. The OD reading was plot against time (h) and the sharpest

increase in OD was recorded.

Co-cultivation

Co-culture of Chlorella sp.-Microcystis sp. and Chlorella sp.-Spirulina sp. was

done six days after the cultivation of pure cultures. The pure cultures were adjusted to

the same optical densities then co-cultivated at three different ratios: 1:1 or 50%

Chlorella sp., 3:2 of 60% Chlorella sp., and 3:1 or 75% Chlorella sp. with a total

12
volume of 2 L per set-up. The set-ups (refer to Table 2) were cultivated under

continuous light and aeration until the end of harvest time.

Extraction and Quantification of Biomass and Lipid

Six days after cultivation of the set-ups, 100 mL of each set-up were collected and

partitioned into eight 15-mL conical tubes containing 12.5 mL volume of the samples

for each tube. The collection was done in triplicates. The 15-mL conical tubes

containing the samples were then centrifuged for 10 minutes set at 3,500 rpm at 25oC.

After centrifugation, the supernatant was decanted and 10 mL of sterile distilled water

was added to the conical tubes. The tubes were centrifuged once again for 10 minutes

at 3,500 rpm. The supernatant was decanted and the algal pellets from the eight

conical tubes per replicate per set-up were pooled in a 50-mL conical tube. 25 mL of

2:1 Chloroform-Methanol solution was then added to the pooled algal pellets, and the

samples were mixed by shaking. The conical tubes containing the samples were

covered with aluminum foil and were left inside a refrigerator overnight. After storage

inside the refrigerator, 5 mL of sterile distilled water was added to each tube. The

tubes were then centrifuged for 10 minutes at 3,500 rpm. After centrifugation, the

samples were separated into three layers: the uppermost methanol layer, the middle

algal layer, and the chloroform layer where the lipids are dissolved (see Figure 4). The

methanol and the algal layer were pipetted out using a 1000-μL pipettor to a separate

15-mL conical tube (see Figure 4). The 15-mL conical tubes containing the methanol

and algal layer were centrifuged for 10 minutes at 3,500 rpm. The algal pellets settled

at the bottom and the methanol was discarded. The conical tubes were left to dry

inside an incubator set at 44oC until the samples were dried completely for about 24-

48 hours. The 50-mL conical tubes containing the lipids dissolved in chloroform were

13
air dried inside a laminar flow hood until the samples were dried completely at about

24-48 hours. Figure 5 shows an image of a completely dried lipid sample. The

samples were then weighed in an analytical balance in the nearest ten thousandths.

Calculations and Statistical Analysis

Biomass expressed in g/L is calculated as:

% Lipid (w/w) is calculated as:

The values of the mono-cultures were calculated as:

Independent t-test was used to assess the significant difference between two

variables at 95% confidence level at α=0.05. Hence, a p-value ≥ 0.05 means that the

difference is significant, and a p-value < 0.05 means that the difference is not

significant.

14
 RESULTS

Growth Curve

Figure 6 shows the graph of the growth curve expressed as absorbance (620 nm)

vs. time (h). The sharpest increase in growth curve as evidenced by the high increase

in absorbance was found between 144-191 hours (6-8 days) in Chlorella sp. and

Microcystis sp. For the Spirulina sp. it was found between 0-47 hours (0-2 days).

However, the subsequent absorbance readings after 0-47 hours for the Spirulina sp.

showed a plateauing pattern until 144 hours, hence the harvest time for all microalgae,

including Spirulina sp., was determined to be at day six.

Biomass Production

Control vs. Co-culture

Figures 7-9 show the biomass (g/L) produced of the control and co-cultures in

different ratio at the sixth, eighth, and tenth day of cultivation. Two controls, the

Chlorella sp., and the particular cyanobacteria for each type of co-culture

(Microcystis sp. for Chlorella sp.-Microcystis sp., and Spirulina sp. for Chlorella sp.-

Spirulina sp. set-ups), are compared with the co-culture biomass yield of the co-

culture. Figure 7 shows the biomass for each set-up during the sixth day of

cultivation. According to this graph, only the 3:1 (75%) Chlorella sp.-Spirulina sp.

co-culture set-up has a significant increase to both controls. Figure 8, which shows the

eighth day of cultivation, four set-ups, the 3:1 (75%) Chlorella sp.-Microcystis sp.,

and all of the Chlorella sp.-Spirulina sp. showed a significant increase compared with

the Chlorella sp. control. However, the 3:1 (75%) C:M and the 3:2 (60%) C:S set-ups

showed a significant decrease compared with their particular cyanobacteria controls,

while the 1:1 (50%), and 3:1 (75%) C:S showed no significant difference with their

15
particular cyanobacteria control. Lastly, Figure 9 shows that all of the set-ups

increased significantly compared with the control. However, for the C:M co-cultures,

the increase is only significant with the Chlorella sp. control but not significant with

the cyanobacteria. For the C:S co-cultures, only the 3:2 (60%) and the 3:1 (75%),

have increased significantly with both controls and the 3:1 (75%) C:S co-culture had

the highest biomass yield among all other set-ups at a ten-day cultivation period.

Mono-cultures vs. Co-cultures

Figure 10 shows the mono-culture biomass (in g/L) and the co-culture biomass

obtained six days after cultivation. The only set-up to show a significant increase in

biomass is the 3:1 (75%) Chlorella sp.-Spirulina sp. co-culture having a biomass of

0.35 g/L which is 2.2 times greater than its calculated mono-culture biomass.

At day eight, only one set-up in the Chlorella sp.-Microcystis sp., the 3:1 (75%)

Chlorella sp.-Microcystis sp., have shown a significant increase in biomass having a

1.3-fold increase compared with the mono-culture value as shown in Figure 11. On

the other hand, all of the set-ups in the Chlorella sp.-Spirulina sp. have shown a

significant increase in biomass with the 3:2 (60%) ratio being the least, followed by

the 1:1 (50%) ratio, and lastly, the 3:1 (75%) ratio having a 1.3, 1.5, and a 2.4-fold

increase, respectively.

At day ten, only the 3:1 (75%) set-up in the Chlorella sp.-Microcystis sp. co-

culture made a significant increase in biomass having a 2.6-fold increase compared

with the mono-culture as shown in Figure 12. In the Chlorella sp.-Spirulina sp. co-

culture however, all of the set-ups made a significant increase relative to the mono-

culture having a 1.2, 1.9, and 4.6 times increase in biomass for the 1:1 (50%), 3:2

(60%), and 3:1 (75%) set-ups, respectively.

16
 Hence, it is the 3:1 (75%) C:S co-culture that yielded the highest biomass among

all other set-ups at a ten-day cultivation period.

Biomass Produced over Time

Figure 13 and Figure 14 shows the amount of biomass (g/L) extracted for three

days on a 48-hour interval of the three different ratios of Chlorella sp. Microcystis sp.

and Chlorella sp.-Spirulina sp. co-cultures, respectively. As shown in Figure 13, the

3:2 (60%), and the 3:1 (75%) Chlorella sp.-Microcystis sp. showed an increase in

biomass production after ten days with the 3:1 (75%) ratio having a linear increase.

Only the 1:1 (50%) ratio showed a decrease in biomass production after eight days. In

Figure 14, only the 3:2 (60%) Chlorella sp.-Spirulina sp. showed an increase in

biomass production after ten days. The 1:1 (50%) and the 3:2 (75%) ratio showed a

decrease in biomass production after eight days.

Overall, the 3:1 (75%) Chlorella sp.-Spirulina sp. set-up has the greatest increase

in biomass atten days of cultivation.

Lipid Content

Control vs. Co-culture

Figures 15-17 show the % lipid (w/w) produced of the control and co-cultures in

different ratio at the sixth, eighth, and tenth day of cultivation. Figure 15 shows %

lipid (w/w) of the control and co-cultures at day six cultivation. For the C:M co-

culture, all of the set-ups showed significant increase with respect to the

cyanobacteria control. However, the 1:1 (50%) C:M set-up showed a significant

decrease with the Chlorella sp. control while the other two set-ups are at par (no

significant difference) with the Chlorella sp. control. The C:S co-culture on the other

17
hand, only the 3:2 (60%), and 3:1 (75%) are significantly greater than the

cyanobacteria control. However, all the set-ups in the C:S co-culture are significantly

lesser compared with the Chlorella sp. control. Figure 16 shows the lipid content of

the set-ups at the eighth day of cultivation. The graph almost shows the same pattern

as the sixth day except for the 1:1 (50%) C:M which shows no significant difference

with the controls, the 1:1 (50%) C:S being significantly greater than the cyanobacteria

control, and the 3:2 (60%) is now at par with (no significant difference) the Chlorella

sp. control. During the tenth day of cultivation, only the 1:1 (50%) C:M showed no

significant difference with both controls as shown in Figure 17, while in the C:S set-

ups, the 1:1 (50%) and the 3:1 (75%) showed a significant difference with the

Chlorella sp. control. Only the 3:2 (60%) remained at par with the Chlorella sp.

control. None of the set-ups on the tenth day showed a significant increase with both

of the controls.

Mono-culture vs. Co-culture

As shown in Figure 18, only two set-ups in the Chlorella sp.-Microcystis sp.,

which are the 3:2 (60%), and the 3:1 (75%) ratio had an increase in % lipid relative to

the calculated mono-culture % lipid, both having a 1.2-fold increase. Interestingly, all

the set-ups in the Chlorella sp.-Spirulina sp. showed a decrease in % lipid relative to

the mono-culture % lipid during the sixth day of cultivation with the 3:1 (75%) ratio

having the greatest decrease, followed by the 1:1 (50%), then the 3:2 (60%) ratio

being the lowest, having 1.8, 1.7, and 1.2-fold decrease in % lipid, respectively.

Still, after eight days, only two set-ups showed an increase in % lipid compared

with the mono-culture % lipid in the Chlorella sp.-Microcystis sp., which are again,

the 3:2 (60%) ratio, and 3:1 (75%) ratio according to Figure 19 but with different

18
increment values with the 3:2 (60%) ratio having a 1.2-fold increase while a 1.6-fold

increase in the 3:1 (75%) ratio. For the Chlorella sp.-Spirulina sp. set-ups, only two

set-ups showed a significant decrease in % lipid in contrary with the first day of

extraction, which are the 1:1 (50%) ratio and the 3:1 (75%) ratio having a 1.5, and

2.4-fold decrease in % lipid, respectively.

At the tenth day of cultivation, the 1:1 (50%), and 3:2 (60%) Chlorella sp.-

Microcystis sp. showed no significant difference with respect to the mono-culture %

lipid according to Figure 20, while the 3:1 (75%) ratio showed a 1.3-fold decrease in

% lipid. For the Chlorella sp.-Spirulina sp., the 1:1 (50%) and the 3:2 (60%) ratio

showed no significant difference with the mono-culture % lipid, while the 3:1 (75%)

ratio showed a 2.5-fold decrease in % lipid.

Lipid Content over Time

Figure 21 and Figure 22 shows a graph of % lipid (w/w) vs. time (h) of the

different ratios of Chlorella sp.-Microcystis sp. and Chlorella sp.-Spirulina sp.,

respectively. All of the co-cultures had their peak % lipid at day eight cultivation and

declined at day ten except for the 1:1 (50%) Chlorella sp.-Spirulina sp. set-up which

maintained its % lipid from the eighth day to the tenth day of cultivation.

In summary, the highest % lipid attained in the duration of the extraction is the 3:1

(75%) Chlorella sp.-Microcystis sp. co-culture eight days after cultivation. The

highest decrease was attained by the 3:1 (75%) Chlorella sp.-Spirulina sp. ten days

after cultivation. All of the set-ups reached their peak % lipid on the eighth day,

except for 1:1 (50%) Chlorella sp.-Spirulina sp. co-culture, which maintained its %

lipid until day ten.

19
 DISCUSSION

Growth Curve

The growth curve of any particular microorganism has four stages: the lag phase,

log phase, stationary phase, and death phase (Brock et al., 2014). The lag phase refers

to the period of adjustment of the cells to the medium, and thus ideally, no growth

happens. The log phase follows the lag phase where there is an exponential increase

in the population of the cells in the culture. The stationary phase is where the cells

stop dividing due to the depleted nutrients, and the death phase is the period when the

cells die off (Engelkirk et al., 2011). The purpose of constructing a growth curve is to

determine the most appropriate harvest time of the cultures which is at the log phase.

A complete growth curve of the three microalgal species can be attained at 25-33 days

(Madkour et al., 2012). However, the time for measuring the growth curve was

limited to eight days due to unavailability of equipment. Fortunately, the log phase for

Chlorella sp., Microcystis sp., and Spirulina sp., is between 0-10 days (Goksan et al.,

2007; Bortoli et al., 2014; Shukri et al., 2014). Thus, to determine the harvest time of

the three microalgae, the sharpest increase in growth curve was identified, which

occurred between the sixth and eighth day of cultivation. Hence, the harvest time for

biomass and lipid was found to be at day six.

Biomass Production

Ideally, to say that the co-culture effectively produces more biomass compared

with the Chlorella sp. and cyanobacteria controls, the actual yield of the co-culture

should significantly be greater than both controls. Two different kinds of graphs were

constructed to show the effect of co-cultivation on biomass production which are: the

20
control vs. co-culture, and the mono-culture vs. co-culture graphs. Both graphs

showed a positive effect of co-cultivation on biomass production. This may be due to

the ability of cyanobacteria to transform nutrients to a more organic and usable form

via metabolic enzyme secretions, hence, hastening the reproducibility of the cells

(Dianursanti & Santoso, 2015).

The highest recorded yield in biomass was found in the 3:1 (75%) Chlorella sp.-

Spirulina sp. co-culture at the tenth day of cultivation. In the Chlorella sp.-

Microcystis sp. co-culture, the highest biomass yield is the 3:1 (75%) C:M at 10 g/L,

which is only about half of the biomass yield of the 3:1 (75%) C:S. This may be due

to the nitrogen-fixing property of Spirulina sp. The process of nitrogen fixation

involves the conversion of N2 into NH3 (ammonia). And in a study by Kim et. al.

(2010), it was found out that Chlorella sp. utilizes ammonia and converts it to

biomass.

It can be observed in Figure 9 that the 3:1 (75%) C:S co-culture, has a linear

increase of biomass from the sixth to the tenth day. This implies the possibility that

the particular co-culture set-up has not yet reached its peak biomass on the tenth day

since the biomass production has not yet slowed down or decreased. Hence, the

maximum amount that can be drawn from the 3:1 (75%) C:S co-culture may be

greater than 20 g/L (refer to Table 3).

Comparing the growth curve of the microalgae and the biomass production, there

seem to be a discrepancy between the sixth and the eighth day of cultivation. While

the biomass of the Spirulina sp. is higher than the Chlorella sp. at the eighth day of

biomass harvest, the absorbance of the Spirulina sp. is lower than the Chlorella sp.

This discrepancy between the growth curve and the biomass may have risen due to the

21
adaptation period of the Spirulina sp. in BG-11 medium since the Spirulina sp. pure

culture obtained was grown in a different culture medium.

Lipid Content

On the first day of extraction, only the 3:2 (60%) and 3:1 (75%) Chlorella sp.-

Microcystis sp. set-ups have a significant increase in the lipid content and reached

their peaks after six days. Although the Chlorella sp.-Spirulina sp. showed a

significant decrease on the first day, it can be noticed that after ten days, two of the

set-ups: 1:1 (50%) and 3:2 (60%) have become at par with the mono-culture lipid

content. Hence, as with the situation in the biomass production, the Chlorella sp.-

Spirulina sp. set-ups, except for the 3:1 (75%) ratio, may have a significant increase

in lipid content beyond ten days. Co-cultivation led to an increase in lipid content,

possibly due to the enhanced nitrogen starvation and synergistic effects brought about

by co-cultivation. Nitrogen starvation may lead to an increase of intracellular acetyl-

CoA—a major requirement for triglyceride biosynthesis as well as other neutral lipids

(Zhao et al., 2013). In addition to that, microalgae in co-cultures released more free

fatty acids in media compared with normal culture conditions and hence, may

stimulate downstream gene transcription for lipid biosynthesis (Dianursanti &

Santoso, 2015).

22
 CONCLUSION

Co-cultivation is a novel method to enhance the accumulation of the biomass

production and lipid content simultaneously, which solves the trade-off between

biomass productivity and lipid content as high lipid content is often counterbalanced

by lower growth rates. The results of the co-cultivation experiments showed that co-

cultivation improves biomass production and lipid content compared with mono-

cultivation with the 3:1 (75%) Chlorella sp.-Microcystis sp. having the highest

increase in both biomass and lipid content. The Chlorella sp.-Spirulina sp., however

shows a promising increase in both biomass and lipid content ten days after

cultivation, and thus, may reach its peak beyond ten days. This study demonstrates the

potential of Microcystis sp. in co-culture with Chlorella sp. to induce lipids for

biodiesel production. This is a promising result since Microcystis sp. is a HAB

causing microalgae. Hence Microcystis from polluted waters can be harvested and

serve as feedstock in co-culture set-ups for biodiesel production providing a dual

benefit to the environment and the industry.

23
 RECOMMENDATIONS

Since some of the set-ups have the potential to increase its biomass and lipid

content beyond 10 days, the cultivation and extraction procedure should be extended

beyond 10 days, to have a better observation and comparison which co-culture

produces more lipid and biomass. Methods in maximizing biomass and lipid

extraction could also be improved through performing more effective cell disruption

procedures such as sonication, heating, and subjection to microwave. Solvents such as

hexane can be added for further purification process. It is also recommended to assess

the quality of lipids as well through spectrometric analyses such as high performance

liquid chromatography or gas-chromatography-mass spectrometry, to determine the

feasibility and the usefulness of the lipids in biodiesel production.

24
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29
 TABLES

Table 1. Components for culture axenization of the three microalgae species:

Chlorella sp., Microcystis sp., and Spirulina sp.

Tube 1 2 3 4

Nystatin 0 mL 1.25 mL 2.5 mL 5 mL

Distilled H2O 45 mL 43.75 mL 42.5 mL 40 mL

Culture 5 mL 5 mL 5 mL 5 mL

Concentration of Nystatin
0 2,500 5,000 10,000
(in units)

30
 Table 2. Green algae-cyanobacteria co-culture and control set-ups for co-cultivation
experiment

1:1 3:2 3:1

Set-up/Ratio
(50% Chlorella sp.) (60% Chlorella sp.) (75% Chlorella sp.)

A C:M C:M C:M

B C:S C:S C:S

Control Chlorella sp. Microcystis sp. Spirulina sp.

31
 Table 3. Algal biomass (g/L) of monocultures of Chlorella sp., Microcystis sp., and Spirulina sp. and co-cultures of Chlorella sp.-Microcystis
sp. and Chlorella sp.-Spirulina sp. in ratios of 1:1 (50% Chlorella sp.), 3:2 (60% Chlorella sp.), and 3:1 (75% Chlorella sp.)
Monocultures Co-cultures
C+M C+S
C M S
50% 60% 75% 50% 60% 75%
Mono-
culture - - - 0.162±0.0724 0.160±0.0729 0.156±0.0738 0.160±0.0774 0.158±0.0770 0.155±0.0763
Day
6
0.015± 0.017± 0.017±
Co-culture 0.102±0.040 0.116±0.009 0.140±0.030 0.126±0.010 0.152±0.028 0.346±0.019
0.043 0.040 0.046
Mono-
- - - 4.240±0.1457 3.891±0.1395 3.367±0.1304 5.776±0.1561 5.119±0.1479 4.134±0.1356
culture
Day
8 0.249±
0.599± 0.906±
Co-culture 0.066 3.988±0.744 4.321±1.142 4.450±0.187 8.465±0.394 6.798±0.376 9.983±1.774
0.102 0.114
Mono-
- - - 4.786±3.0415 4.409±2.4645 3.845±1.5990 5.981±0.1120 5.366±0.1209 4.443±0.1342
culture
Day
10 0.291± 0.667± 0.906±
Co-culture 0.039 7.128±0.146 8.440±0.538 10.676±0.039 12.511±0.867 11.845±0.538 20.433±0.505
0.146 1.710
*C=Chlorella sp., M=Microcystis sp., S=Spirulina sp.

32
 Table 4. Lipid content (w/w%) of monocultures of Chlorella sp., Microcystis sp., and Spirulina sp. and co-cultures of Chlorella sp.-Microcystis
sp. and Chlorella sp.-Spirulina sp. in ratios of 1:1 (50% Chlorella sp.), 3:2 (60% Chlorella sp.), and 3:1 (75% Chlorella sp.)
Monocultures Co-cultures
C+M C+S
C M S
50% 60% 75% 50% 60% 75%
Mono- 39.439± 27.081± 10.183± 33.260± 34.495± 36.349± 24.811± 27.736± 32.125±
Day culture 1.893% 0.665% 1.090% 0.502% 0.583% 0.715% 0.546% 0.608% 0.723%
6 Co- 33.406± 41.379± 43.502± 14.248± 22.319± 17.942±
culture 0.983% 1.317% 2.179% 2.418% 1.737% 1.158%
Mono- 58.316± 28.415± 12.277± 43.365± 46.355± 50.840± 35.296± 39.900± 46.806±
Day culture 9.130% 1.771% 1.100% 2.325% 2.762% 3.431% 2.299% 2.748% 3.427%
8 Co- 43.436± 57.741± 79.579± 24.166± 35.239± 19.290±
culture 14.402% 2.039% 3.627% 1.559% 4.073% 1.246%
Mono- 35.013± 19.185± 10.069± 27.099± 28.682± 31.056± 22.541± 25.035± 28.777±
Day culture 2.014% 1.914% 0.923% 0.695% 0.715% 0.792% 0.554% 0.632% 0.764%
10 Co- 29.213± 26.789± 24.747± 23.778± 28.189± 11.715±
culture 7.222% 1.832% 1.412% 2.569% 3.140% 1.931%
*C=Chlorella sp., M=Microcystis sp., S=Spirulina sp.

33
 FIGURES

25μm

Figure 1. Chlorella sp. at x40 magnification inoculated from pure culture seed stock of
Chlorella sp.

34
 25μm

Figure 2. Microcystis sp. at x40 magnification inoculated from pure culture seed stock of
Microcystis sp.

35
 25μm

Figure 3. Spirulina sp. at x40 magnification inoculated from pure culture seed stock of
Spirulina sp.

36
 Figure 4. Left: Phase separation into three layers from up to bottom: methanol layer, algal
layer, chloroform layer. Right: Methanol and algae layer (15-mL conical tube) separated from
chloroform layer (50-mL conical tube).

37
Figure 5. Bottom part of a 50-mL conical tube containing dried lipid residue. Left:
taken from outside the tube; right: taken frominside the tube.

38
Figure 6. Growth curve of the three microalgae species expressed as absorbance vs time (h). Blue line corresponds to Chlorella sp., red line for
Microcystis sp., and green line for Spirulina sp.

39
Figure 7. Graph showing comparison of biomass (g/L) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and the actual biomass (g/L) harvested from each different co-culture set-up, six days after cultivation (red). Error bars are represented as
standard error of the mean. Significant differences are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

40
Figure 8. Graph showing comparison of biomass (g/L) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and the actual biomass (g/L) harvested from each different co-culture set-up, eight days after cultivation (red). Error bars are represented as
standard error of the mean. Significant difference are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

41
Figure 9. Graph showing comparison of biomass (g/L) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and the actual biomass (g/L) harvested from each different co-culture set-up, ten days after cultivation (red). Error bars are represented as
standard error of the mean. Significant difference are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

42
Figure 10. Graph showing the biomass (g/L) produced by the mono-culture vs. co-culture of varying ratios of Chlorella sp.-Microcystis sp. and
Chlorella sp.-Spirulina sp. six days after cultivation. Error bars are represented as standard error and asterisks were shown to indicate
significant difference with respect to the corresponding mono-culture biomass.

43
Figure 11. Graph showing the biomass (g/L) produced by the mono-culture vs. co-culture of varying ratios of Chlorella sp.-Microcystis sp. and
Chlorella sp.-Spirulina sp. eight days after cultivation. Error bars are represented as standard error and asterisks were shown to indicate
significant difference with respect to the corresponding mono-culture biomass.
44
Figure 12. Graph showing the biomass (g/L) produced by the mono-culture vs. co-culture of varying ratios of Chlorella sp.-Microcystis sp. and
Chlorella sp.-Spirulina sp. ten days after cultivation. Error bars are represented as standard error and asterisks were shown to indicate
significant difference with respect to the corresponding mono-culture biomass.
45
 Figure 13. Biomass (g/L) vs. time (h) graph of the three different ratios of the Chlorella sp.-Microcystis sp. co-culture. Blue line represents 1:1
(50%) ratio, red line represents 3:2 (60%) ratio, and green line represents 3:1 (75%) ratio. Error bars are expressed as standard error of the mean.
Similar letters indicate no significant difference between data points of each ratio.

46
 Figure 14. Biomass (g/L) vs. time (h) graph of the three different ratios of the Chlorella sp.-Spirulina sp. co-culture. Blue line represents 1:1
(50%) ratio, red line represents 3:2 (60%) ratio, and green line represents 3:1 (75%) ratio. Error bars are expressed as standard error of the mean.
Similar letters indicate no significant difference between data points of each ratio.

47
 Figure 15. Graph showing comparison of % lipid (w/w) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and lipid (w/w%) harvested from each different co-culture set-up, six days after cultivation (red). Error bars are represented as standard error of
the mean. Significant difference are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

48
 Figure 16. Graph showing comparison of % lipid (w/w) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and lipid (w/w%) harvested from each different co-culture set-up, eight days after cultivation (red). Error bars are represented as standard error
of the mean. Significant difference are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

49
 Figure 17. Graph showing comparison of % lipid (w/w) between two controls—Chlorella sp. (blue) and Microcystis sp./Spirulina sp. (green),
and lipid (w/w%) harvested from each different co-culture set-up, ten days after cultivation (red). Error bars are represented as standard error of
the mean. Significant difference are represented as a for the Chlorella sp. control, and b for the cyanobacteria control.

50
Figure 18. Graph showing the lipid content (% lipid w/w) of the mono-culture vs. the co-culture of varying ratios of Chlorella sp.-Microcystis
sp. and Chlorella sp.-Spirulina sp. six days after cultivation. Error bars are represented as standard error and asterisks were shown to
indicate significant difference with respect to the corresponding mono-culture biomass.

51
Figure 19. Graph showing the lipid content (% lipid w/w) of the mono-culture vs. the co-culture of varying ratios of Chlorella sp.-Microcystis
sp. and Chlorella sp.-Spirulina sp. eight days after cultivation. Error bars are represented as standard error and asterisks were shown to
indicate significant difference with respect to the corresponding mono-culture biomass.

52
Figure 20. Graph showing the lipid content (% lipid w/w) of the mono-culture vs. the co-culture of varying ratios of Chlorella sp.-Microcystis
sp. and Chlorella sp.-Spirulina sp. ten days after cultivation. Error bars are represented as standard error and asterisks were shown to
indicate significant difference with respect to the corresponding mono-culture biomass.

53
 Figure 21. Lipid content (w/w%) vs. time (h) graph of the three different ratios of the Chlorella sp.-Microcystis sp. co-culture. Blue line
represents 1:1 (50%) ratio, red line represents 3:2 (60%) ratio, and green line represents 3:1 (75%) ratio. Error bars are expressed as standard
error of the mean. Similar letters indicate no significant difference between data points of each ratio.

54
Figure 22. Lipid content (w/w%) vs. time (h) graph of the three different ratios of the Chlorella sp.-Spirulina sp. co-culture. Blue line represents
1:1 (50%) ratio, red line represents 3:2 (60%) ratio, and green line represents 3:1 (75%) ratio. Error bars are expressed as standard error of the
mean. Similar letters indicate no significant difference between data points of each ratio.

55
 APPENDICES

Appendix A. Raw Data for Biomass Extraction -Day 6

Corrected Corrected Error Bar Error Bar

Set-up g/L Ave FW Ave DW
FW DW FW DW
1a 0.2703 0.0237 0.2370
1b 0.3603 0.0113 0.1133 0.326967 0.0150 0.049328829 0.007521254
1c 0.3503 0.0101 0.1010
2a 0.5103 0.0248 0.2480
2b 0.4503 0.0162 0.1623 0.466967 0.017356 0.037859389 0.006951605
2c 0.4403 0.0110 0.1103
3a 0.5203 0.0251 0.2507
3b 0.5403 0.0169 0.1687 0.536967 0.017022 0.015275252 0.007967806
3c 0.5503 0.0091 0.0913
4a 0.4103 0.0102 0.1017
4b 0.5203 0.0172 0.1717 0.446967 0.010178 0.06350853 0.00698334
4c 0.4103 0.0032 0.0320
5a 0.3603 0.0107 0.1070
5b 0.4303 0.0134 0.1340 0.4503 0.0116 0.101488916 0.001558846
5c 0.5603 0.0107 0.1070
6a 0.4903 0.0085 0.0847
6b 0.5603 0.0150 0.1500 0.4803 0.014022 0.085440037 0.00513694
6c 0.3903 0.0186 0.1860
7a 0.4903 0.0145 0.1450
7b 0.3703 0.0113 0.1130 0.386967 0.012633 0.096090235 0.001665333
7c 0.3003 0.0121 0.1210
8a 0.3603 0.0143 0.1430
8b 0.3103 0.0110 0.1100 0.343633 0.015233 0.028867513 0.004768997
8c 0.3603 0.0204 0.2040
9a 0.6203 0.0308 0.3080
9b 0.7003 0.0373 0.3730 0.663633 0.034556 0.040414519 0.003365897
9c 0.6703 0.0356 0.3557

56
 Appendix B. Raw Data for Biomass Extraction -Day 8

Corrected Corrected Error Bar Error Bar

Set-up g/L Ave FW Ave DW
FW DW FW DW
1a 0.44 0.249 2.49
1b 0.3422 0.261 2.61 0.4774 0.249333 0.157271358 0.011503623
1c 0.65 0.238 2.38
2a 2.3977 0.6011 6.011
2b 0.5956 0.5799 5.799 1.49665 0.598633 0.90105 0.017629899
2c 1.49665 0.6149 6.149
3a 2.05 0.9017 9.017
3b 2.8 0.9272 9.272 2.425 0.905767 0.375 0.019717082
3c 2.425 0.8884 8.884
4a 1.98 0.33 3.3
4b 1.94 0.31875 3.1875 2.016667 0.39875 0.100166528 0.128944029
4c 2.13 0.5475 5.475
5a 1.264 0.6582 6.582
5b 0.7782 0.291 2.91 1.0119 0.4321 0.243422123 0.197807255
5c 0.9935 0.3471 3.471
6a 1.0299 0.4306 4.306
6b 1.0294 0.4821 4.821 1.004233 0.444967 0.044023668 0.032431518
6c 0.9534 0.4222 4.222
7a 1.5779 0.796 7.96
7b 1.5394 0.9241 9.241 1.538867 0.8465 0.039302714 0.068214441
7c 1.4993 0.8194 8.194
8a 1.2448 0.7179 7.179
8b 1.1499 0.6047 6.047 1.226167 0.679833 0.068867288 0.065069296
8c 1.2838 0.7169 7.169
9a 1.6714 1.2968 12.968
9b 1.7132 1.015 10.15 1.68 0.998267 0.029844262 0.307241946
9c 1.6554 0.683 6.83

57
 Appendix C. Raw Data for Biomass Extraction-Day 10

Corrected Corrected Error Bar Error Bar

Set-up g/L Ave FW Ave DW
FW DW FW DW
1a 0.74 0.311696 3.116961
1b 0.3422 0.261513 2.615134 0.6474 2.881177 0.27103557 0.015644914
1c 0.86 0.291144 2.911438
2a 1.8 0.433152 4.331516
2b 1.72 0.341111 3.411113 1.733333 5.560535 0.061101009 0.592657147
2c 1.68 0.893897 8.938974
3a 1.98 0.906821 9.06821
3b 2.73 0.902753 9.027526 2.355 9.085086 0.375 0.006750062
3c 2.355 0.915952 9.159521
4a 1.98 0.557096 5.570959
4b 1.94 0.755746 7.557461 2.016667 7.21436 0.100166528 0.129639517
4c 2.13 0.851466 8.514659
5a 2.4621 1.019106 10.19106
5b 2.1153 1.141548 11.41548 2.188933 10.39791 0.244801396 0.241234844
5c 1.9894 0.958718 9.587179
6a 2.5178 0.90431 9.043104
6b 2.0932 0.98771 9.877103 2.4396 9.015779 0.31467399 0.245757584
6c 2.7078 0.812713 8.127129
7a 2.8917 0.986163 9.861628
7b 2.4214 1.2576 12.576 2.697867 10.79009 0.245798135 0.270711919
7c 2.7805 0.993266 9.932656
8a 1.5012 1.217286 12.17286
8b 1.8273 1.216758 12.16758 1.7064 11.98463 0.178648174 0.045334865
8c 1.7907 1.161344 11.61344
9a 4.5081 2.383964 23.83964
9b 3.658 1.738432 17.38432 4.08305 20.67589 0.42505 0.40485
9c 4.08305 2.080372 20.80372

58
Appendix D. Raw Data for Lipid Extraction-Day 6

Corrected Ave Ave % Error Bar

Set-up % Lipid Ave DW
DW Biomass Lipid DW
1a 0.0065 0.015044444 43.20532
1b 0.0056 0.015044444 37.22304 39.4387 0.005933 0.000493288
1c 0.0057 0.015044444 37.88774
2a 0.0047 0.017355556 27.08067
2b 0.0049 0.017355556 28.23303 27.08067 0.0047 0.0002
2c 0.0045 0.017355556 25.9283
3a 0.0021 0.017022222 12.33681
3b 0.0015 0.017022222 8.81201 10.18277 0.001733 0.000321455
3c 0.0016 0.017022222 9.399478
4a 0.0035 0.010177778 34.38865
4b 0.0032 0.010177778 31.44105 33.40611 0.0034 0.000173205
4c 0.0035 0.010177778 34.38865
5a 0.0047 0.0116 40.51724
5b 0.0046 0.0116 39.65517 41.37931 0.0048 0.000264575
5c 0.0051 0.0116 43.96552
6a 0.0067 0.014022222 47.7813
6b 0.0059 0.014022222 42.07607 43.50238 0.0061 0.00052915
6c 0.0057 0.014022222 40.64976
7a 0.0014 0.012633333 11.08179
7b 0.0016 0.012633333 12.66491 14.24802 0.0018 0.00052915
7c 0.0024 0.012633333 18.99736
8a 0.0033 0.015233333 21.66302
8b 0.003 0.015233333 19.69365 22.31947 0.0034 0.000458258
8c 0.0039 0.015233333 25.60175
9a 0.007 0.034555556 20.25723
9b 0.0058 0.034555556 16.78457 17.94212 0.0062 0.00069282
9c 0.0058 0.034555556 16.78457

59
Appendix E. Raw Data for Lipid Extraction-Day 8

Corrected Ave Error Bar

Set-up % Lipid Ave DW
DW Biomass DW
1a 0.1015 0.249333333 40.70856
1b 0.1569 0.249333333 62.92781 0.1454 0.039428543
1c 0.1778 0.249333333 71.31016
2a 0.1889 0.598633333 31.55521
2b 0.1522 0.598633333 25.42458 0.1701 0.018366546
2c 0.1692 0.598633333 28.26438
3a 0.0963 0.905766667 10.63188
3b 0.1301 0.905766667 14.36352 0.1112 0.017251377
3c 0.1072 0.905766667 11.83528
4a 0.2337 0.39875 58.60815
4b 0.2275 0.39875 57.05329 0.1732 0.099468035
4c 0.0584 0.39875 14.64577
5a 0.2558 0.4321 59.19926
5b 0.2321 0.4321 53.71442 0.2495 0.015258768
5c 0.2606 0.4321 60.31011
6a 0.3756 0.444966667 84.41082
6b 0.3225 0.444966667 72.47734 0.3541 0.027953712
6c 0.3642 0.444966667 81.84883
7a 0.1803 0.8465 21.29947
7b 0.2257 0.8465 26.66273 0.204567 0.022861613
7c 0.2077 0.8465 24.53633
8a 0.189 0.679833333 27.80093
8b 0.2453 0.679833333 36.08237 0.239567 0.047957724
8c 0.2844 0.679833333 41.83378
9a 0.2141 0.998266667 21.44718
9b 0.171 0.998266667 17.12969 0.192567 0.021550019
9c 0.1926 0.998266667 19.29344

60
Appendix F. Raw Data for Lipid Extraction-Day 10

Corrected Ave Error

Set-up % Lipid Ave DW
DW Biomass Bar DW
1a 0.1125 0.290467 38.73078
1b 0.1002 0.290467 34.49621 0.1017 0.010134
1c 0.0924 0.290467 31.81088
2a 0.1498 0.666667 22.47
2b 0.1056 0.666667 15.84 0.1279 0.022103
2c 0.1283 0.666667 19.245
3a 0.0745 0.905769 8.225053
3b 0.1001 0.905769 11.05138 0.0912 0.014473
3c 0.099 0.905769 10.92994
4a 0.1606 0.712821 22.53019
4b 0.3111 0.712821 43.64347 0.208233 0.089166
4c 0.153 0.712821 21.464
5a 0.2056 0.844 24.36019
5b 0.2564 0.844 30.37915 0.2261 0.02678
5c 0.2163 0.844 25.62796
6a 0.2863 1.0676 26.81716
6b 0.2354 1.0676 22.04946 0.2642 0.026103
6c 0.2709 1.0676 25.37467
7a 0.3614 1.251133 28.88581
7b 0.2717 1.251133 21.71631 0.2975 0.05568
7c 0.2594 1.251133 20.7332
8a 0.3928 1.1845 33.16167
8b 0.3438 1.1845 29.02491 0.3339 0.064423
8c 0.2651 1.1845 22.38075
9a 0.3077 2.04325 15.05934
9b 0.171 2.04325 8.36902 0.239367 0.06835
9c 0.2394 2.04325 11.71663

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