Abstract
Algae are large and diverse group of microorganisms that can carry out photosynthesis since they
are able to capture energy from sun light. Algae may range in size from single cells as small as
one micrometer to large seaweeds that grow to over 50 meters. Algae grow in the wide and are
prompt to attack by predators and can easily be invaded which could result to competition that
will eventually lead to low production of biomass which are important to organisms and the
ecosystem. Chlorella viriabilis recently renamed Chlorella viriabilisNC64A that is a bona fide
member of the true Chlorella genus, belonging to the Trebouxiophyceae was used in this present
research. Chlorellaviriabilis was propagated in BG11 media enriched with 0.5g/L of glucose for
mixotrophic growth and in autotrophic growth condition. The cell culture was monitored using
the hemocytometer for increase in cells concentration. At the end of three weeks, the cells were
harvested after centrifugation and dried in the oven. The mixotrophic dried biomass weighed
0.5g/L and that of the autotrophic weighed 0.1g/L. The results for protein analyses for both
mixotrophic and autotrophic yielded 1.118g/L &0.07g/L respectively. Also, the results for the
glucose was obtained using the Mercz protocol, the mixotrophic had higher glucose content than
the autotrophic with 0.0564g/L & 0.0266g/L respectively. The cell concentration was more in the
autotrophic than in mixotrophic but the mixotrophic cell culture had bigger cell size which
showed the presence of accumulated materials. Glucose enhanced the production of algal
biomass.
vi
TABLE OF CONTENT
Title page i
Approval page ii
Dedication iii
Acknowledgement iv
Abstract v
Table of content vi-viii
List of table ix
List of figures x
CHAPTER ONE
1.1 BACKGROUND OF STUDY 1
1.2 Relevance of Study 3
1.3 Statement of Problem 3
1.4 Aim and Objectives 4
1.5 Hypothesis 4
1.6 Scope of the Study 5
CHAPTER TWO
2.1 Algae 5
2.2 The origin and evolution of green algal and plant actions 7
2.3 Algal phylogeny and the origin of land plants 7
vii
2.4 Single Cell Protein (SCP) 8
2.5 New phylogenetic classification 10
2.6 Life cycle patterns found in the algae 12
2.7 Habit and habitat 14
2.8 Freshwater algae 18
2.9 Factors required for cultivation of algal biomass 20
2.10 Methods for cultivation of algal biomass 22
2.11Comparison of different cultivation techniques 25
2.12 Harvesting of algal biomass 25
2.13 Protein extraction method 28
2.14 Importanceof algae 31
CHAPTER THREE
3.1 Chlorella viriabilis 32
3.2 Sample collection 32
3.3 Cultivation of Chlorella viriabilis 33
3.4 Harvesting of cells 34
3.5 Drying 35
3.6 Protocol for chlorophyll quantification 35
3.7 Protocol for carbohydrate quantification 35
3.8 Protocol for protein quantification 36
CHAPTER FOUR
viii
4.1 Growing chlorella viriabilis for a period of three weeks. 39
4.2 Growth rate of chlorella viriabilis 41
4.3 Determination of chlorophyll content present in the cell biomass 44
4.4 A glucose standard curve for glucose determination 45
4.5 A protein standard curve for glucose determination 48
CHAPTER FIVE
5.1 Discussion 51
5.2 Conclusion and recommendation 53
REFERENCES
APPENDIX
ix
LIST OF TABLES
Table 1: Average Different Composition of the Main Groups of Microorganisms (% Dry
Weight). 9
Table 2: Growth mode of algae (microalgae) cultivation. 24
Table 3: Glucose standard curve 36
Table 4: Values for protein curve. 37
Table 5: Growth of Chlorella under three growth conditions 39
Table 6: Comparison between the growth rates of Chlorella variabilis 42
Table 7: Result of Chlorophyll Content Obtained from the Biomass Produced 44
Table 8: Glucose Standard Curve 45
Table 9: Glucose Content obtained from the standard curve for both autotrophic and mixotrophic
growth 47
Table 10: Datas obtained for Protein Standard Curve 48
Table 11: Protein Content obtained from algal biomass. 50
x
LIST OF FIGURES
Figure 1: A diagram showing the life cycle of algae 14
Figure 2: Diagrammatic Structure of Some Algae revealing some vital parts.
19
Figure 3: A bar chart showing the growth rate of chlorella viriabilis 40
Figure 4: A bar chart showing the specific growth of chlorella viriabilis during different growth
condition. 43
Figure 5: Glucose Standard Curve 46
Figure 6: Protein Standard Curve 49
1
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF STUDY
Smith (1955) defined algae based on characters of the sex-organs. He said-in algae the sex
organs are usually unicellular and when they are multicellular as in most brown algae, all cells
are fertile (Smith, 1995). There are approximately 1800 genera with 21,000 species which are
highly diverse with respect to habitat, size, organization, physiology, biochemistry and
reproduction (Pandey, 2009).
Algae may range in sizes from single cell as small as one micrometer to large seaweeds that may
grow to over fifty meters (Vymagal, 1995). Algae are ubiquitous, they occur in almost every
habitable environment on earth, soil, permanent ice, snow fields, hot and cold desert.
Biochemically and physiologically, algae are similar in many aspects to other plants.
Furthermore, algae are the major primary producers of organic compounds and play a central
role as the base of the food chain in aquatic systems. Besides forming the basic food source for
these food chains, they also produce oxygen necessary for the metabolism of the consumer
organism (Lee,et al., 1989).
Algal biomass is always made up of these three main components: Carbohydrates, Protein and
Natural oils. The most important component for biodiesel production is the natural oils that can
be converted to biodiesel. The percentage lipid composition varies and so the fatty acid
2
composition varies according to the algae strain within a range of 10 to 40% under natural
conditions. The lipids present are mainly made up of polyunsaturated lipids (John,et al., 1998).
The algae Spirulina has been considered for use as a supplementary protein (Raja,et al., 2008),it
is a blue green algae having strong antioxidant activity and provokes a free radical scavenging
enzyme system.
In addition, the presence of algae leads to reduced erosion by regulating the water flow into soils.
Similarly, they play a role in soil fertility, soil reclamation, and bio-controlling of agricultural
pest, formation of microbiological crust, agricultural wastewater treatment and recycling of
treated water. Human civilization depends on agriculture for its existence.
They are aquatic, both marine and fresh water, and occur on or within soil and on moist stones
and woods as well as in association with fungi and certain animals. The algae are of great
importance as primary producers of energy rich compounds which form the basis for this
purpose, the planktonic algae are of special importance, since they serve as food for many
animals. It is thought that 90% of the photosynthesis on earth is carried on by aquatic or by
aquatic plants, the planktonic (suspended) algae are chiefly responsible this while
photosynthesizing, they oxygenate their habitat, thus increasing the level of dissolved oxygen in
their environment. Certain blue-green algae like some bacteria can use gaseous nitrogen from the
atmosphere in building their protoplasm and in this way; they increase the nitrogenous
compounds in water and soils of their habitat.
Light conditions affect directly the growing and photosynthesis of microalgae (duration and
intensity). Microalgae needs a light/dark regime for productive photosynthesis, it needs light for
a photochemical phase to produce Adenine triphosphate(ATP), Nicotinamide adenine
3
dinucleotide phosphate oxidase (NADPH) and also needs dark for biochemical phase to
synthesize essential molecules for growth (Belcher, 1982).
1.2 RELEVANCE OF STUDY
The relevance of this project was to grow algae using glucose in a sterile bioreactor for better
yield of protein which can be used for nutritional enrichment of cereals, pharmaceutical, animal
feeds and other purposes.
1.3 STATEMENT OF PROBLEM
World Health Organization (WHO) report in 2012 states that malnutrition is the underlying
contributory factor in over one third of all child deaths, making children more vulnerable to
severe diseases. The increasing world deficiency of protein is becoming a main problem of
humankind. Since the early fifties, intense efforts have been made to explore new, alternate and
unconventional protein. Research has shown that the chance of infection with HIV virus might
be reduced in individuals who have good nutrition status with micro nutrients (Egal & Valstar,
1999).
Algae in the oceans, rivers, and lakes of the world are thought to produce about half of all the
oxygen produced on the planet. Given that the total biomass of the world’s algae is but a tenth of
the biomass of all the other plants, the efficiency of the algae is impressive and of interest in
terms of producing biofuels. Cyanobacteria currently cultivated in large scale systems are
economically viable sources of protein used in food because they often meet the requirements of
nutrient in the diets. Moreover, through them you can get other human consumer products
4
(Kuhad et al., 1997). A cyanobacterium as a source of single-cell protein has certain advantages
over the use of other microorganisms because of its rapid growth and quantity and quality of
protein (Molina et al., 2002). Among the microalgae, the genus Spirulina contains about 60 to
70% of proteins, nucleic acids and amino acids recommended by the Food and Agriculture
Organization (Pelizer,et al., 2003). It also contains betacarotene and absorbable iron, and other
minerals and high levels of vitamins, phenolic compounds, gammalinolenic acid and other
essential fatty acids (Belayet al., 1993: Von et al., 2000).
The protein content of Spirulina varies between 50% and 70% of its dry weight. These levels are
quite exceptional, even among microorganisms. Moreover, the best sources of vegetable protein
achieve only half these levels; for example, soya flour contains “only” 35% crude protein.
However, the protein content varies by 10-15% according to the time of harvesting inrelation to
daylight. The highest values being obtained at early daylight (Association française pour
l’a1gologie appliquée (AFAA) (1982).
1.4 AIM AND OBJECTIVES
The broad aim of this research was to grow algae mixotrophically using glucose as organic
substrate with the following objectives:
To culture and compare the algal growth using glucose
To harvest and purify algal biomass.
To evaluate the nutritional content of the algal biomass.
1.5 HYPOTHENSIS
5
There is no significant difference in the amount of algal biomass cultivated in glucose
and ethanol.
There is a significant difference in the nutritional content of algae cultivated in glucose
and ethanol.
1.6 SCOPE OF THE STUDY
The scope of this study was governed around Chlorellaviriabilis,a strain of Chlorella spp
isolated from our local environment
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