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Fred Attah

EFFECTS OF

PRODUCTIVITY OF

Digitally Signed by: Content manager’s

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Fred Attah

Faculty of Biological Science

Department of Microbiology

EFFECTS OF VARIOUS PHYTOHORMONES ON GROWTH AND

PRODUCTIVITY OF Chlorella sorokinia and Spirulina

OZIOKO, FABIAN UCHECHUKWU

PG/M.Sc./08/49407

i

: Content manager’s Name

Weabmaster’s name

a, Nsukka

Sciences

GROWTH AND

Spirulina platensis

FABIAN UCHECHUKWU

ii

EFFECTS OF VARIOUS PHYTOHORMONES ON GROWTH AND

PRODUCTIVITY OF Chlorella sorokinia and Spirulina platensis

BY


PG/M.Sc./08/49407

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

APRIL, 2014.

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TITLE PAGE

EFFECT OF VARIOUS PHYTOHORMONES ON GROWTH AND

PRODUCTIVITY OF Chlorella sorokinia and Spirulina platensis

BY


PG/M.Sc./08/49407

TO THE SCHOOL OF POST GRADUATE STUDIES

UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD

OF MASTER’S DEGREE (M.Sc.) IN INDUSTRIAL MICROBIOLOGY

SUPERVISOR: PROF. J.C OGBONNA.

APRIL, 2014.

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CERTIFICATION

Mr. Ozioko, Fabian Uchechukwu, a postgraduate student in the Department of

Microbiology, majoring in Industrial Microbiology, has satisfactorily completed the

requirements for the course work and research for the degree of Master of Science

(M.Sc.) in Microbiology. The work in embodied in his dissertation original and has

not been submitted in part or full for either diploma or degree of this University or

any other University.

……………………………… ……………………………….. Prof. J.C. Ogbonna Prof. A.N. Moneke Supervisor Head Department of Microbiology Department of Microbiology University of Nigeria, Nsukka University of Nigeria, Nsukka

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DEDICATION

This work is dedicated to Mum, Mrs. Christiana Ozioko, for the uncompromising

moral philosophy that nurtured the spirit of doggedness in me

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ACKNOWLEDGEMENTS

My gratitude goes to God Almighty for his gift or life and other invaluable

blessings.

I acknowledge with deep sense of indebtedness and appreciation, the father by

support of my supervisor Prof. J.C. Ogbonna. Words are not adequate to express my

gratitude for the kind attention, support, motivation and mentoring I received from

him. His patience in going through this work in quite exemplary as he was always

there, ever read, to guide and get me back on the track anytime I derailed.

I also owe a great debt or gratitude to Mrs. Joy Oziolo, my wife, Mr. Evaristus

Ozioko, my elder brother, and Mr. Akpu Geoffrey who had always had my welfare at

heart from the very beginning of this study. They made sure I lacked nothing

throughout my study. My thanks also go to Dr. E.A Eze and Dr. C. Nwuche of

Microbiology Department, Dr. O. Eze of Biochemistry Department and Dr. J. C.

Ugwuoke, for their support and encouragement throughout my research work.

My gratitude goes to the following persons for their various contributions

towards the actualization of this work, Dr. M. Wogu of Anatomy Department, Faculty

of Veterinary Medicine, and Prof. J.O Ugwuanyi of Department of Microbiology.

Finally, my thanks go to Mr. Aloka and Miss Ngozi Asogwa of Anatomy

Department, Faculty of Veterinary Medicine UNN, all the 2008/2009 session PG

students, Mr. C. Ugwoke, and Director, National Centre for Energy Research and

Development(NCERD) for allowing me space at the Centre to carry out my research

work.

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ABSTRACT The effects of indoleacetic acid, indolebutyric acid, gibberellic acid and kinetin on growth and biomass productivity of Chlorella sorokiniana and Spirulina platensis were investigated. The optimum concentration of the phytohormones for Chlorella

sorokiniana cell enlargement was 20ppm for GA3, Kinetin, IAA, and IBA. At this concentration, the Chlorella cell sizes were 81.07µm, 78.67µm, 78 .07µm and 66.90µm respectively. The effectiveness of the phytohormones in increasing the size of the cells can be ranked as GA3 > kinetin > IAA > IBA. Treatment with IAA at concentration of 10ppm had the highest effect on Chlorella sorokiniana cell number with a value of 7.94x 109 cells/ml, followed by IBA at 15ppm with a value of 4.36 x 109 cells/ml. GA3 and kinetin had no significant effects (P< 0.05) on cell number. The effects of the phytohormones on dry cell weight of the two microalgae species followed the same trend as the cell number with 10ppm of IAA giving the highest value of 4.825g/l in Chlorella sorokiniana and 1.10g/l in Spirulina platensis. The optimal concentrations of the phytohormones for Chlorella chlorophyll contents were 15ppm for IAA, GA3, IBA and kinetin. At these concentrations, the values of extractable chlorophyll were 594.20 mg/g, 238.60 mg/g, 141.65 mg/g and 140.90 mg/g respectively. The effectiveness of the phytohormones on chlorophyll contents can be ranked as IAA > GA3 > IBA > kinetin. In the case of Spirulina platensis, the optimal concentrations were 10ppm for IAA and 15ppm for GA3. At these concentrations, the extractable chlorophyll contents were 444.14 mg/g and 156.92 mg/g respectively. There were no significant effects (P > 0.05) of phytohormones on protein content of the two strains of microalgae in all the treatments. Combination of the phytohormones exhibited synergistic effect on growth and productivity of Chlorella sorokiniana. IBA (12.5ppm) + GA (2.5ppm), IBA (10ppm) + GA (5ppm), and IAA (12.5ppm) + GA (2.5ppm) gave the highest dry cell weight of 3.368g/l, 3.02g/l and 1.688g/l respectively. These values were much higher than the 0.552g/l obtained in the control experiment (without phytohormone). The optimal concentrations of the combined pyhtohormones for Chlorella chlorophyll contents were IBA(12.5ppm) + GA(2.5ppm), IAA(7.5ppm ) + kinetin(7.5ppm) and IAA(5ppm) + kinetin(10ppm.). At these concentrations, the chlorophyll contents were 425 mg/g, 136.26 mg/g and 122.60 mg/g respectively

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TABLE OF CONTENTS

Title page: i

Certification: ii

Dedication: iii

Acknowledgement: iv

Abstract: v

Table of contents: vi

List of table: ix

List of figures: - x

CHAPTER ONE: INTRODUCTION 1

LITERATURE REVIEW: 4

1.0 Production Techniques 4

1.1 Open pond production systems 5

1.2 Closed Photobioreactor systems 7

1.2. Flat-plate photobioreactor: 8

1.2.2 Tubular photobioreactor: 8

1.2.3 Column photobioreactor: 9

1.3 Hybrid production systems: 10

1.4 Heterotophic production: 10

1.5 Common phytohormones and their physiological roles in algae: 12

1.5.1 Auxins: 12

1.5.2: Cytokinin: 14

1.5.3 Gibberellins: 16

Page number

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CHAPTER TWO: MATERIALS AND METHODS: 17

2.0 Strains of microalgae: 17

2.1 Selection of the phytohormones: 17

2.2 Effects of phytohormones in growth of microalgae: 17

2.2.1 Photohormone preparation: 17

2.2.2 Synergistic study: 18

2.3 Cultivation of the microalgal: 19

2.3.1 Preparation of basal growth media: 19

2.3.2 Effects of phytohormones on productivity of microalgae: 20

2.4 Determination of cell number: 20

2.5 Determination of cell size; 20

2.6 Determnation of by cell weight: 20

2.7 Determination of cholorophyll contents: 21

2.8 Determination of protein content of the cell: 21

2.8.1 Estimation of the percentage nitrogen of the Biomass: 21

CHAPTER THREE: RESULTS: 22

3.1 Effect of phytohormones on Chlorella snokinia cell number: 22

3.1.2 Effect of phytohormones on cell size of Chlorella sorokinina: 24

3.1.3 Effect of phytohormones on dry weight of Chlorella sorokiniana

and Spirulina platensis after 8 days cultivation: 26

3.1.4 Effect of phytohormones on chlorophyll contents of Chlorella

sorokinia and Spirulina platensis after 8 days cultivation: 31

3.1.5 Effect of phytohormones on protein contents of Chlorella sorokinia

and Spirlina platensis after 8 days cultivation: 36

3.2.1 Effect of combined phytohormones on the day cell

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weight of Chlorella sorokinia: 39

3.2.2 Effect of combined phytohormones on the chlorophy

Content of Chlorella sorokinia 41

CHAPTER FOUR: DISCUSSION: 43

Conclusion: 47

Reference: 48

Appendices: 52

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LIST OF TABLES

Table 1: Preparation of media with different concentration

of phytohormones: 18

Table 2: Preparation of culture media with combinations of

Phytohormones: 19

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LIST OF FIGURES

Fig 1: Effect of Phytohormones on cell number of Chlorella sorokinia

after 8 days cultivation: 23

Fig 2: Effect of Phytohormones on cell size of Chlorella sorokinia

after 8 days of cultivation: 25

Fig 3: Effect of Phytohormones on dry weight of Chlorella sorokinia


Fig 4: effects of different Phytohormones on dry weight

of Spirulina platesis after 8 days cultivation: 28

Fig 5: Effect of different Phytohormones on dry weight of Chlorella

Sorokinia at 15 ppm concentration: 29

Fig 6: Effect of different Phytohormones on dry weight of

Spirulina platensis at 15 ppm concentration: 30

Fig 7 Effect of Phytohormones on chlorophyll content of Chlorella sorokinia

after 8 days cultivation: 32

Fig 8: Effect of Phytohormones on chlorophyll content of Spirulina

platensis ater 8 days cultivation: 33

Fig 9: Effect of different Phytohormones on chlorophyll

content of Chlorella sorokinia at 15 ppm concentration: 34

Fig 10: Effects of different phytohormones on chlorophyll

content of Spinulina platensis at 15ppm concentration: 35

Fig 11: Effect of Phytohormones on protein content of Chlorella sorokinia

after 8 days cultivation : 37

Fig 12: Effect of Phytohormones on protein content of Spirunlina platensis


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Fig 13: Effect of combined Phytohormones on dry cell weight of

Chlorella sorokinia After 8 days cultivation: 40

Fig 14: Effect of combined Phytohormones on chlorophyll content of

Chlorella sorokinia after 8 days cultivation: 42

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CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

INTRODUCTION

There are numerous applications for microalgae and microalgal derived valued-

added products, including, pharmaceuticals, biomedicals, diagnostics, cosmetics,

aquaculture, food and animal feeds (Borowitzka, 1997). With increasing interest in

environmental policy, global oil price increase and climate change, the potential for

microalgal biofuel production is also of commercial and environmental interest

(Butler, N., 2006).

In view of this, judicious exploitation of microalgal cultivation biotechnology

for enhanced biomass productivity to meet up with the demand for provision of

nutraceutical, pharmaceutical and environmental benefits is technically and

economically viable and imperative. In recent years, metabolic engineering and

application of synthetic biology to potentially enhance living systems especially

microbes for use in medicine, agriculture, industry and bioremediation have gained

considerable attention. Genetic manipulation which invariably leads to inheritable

changes in a species might bring about adverse developmental changes in the

ecosystem when used for environmental and agricultural applications (Hunt et al.,

2009). Alternative means such as phytohormones and micronutrients have been used

to improve productivities in higher plants since the 1930s (Piotrowska et al., 2008).

Microalgae share physiological similarities with higher plants. Although

contemporary research on phytohormone physiological actions remain almost

completely focused on the higher plants, there are few studies devoted to auxins and

other classes of phytohormones in green algae from Chlorella, Scenedesmus, and

Spirulina species (Czepark et al., 1999). Studies with Chlorella species showed that

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the use of phytohormones have considerable stimulating effects on algal growth and

productivity (Czepark et al., 1999).

Experimental studies on the physiological effects of selected phytohormones on

microalgae biomass production and the concentrations which might lead to the

reduction in the cost of large-scale algae cultivation are thus needed. Of paramount

importance is the need to identify whether combinations of these phytohormones

would have any synergistic effect on enhancing the metabolites productivities and

yield.

Historically, microalgal culture has been carried out in a variety of ways for

mariculture and natural products production. Microalgae are very efficient solar

energy converters and they can produce a great variety of metabolites. Man has used

this natural process of harvesting the sun in the development of algal cultivation

systems for secondary waste water treatment (Oswald, 1998), for animal feeds and

chemical and secondary metabolites of pharmaceutical potential (De Pauw Persoone,

1998).

The finger that stirred the “honeycomb” of activities in algal biotechnology

was the publication, “Algal culture from laboratory to pilot plant” produced by the

Carnegie Institution of Washington in 1952. In that document, many workers foresaw

the great potential of algae as a product different from the fermentation industry and

as a potential source for agricultural and chemical commodities. This work stung the

“honey bee venom” of interest in many research groups during the sixties and the

seventies, most notably in the USA, Germany, Israel, Japan, Thailand and France.

With the onset of energy crisis, microalgae were then suggested as a source of

biomass for methane.

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Eicosapentaenoic acid (EPA) has a significant effect on the vascular status of

humans because of the antithrombotic and antiaggregatory effects (Apt and Behrens,

1999). In addition, docosahexaenoic acid (DHA) is a dominant fatty acid in

neurological tissue, i.e. the gray matter of the human brain. These compounds are

very interesting as nutritional supplements. Other nutraceuticals already derived from

microalgae are β- carotene and astaxanthin and these two processes have been scaled

to a commercial scale (Olaizola, 2000). Microalgae produce a range of valuable

compounds including carbohydrates, proteins, essential amino acids, pigments and

vitamins (Olaizola, 2003). The pigments include chlorophyll a, b, and c, phcocyanin,

xyanthophylls (astaxanthin, canthaxanthin, lutein); these pigments have existing

applications in foods and feeds (Apt and Behrens, 1999). Because microalgae

incorporate inorganic carbon (CO2 and HCO3), they are very useful for production of

isotopically labelled 13 C-compounds. In addition to carbon, it is easy to produce

labeled 2H- or 15N- Compound from nitrate (15NO3-) and (2H20) (Apt and Behrens,

1999). Furthermore, microalgae contain sterols, which could be used as building

blocks for pharmaceuticals (hormones). Moreover, microalgae are potential sources of

compounds with biomedical applications (antimicrobial, antiviral, anticancer) (Apt

and Behrens, 1999).

Many applications of microalgae demand the use of monocultures and

controlled cultivation systems. This has led to increased emphasis on development of

cultivation strategies that will complement the controlled cultivation method in

photobioreactors for cost-effective biomass production. A lot of work has been on

photobioreactor design and optimization for efficient cultivation of microalgae but

productivities are still low due to the technical problems with light supply and

distribution inside photobioreactors. Efforts to develop strains with high growth rates

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and productivities through genetic engineering approach have not yielded desired

results. Although, phytohormones and micronutrients have been used to increase

productivities in higher plants, work on application of phytohormones to improve

productivity in microalgae is scarce. There is therefore, a need to study

phytohormones-mediated stimulation of microalgae growth for enhanced and cost-

effective biomass productivity.

This research work sets to:

1) Investigate the physiological effects of selected phytohormones on growth and

productivity of microalgae-Chlorella sorokiniana (a green alga) and Spirulina

platensis (cyanobacteria).

2) Determine optimum concentration of phytohormones that will lead cost effective

and optimal biomass production.

3) Determine whether there is synergy between the phytohormones for enhanced

biomass production.

LITERATURE REVIEW

1.0 PRODUCTION TECHNIQUES

The traditional methods used for microalgae production are large-scale ponds

for natural products production or photobioreactors for fine chemicals (Lee, 2001).

Currently, phototrophic production is the only method which is technically

and economically feasible for large-scale production of algae biomass for natural

products production (Borowitzka, 1999). Two systems that have been deployed are

based on open pond and closed photobioreactor technologies (Borowitzka, 1999). The

technical viability of each system is influenced by intrinsic properties of the algae

strain used as well as climatic conditions and the costs of land and waters

(Borowitzka, 1992).

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1.1 OPEN POND PRODUCTION SYSTEMS.

Algae cultivation in open pond production systems has been used since 1950s

(Borowitzka, 1999). These systems can be categorized into natural waters (lakes,

lagoons, and ponds) and artificial ponds or containers. Raceway ponds are most

commonly used artificial systems (Jiménez et al., 2003). They are typically made of a

closed loop oval -shaped recirculation channels generally between 0.2 and 0.5m deep,

with mixing and circulation required to stabilize algal growth and productivity.

Raceway ponds are usually built in concrete, but compacted earth-lined ponds with

white plastic have also been used.

Plane view of a raceway pond. Algae broth is introduced after the paddlewheel, and completes a cycle while being mechanically aerated with CO2. It is harvested before the paddlewheel to start the cycle again (adapted from Chisti, 2007).

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Open pond is the cheaper method of large-scale algal biomass production. Open pond

production does not necessarily compete for land with existing agricultural crops,

since they can be implemented in areas with marginal crop production potential

(Jiménez et al; 2003). They also have lower energy input requirement (Pulz, 2001),

and regular maintenance and clearing are easier (Ugwu et al., 2008).

Open pond system, require highly selective environments due to inherent

threat of contamination and pollution from other algae species and protozoa (Pulz,

2001). Monoculture cultivation is possible by maintenance of extreme culture

environment, although only a small number of algae strains are suitable. For example,

the species Chlorella (adaptable to nutrient-rich media), Dunaliela salina (adaptable

to very high salinity and Spirulina (adaptable to high alkalinity) thrive under such

extreme environments (Borowitzka, 1999).

In respect to biomass productivity, open pond systems are less efficient when

compared with closed photobioreactors (Christi, 2007). This can be attributed to

several determining factors, including, evaporation losses and temperature fluctuation

in the growth media, CO2 deficiencies, inefficient mixing, and light limitation.

Although evaporation losses make a net contribution to cooling, it may also result in

significant changes in ionic composition of the growth medium with detrimental

effects on algal growth (Doucha and Livansky, 2006). Temperature fluctuations due

to diurnal cycles and seasonal variations are difficult to control in open ponds (Christi,

2007). Potential CO2 deficiencies due to diffusion into the atmosphere may result in

reduced biomass productivity due to less efficient utilization of CO2. Also, poor

mixing by inefficient stirring mechanisms may result in poor mass CO2 transfer rates

causing low biomass productivity (Ugwu et al; 2008). Light limitation due to top

layer thickness may also lead to reduced biomass productivity.

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High algae biomass production rates are achievable with open pond systems.

However, there are still inconsistencies in the production rates reported in literature.

Jeménez et al., (2003) extrapolated an annual dry weight biomass production rate of

30 tonnes per hectare using data from a 450m2 and 0.30m deep raceway pond system

producing biomass dry weight of 8.2gm-2 per day in Malaga, Spain. Using similar

depth of culture, and biomass concentrations of up to 1 g L-1, Becter (2007), estimated

dry biomass productivity in the range of 10-25gm-2 per day. However, the only open

pond system for large scale production that has achieved such high biomass

productivity is the inclined system developed by Setlik, et al., (2002) for the

production of Chlorella. In this system, a biomass concentration of higher than 10 g

L-1 was achieved.

1.1 CLOSED PHOTOBIOREACTOR SYSTEMS

Microalgae production based on closed photobioreactor technology is

designed to overcome some of the major problems associated with the described open

pond production systems. For example, pollution and contamination risks with open

pond systems for the most parts precludes their use for the preparation of high-value

products for use in the pharmaceutical and cosmetics industries (Ugwu et al., 2008).

Also, unlike open pond production, photobioreactors permit culture of simple-species

of microalgae for prolonged durations with lower risk of contamination (Christi,

2007). Closed systems include the tubular, flat plate, and column photobioreactors.

These systems are more appropriate for sensitive strains as the closed configuration

makes the control of potential contamination easier. Owing to the higher cell mass

productivities attained, harvesting costs can also be significantly reduced. However,

the costs of closed system are substantially higher than open pond systems (Christi,

2007).

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1.1.1 Flat-plate Photobioreactor

Some of the earliest forms of closed systems are flat-plate photobioreactors

(Samson et al., 1985) which have received much research attention due to the large

surface area exposed to illumination (Ugwu et al., 2008) and high densities of

photoautotrophic cells (780 g l-1) observed (Hu et al., 1998). The reactors are made of

transparent materials for maximum solar energy capture, and a thin layer of dense

culture flows across the flat plate (Hu et al., 1998, Richmond et al., 2003) which

allows radiation absorbance in the first few millimeters thickness. Flat plate

photobioreators have low dissolved oxygen concentration and high photosynthetic

efficiency is achieved when compared to tubular version (Ugwu et al., 2008).

1.1.2 Tubular Photobioreactor

Tubular photobioreactor have design limitation on length of the tubes, which

is dependent on potential O2 accumulation, C02 depletion, and pH variation in the

systems (Becker, 2007). Therefore, they cannot be scaled up indefinitely; hence large-

scale production plants are based on integration of multiple reactor units. However,

tubular photobioreactors are deemed to be more suitable for outdoor mass cultures

since they expose their larger surface area to sunlight.

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Basic design of a horizontal tubular photobioreactor (adapted from Becker, 2007). Two main sections: airlift system and solar receiver; the airlift systems allow for the transfer of O2 out of the systems and transfer of CO2 into the system as well as providing ameans to harvest the biomass. The solar receiver provides a platform for the algae to grow by giving a high surface area to volume ratio.

1.1.3 Column Photobioreactor

Column photobioreactors offer the most efficient mixing, the highest

volumetric mass transfer rates and the best controllable growth conditions (Eriksen,

2008). They are low-cost, compact and easy to operate. The vertical columns are

aerated from the bottom, and illuminated through transparent walls (Eriksen, 2008), or

internally (Suh and Lee, 2003) illuminated. Their performance compares favourably

with tubular photobioreactors (Sanchez et al., 2002).

Closed photobioreactors have received major research attention in recent

years. The proliferation of pilot-scale production using closed photobioreactors

xxiii

compared to open raceway ponds could be attributed to more rigorous process control

and potentially higher biomass production rates.

1.2 HYBRID PRODUCTION SYSTEMS

The hybrid two-stage cultivation is a method that combines distinct growth

stages in photobioreactors and in open ponds. The first stage is in a photobioreactor

where controllable conditions minimize contamination from other organisms and

favour continuous cell division. The second production stage is aimed at exposing the

cells to nutrient stresses, which enhances synthesis of the desired products (Rodolfi et

al., 2008, Huntley and Redalji, 2007). This stage is ideally suited to open pond

systems, as the environmental stresses that stimulated production can occur naturally

through the transfer of the culture from photobioreactors to the open pond.

Huntley and Redalje, (2007) used such a two-stage system for the production

of both oil and astaxanthin (used in salmon feed) from Haematococcus pulvalis and

achieved an annual average microbial oil production rate > 10 tonneha-1 per annum

with a maximum rate of 24 tonneha-1 per annum.

1.3 HETEROTROPIC PRODUCTION

Heterotrophic production has also been successfully used for algae biomass

and metabolites productions (Miao and Wu, 2006). In this process, microalglae are

grown on organic carbon substrates such as glucose in the stirred tank bioreactors or

fermenters. Algae growth is independent of light energy which allows for much

simpler scale-up possibilities since smaller reactor surface to volume ratios may be

used (Erikson, 2008). These systems provide a high degree of growth control and also

lower harvesting costs due to the higher cell densities achieved (Chen and Chen,

2006). The set-up costs are minimal although the system uses more energy than the

production of photosynthetic microalgae because the process cycle includes the initial

xxiv

production of organic carbon sources via the photosynthetic process (Christi, 2007).

Ogbonna et al., (1997) investigated the feasibility for large-scale bio mass production

based on sequential heterotrophic/autotrophic cultivation of Chlorella sorokiniana but

concluded that biomass productivity in terms of chlorophyll and protein contents were

less efficient.

Technical and biological limitations of these culture systems have given rise to

the development of other biotechnological strategies to enhance bulk/large biomass

production. They are considered as a complementary way of algae mass culture which

leads directly to reduction in the high cost of production associated with these

traditional algal culture systems. The cost of production has been very high, with

either the volume of end product being very low or the value of the end product being

very high.

Currently, one of the most promising avenues in improving algal biomass

production for bioactive molecules production and bio fuel production is nutrient

deprivation (Miao and Wu, 2006). This method involves starving the algae of

essential nutrients, which triggers a stress response that leads to an increase in

bioactive molecules production and lipid for biofuel production. This stress response

however, also slows down the growth of the algal cells. In order to avoid this

compromise between bioactive molecules yield and growth rate, the actual signaling

pathways being affected by nutrient deprivation must be directly manipulated. A

novel way that could potentially target these algal growth and metabolic pathways

directly and without compromise involves the exogenous application of

phytohormones or plant hormones.

The exploration of phytohormone signaling mechanisms has produced several

lines of findings. Signaling pathways have been established for several

xxv

phytohormones including kinetin and different types of auxins and gibberellins (Nam

and Li, 2002). To understand the observed effects of phytohormones, investigations

have been focused on molecular mechanisms of signaling. Leading models suggest

two primary mechanisms of signaling for most phytohormones. Phytohormone effects

begin with receptors with kinase activity. In the first model, these receptors produce

signal cascades which employ phosphorylation events to eventually control

transcriptional activators and enhancers for growth-regulating genes. Cytokinins, for

instance, employ a two-part system with a histidine protein kinase receptor plant

(Hwang and Sheen, 2001). The induced signal cascade increases transcription of

genes involved in growth regulation (Nam and Li, 2002).

In the second mechanism of phytohormone signaling, active signal cascades

can act on intracellular proteins rather than acting at the transcriptional level. This

activity promotes an increase in growth of the cell (Fu et al., 2003). Auxins affect the

activities of the cell by a similar means. At the transcription level, auxin response

factors (ARFS) dimerize and bind to DNA to allow transcriptional control

(Friedrichsen et al., 2000). However, auxins also bind to receptors that generate signal

cascades to induce ancillary proteins to regulate the dimerization of ARFS. This

controls the transcription of growth-regulating genes. In the end, both models produce

similar effects of altering the transcription of growth-regulating genes.

1.5 COMMON PHYTOHORMONES AND THEIR PHYSIOLOGICAL ROLES

ON ALGAE

1.5.1 Auxins

Auxins (Indoleacetic acid, Phenylacetic acid, Indolebutyric acid and

Naphthalene acetic acid) are a class of phytohormones that primarily increase growth

in plants (Woodward and Bartel, 2005). Plants can synthesize their own auxins from

xxvi

tryptophan or indole-3- butyric acid, or they can obtain them from their surrounding

environments (Piotrrowska et al., 2008).

Indole-3- acetic acid (IAA) is the most common naturally occurring auxin.

Originally, the term auxin was used to classify phytohormones that induce elongation

in shoot cells. After extensive study of auxin response, however, they have been

found to promote root initiation and inhibit root elongation (Woodward and Bartel,

2005) by regulating the activity of the types cyclin-dependent kinase A during the

Gap (G1) and synthesis (s) phases of the cell cycle (Himanen et al., 2002).

Additionally, auxins delay leaf abscission (shedding), inhibit lateral bud formation,

induce callus formation and promote an epinastic (downward-bending) response on a

cellular level. Auxins accomplish these tasks by increasing cell wall plasticity,

increasing water intake, altering respiratory patterns and altering nucleic acid

metabolism (Woodward and Bartel, 2005). Auxins cause these profound changes due

to their activity at the transcriptional level (Himanen et al., 2002). Their effects can be

observed as early as 3 minutes after binding to cellular phytohormones receptors

(Piotrrowska et al., 2008).

Clearly, extensive auxin activity has been documented in plant species. Unlike

many other phytohormones, it is known to exist in certain algal species as well but

uncertainty concerning its function remains. Auxins are one of the few families of

phytohormones that are naturally secreted in algae (Lou et al., 2009). The most

common auxin found in brown algae, red algae, green algae, and diatoms is IAA

(Hunt et al., 2011). However, the concentration of this auxin is much lower than

concentration common in higher plants. (Lou et al., 2009). In certain algae of the

chlorophyceae class, low concentrations of IAA stimulate an inhibitory effect on

growth, while high concentrations have proven toxic (Hunt et al., 2011). However,

xxvii

IAA has a positive effect on growth rate and cell size in Chlorella species and Ocystis

while having no effect on Alaria esculenta (Piotrrowska et al., 2008). Auxin was

shown to stimulate rhizoid formation in the green algae Bryopsis plumosa and activate

the growth of some cultured microalgae and cyanobacteria (Hunt et al., 2011). In red

macrophytes, treatment with natural or synthetic auxins accelerated tissue growth in

the culture and callus development (Hunt et al., 2011). Exogenous IAA stimulated

zygote polarization and germination in Fucacean (Basu et al., 2002; Tarakhovskaya

et al., 2003). The action of endogenous and exogenous auxins on algal growth (thalus

branching, rhizogenesis, polarization) and development (induction of division, the

formation of reproductive structures) indicate that its functions correspond to those

fulfilled by this phytohormone in higher plants. The different responses of algae

show that these algal species may possess different auxin signaling pathways (Lou et

al., 2009). Although auxins are responsible for promoting morphological changes in

plants, there is currently few evidence suggesting parallel effects in algae (Tromas et

al., 2009).

1.5.2 Cytokinin

Cytokinins are plant growth substances which play a role in senescence and

chloroplast development, primarily by promoting cell division (Tarakhovskaya et al.,

2007). An example of a cytokinin is trans-zeatin and kinetin. It has been shown that

plants with lower levels of cytokinins develop stunted shoots, with leaf cell

production at 3-4% of that for plants with regular levels of cytokinins (Werner et al.,

2001). These phytohormones also impose upper limits on the rate of growth in order

to prevent overgrowth in plants (Werner et al., 2001). There is a clear relationship

between auxin and Cytokinins with the combination playing an essential role in the

formation of roots and their growth (Riou-Khamlichi et al., 1999). Endogenous

xxviii

Cytokinin-like activity has been documented in various microalgae (Stirk et al.,

2002). While the signaling features are present, they are not as common as in normal

plants. This would likely lead to a less pronounced effect of cytokinins in algae due to

fewer receptors. Effects of cytokinins have been determined in higher plants by

exogenous addition of cytokinins. The data concerning the effects of exogenous

cytokinins on algal growth and development were obtained mainly on the members of

the division Rhodophyta. Cytokinins (alone or in combination with auxins) were

shown to accelerate red algal growth in the culture and in some cases, facilitate callus

formation (Yokoya et al., 1999). In the tissue culture of Grateloupta doryphora,

cytokinins suppressed morphogenetic processes (Sheen, 2001). Algae treatment with

cytokinins activitated cell division and protein accumulation and stimulated

photosynthetic processes (activation of photosystems I and II) (Tarakhovskaya &

Maslov, 2004). These functions correspond completely to cytokinin functions in

higher plants. Similar methods in algae, if resulting in marked growth, would further

improve the biomass productivity efficiency of the culture systems to which

exogenous addition of phytohormones is complementary.

Cytokinin signal transduction pathway begins with binding to a two-

component receptor system, involving the cytokinin receptor, CR2 (Inoue et al.,

2001). Along these pathways, regulatory proteins play a critical role in increasing and

decreasing the cytokinin signal. The effect of increased growth from cytokinins is a

product of the activation of these regulators of the cell division cycle and

differentiation (Sheen, 2001, Rióu-khamlichi et al., 1999). Thus, cytokinins, as cell-

division promoting substances, may induce a faster growth rate in algae cells as they

do in higher plant species. This fact, along with the detection of cytokinin-like activity

xxix

in algae cells, is encouraging and highlights the potential for these substances to

promote enhanced bioactive molecules and biofuel productions from algae.

1.5.3 Gibberellins (GA3)

Gibberellins are diterpenoid acids that affect many areas of plant growth. They

promote stem elongation and fruit generation and allow seed germination (Nakajima

et al., 2006). Application of Gibberellins caused cells to increase in size (Gonai et al.,

2004). Little evidence for endogenous gibberellins activity has been observed in green

algae. Although increased growth in response to gibberellins has been documented in

algae, there is scarce evidence for its actions beyond those in higher plants. In the

presence of exogenous Gibberellins, heterotrophic growth of Westiellopsis prolifica

was accelerated (Rióu-khamlichi et al., 1999). In these experiments, the effect of the

phytohormone depended on the organic substrate used, which indicates a possibility

that gibberellins are involved in the control of the assimilation of the exogenous

sources of organic carbon by the cells. Gibberellic acid suppressed callus formation

and organogenesis in the tissue culture of the red alga Grateloupta doryphora (Sheen,

2001). In brown and red macrophytes, exogenous gibberellins accelerated growth and

increased the thalus length. (Yokoya et al., 1999).Thus it seems likely that like in

higher plants, gibberellins control growth of axial structures in both micro and

macroalgae.

xxx

CHAPTER TWO

MATERIALS AND METHODS

2.1 STRAINS OF MICROALGAE

Axenic strains of Chlorella sorokiniana IAM-C212 and Spirulina platensis NIES-46

used in this study were obtained from the Culture Collection Centre, University of

Tokyo, Japan.

2.2 SELECTION OF THE PYTOHORMONES

The four classes of Phytohormones that were used in this study are

• Indoleacetic acid (IAA)

• Indolebutyric acid (IBA)

• Gibberellic acid (GA3)

• Kinetin

These phytohormones were selected based on literature survey on their specific

physiological effects on higher plants. Samples of IAA, IBA, GA3 and Kinetin were

obtained from Wako Pure Chemical industrial Ltd, Tokyo, Japan.

2.2 EFFCETS OF PHYTOHORMONES ON GROWTH OF MICROALGAE

2.2.1 Phytohormone Preparation

Twenty milligrams of each of the phytohormones was first dissolved in

appropriate solvent (GA3 in 5.0 ml of deionized water, IAA and IBA in 0.5 ml of

95% of ethanol, and Kinetin in 0.1N hydrochloric acid) and then added to 200 ml

of de-ionized water to obtain 100 ppm which served as the stock solution. Desired

concentrations (5 ppm, 10 ppm, 15 ppm, and 20 ppm) were obtained using the

dilution formula: C1V1=C2V2 as shown in Table 1.

For 5 ppm: C1 = 100 ppm, V1 = ? C2 = 5 ppm, C2 = 300 ml.

V1 = 5 ppm * 300 ml ⁄ 100 pm

xxxi

= 15 ml (this volume was pippetted out from the stock solution).

For 10 ppm, 15 ppm and 20 ppm, the trend was the same.

TABLE 1: Preparation of media with different concentration of phytohormones

Growth culture vol.

(ml)

Phyto stock culture

vol. (ml)

Vol. of distilled water

(ml)

Phyto concentration

(ppm)

240 15 45 5

240 30 30 10

240 45 15 15

240 60 Nil 20

Control Nil 60 Nil

2.2.2 Synergistic Study

The effect of combined phytohormones on growth and productivity of the

microalgae were studied using the following phytohormone combinations: IBA

combined with GA3, IAA combined with GA3 and IAA combined with Kinetin.

Table II shows volume combinations of growth medium, combined phytohormones

and distilled water bringing the total culture volume to 100ml.

xxxii

Table II. Preparation of culture media with combinations of phytohormones

Growth medium(ml)

Phytohormone A

Vol. of phytohormone

(ml)

Phytohormone B

Vol. of phytohormone

(ml)

Vol. of water (ml)

Total culture Volume(ml)

80 IAA(2.5ppm) 2.5 GA(12.5ppm) 12.5 5.0 100

80 IAA(2.5ppm) 2.5 Kinetin(12.5ppm) 12.5 5.0 100

80 IBA(2.5ppm) 2.5 GA(12.5ppm) 12.5 5.0 100

80 IAA(5ppm) 5.0 GA(10ppm) 10.0 5.0 100

80 IAA(5ppm) 5.0 Kinetin(10ppm) 10.0 5.0 100

80 IBA(5ppm) 5.0 GA(10ppm) 10.0 5.0 100

80 IAA(7.5ppm) 7.5 GA(7.5ppm) 7.5 5.0 100


80 IBA(7.5ppm) 7.5 GA(7.5ppm) 7.5 5.0 100

80 IAA(10ppm) 10.0 GA(5ppm) 5.0 5.0 100

80 IAA(10ppm) 10.0 Kinetin(5ppm) 5.0 5.0 100

80 IBA(10ppm) 10.0 GA(5ppm) 5.0 5.0 100

80 IAA(12.5ppm) 12.5 GA(2.5ppm) 2.5 5.0 100


80 IBA(12.5ppm) 12.5 GA(2.5ppm) 2.5 5.0 100

2.3 CULTIVATION OF THE MICROALGAE

2.3.1 Preparation of basal growth media

The basal growth media for the two species of microalgae were prepared

according to Ogbonna et al., 1997, by dissolving (g/l) Urea, 1.2; KH2PO4, 0.3;

MgSO4.7H2O, 0.3; CaCl2, 0.02; Sodium citrate, 0.05; Fe-solution, 0.16 ml; and A5

solution, 0.8 ml for Chlorella sorokiniana and NaNO3, 5.0; NaHCO3, 13.6; K2SO4,

1.0; NaCl, 1.0; MgSO4.7H2O, 0.2; CaCl2.H2O, 0.04; FeSO4.7H2O, 0.01; EDTA-Na2,

0.08; K2HPO4, 0.5; Na2CO3, 7.6; A5-solution, 1.0 ml and distilled water, 999.0 ml for

Spirulina platensis. The Fe-solution was composed of 25 g FeSO4.7H2O and 33.5 g

EDTA per litre of distilled water. A5-solution was composed of 2.86 g H3BO3, 1.81 g

xxxiii

MnCl2.4H2O, 0.22 g ZnSO4.7H2O, 0.08 g CuSO4.7H2O and 0.015 g MoO4 per litre of

distilled water. The media were autoclaved at 121oC for 15 minutes and allowed to

cool before adding appropriate volumes of the phytohormone stock solutions.

2.3.2 Effects of phytohormones on productivity of microalgae

Five (500 ml) Erlenmeyer flasks containing 300 ml the of basal growth

medium supplemented with various concentrations of phytohormones were inoculated

with 5.00x108 cells/ml each of the test organisms and incubated for 8 days in a growth

chamber illuminated by six-12 watts energy-saving bulbs fixed on two parallel

rectangular wooden boxes. Ten millitre of the culture broth was aseptically drawn on

48 hourly bases for assay.

2.4 Determination of cell number

The cell concentration of Chlorella sorokiniana was measured on 48 hourly

bases by counting the cell number, of the using microscope and Neubaur counting

chamber.

2.5 Determination of cell size

Cell sizes of the test organism were measured using a micrometer rule fixed on a

microscope. The determination of cell size was done using replicate samples.

2.6 Determination of dry Cell weight

These were made using triplicate samples of the culture. A 10ml of algal

culture was filtered through a preweighed Whatman filter paper after centrifuging at

3000rpm for 15min to concentrate the cells and remove some quantity of water. The

filter paper was washed with 5ml dilute 0.1N HCL to remove the precipitated salts

and dried overnight at 800C in an oven. Dried filter paper with biomass was cooled

and weighted again to estimate the final dry weight of the algae (Ogbonna et al.,

1997).

xxxiv

2.7 Determination chlorophyll contents

A 10 ml of algal culture broth was centrifuged at 3000rpm for 20min, and the

algal pellet, extracted with 4 ml of methanol (95%). The amount of chlorophyll

extracted in the methanol was determined spectrophotometrically according to the

method described by Ogbonna et al., 1997, using the following equation:

Chlorophyll (mgml-1) = 25.5 (A650-A 750) + 4.0 (A 665 – A 750).Where A650, A665 and

A750 are absorbance at 650 nm, 665 nm, and 750 nm respectively.

2.8 Determination of protein content of the cells

The protein content was determined using 0.2g of dry algal sample to

estimate the nitrogen content of the biomass. Measured percentage values of nitrogen

were multiplied with the nitrogen – to – protein conversion factor of 6.25.

2.8.1 Estimation of the percentage nitrogen of the biomass.

A 0.2g weight of the each microalga was added into a clean and dry digestion flask

(kjedhal flask). Selenium powder (0.05g), copper sulphate (0.5g) and sodium sulphate

(2g) were added. This was followed by the addition of 20 ml of conc. H2S04. The

solution was swirled until it darkened and then heated in a fume cabinet until it

became clear. The digested sample was diluted to 100 ml with distilled water and 5ml

taken for distillation. A 10 ml of 50% Sodium hydroxide was added to 5 ml of the

sample in a Markham apparatus and the solution allowed to distil over 10 ml of boric

acid mixed indicator until the indicator turned light green. 50ml of the distillate was

titrated against 25 ml of 0.01N HCl until the first pink appearance occurs. The

percentage nitrogen was calculated using the formula:

%Nitrogen = DF x M x Tv x Mwt of Nitrogen x100 Weight of sample x 1000mg

Where: DF = Dilution factor M = Molarity of the acid used

Tv = Titre value Mwt = Molecular weight of Nitrogen

xxxv

CHAPTER THREE

RESULTS

3.1 Effect of phytohormones on Chlorela sorokiniana cell number.

The result of the effect of different concentrations of phytohormones on cell

number of Chlorella sorokiniana after 8 days of cultivation is shown in Fig 1.

Amongst the phytohormones (IAA, IBA, GA3 and Kinetin), IAA at a concentration of

15ppm gave the highest cell number with an average value of 7.83x109 cells/ml. This

compares with the control (without phytohormone) which had an average value of

2.43x109cells/ml. The effectiveness of the phytohormones on the cell number of

Chlorella sorokiniana can be ranked as IAA (7.83x109 cells/ml) > IBA (4.36 x109) >

Kinetin (2.27 x109 cells/ml) > GA3 (2.19 x109 cells/ml) and the effectiveness of

various concentrations was ranked as 15ppm > (10ppm=20ppm) > 5ppm.

xxxvii

C- control; IAA- Indoleacetic acid; IBA- Indolebutyric acid; GA3 – Gibberellic acid.

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

6.00E+09

7.00E+09

8.00E+09

9.00E+09

Control 5ppm 10ppm 15ppm 20ppm 5ppm 10ppm 15ppm 20ppm 5ppm 10ppm 15ppm 20ppm 5ppm 10ppm 15ppm 20ppm

C GA3 GA3 GA3 GA3 IAA IAA IAA IAA IBA IBA IBA IBA Kinetin Kinetin Kinetin Kinetin

Ce

ll n

um

be

r(ce

lls/m

L)

Day 8

Fig 1 :Effect of phytohormones on cell number of Chlorella sorokiniana after

8 days of cultivation

C…

xxxviii

3.1.2 Effect of phytohormones on cell size of Chlorella sorokiniana.

The result of the effect of different concentrations of phytohormones on cell size of

Chlorella sorokiniana after 8 days of cultivation is shown in Fig 2. The optimum

concentration of phytohormones for Chlorella sorokiniana cell enlargement was

20ppm for each of the phytohormones (GA3, Kinetin, IAA, and IBA). At this

concentration, the average values of the cell sizes were 81.07 µm, 78.67 µm, 78.07

µm, and 66.90 µm for GA3, Kinetin, IAA, and IBA, respectively. This compares with

the control (without phytohormone) which had an average value of 64.43 µm. The

effectiveness of the phytohormones in increasing the size of the cells can be ranked as

GA3 > Kinetin = IAA >IBA.

xxxix

C- Control; IAA- Indoleacetic acid; IBA- Indolebutyric acid; GA3 – Gibberellic acid.

0

10

20

30

40

50

60

70

80

90



Ce

ll s

ize

in

mic

rom

etr

e

Day 8

Fig 2 :Effects of phytohormones on cell size of Chlorella sorokiniana after 8

days of cultivation

C…

xl

3.1.3 Effect of phytohormones on dry weight of Chlorella sorokiniana and

Spirulina platensis after 8 days of cultivation

The effects of the phytohormones on dry weight of the two microalgae species

followed the same trend as the cell number with IAA at a concentration of 10ppm

giving the highest value of 4.825 g/l in Chlorella sorokiniana and 1.10 g/l in Spirulina

platensis as shown in figs 3 and 4. These compare with the control (without

phytohormone) which had an average value of 0.519 g/l for Chlorella and 0.574 g/l

for Spirulina platensis. The effectiveness of the phytohormones in increasing the dry

weight of the microalgae can be ranked as IAA (4.825 g/l) > IBA (1.664 g/l) >

Kinetin (0.621 g/l) > GA3 (0.471 g/l). The effectiveness of the concentrations of these

phytohormones can be ranked as 15ppm > (10ppm = 20ppm) > 5ppm (L.S.D =

0.2317). However, on Kinetin, highest effect on dry weight of Chlorella sorokiniana

was exhibited at 5ppm concentration with a value of 0.621 g/l. The time courses of

the growth of Chlorella in response to the phytohormones are shown in fig 5. As is

evidenced in fig 5, IAA did not have a higher impact in the first 3 days and recorded

an average value of 0.157 g/l in biomass production over the control 0.101 g/l during

that period. In contrast, IBA increased in biomass productivity in the first 3 days, but

maintained a gradual and steady increase thereafter resulting in an 8-day average of

1.664 g/l over the control 0.519 g/l. The same trend follows in Spirulina platensis as

shown in fig 6 with the effect of IAA being low until after the first six days.

xli

C- Control; IAA- Indoleacetic acid; IBA- Indolebutyric acid; GA3 – Gibberellic

acid.

0

1

2

3

4

5

6



Dry

we

igh

t (

g/L)

Day 8

Fig 3 : Effects of phytohormones on dry weight of Chlorella sorokiniana after

8 days of cultivation

C…

xlii

C- Control; GA3- Gibberellic acid; IAA- Indoleacetic acid.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control 5ppm 10ppm 15pmm 20pmm 5ppm 10ppm 15pmm 20pmm

C GA3 GA3 GA3 GA3 IAA IAA IAA IAA

Dry

we

igh

t (g

/L)

Day 8

Fig 4:Effects of different phytohormones on dry weight of Spirulina platensis

after 8 days cultivation

C…

xliii


acid.

Fig 5 :Effect of different phytohormones on dry weight of Chlorella sorokiniana at 15ppm

concentration

0.08 0.145 0.1940.356

0.469

0.08 0.157

1.462

2.256

4.684

0.08

0.633

1.1981.377

1.664

0.080.228

0.3710.473

0.584

0.08 0.162

0.3890.49 0.519

0

1

2

3

4

5

6

Zero Day Day 2 Day 4 Day 6 Day 8

Days

Dry

weig

ht

(g/L

)

GA3 15ppm

IAA 15ppm

IBA 15ppm

Kinetin 15ppm

C Control

xliv


0.08

0.146

0.301

0.52

0.831

0.080.117

0.228

0.335

1.046

0.08

0.13

0.252

0.409

0.607

0

0.2

0.4

0.6

0.8

1

1.2


Dry

weig

ht

(g/L

)

Days

Fig 6 :Effect of different phytohormones on dry weight of Spirulina platensis at 15ppm concentration

GA3 15pmm

IAA 15pmm

C control

xlv

3.1.4 Effect of phytohormones on chlorophyll contents of Chlorella sorokiniana

and Spirulina platensis after 8 days cultivation

The effects of the phytohormones on chlorophyll contents of Chlorella sorokiniana

followed the same trend as the dry weight with IAA at a concentration of 15ppm

giving the highest value of 594.09 mg/g in Chlorella sorokiniana as shown in fig 7.

This compares with the control (without phytohormone) which had an average value

of 154.0 mg/g. This represents about 4-fold increase in chlorophyll contents yield

over the control. The effectiveness of the phytohormones in increasing the chlorophyll

contents of the microalgae can be ranked as IAA (594.09 mg/g) > GA3 (253.7 mg/g) >

IBA (143 mg/g) > Kinetin (141mg/mg). The effectiveness of various concentrations

of these phytohormones in increasing the chlorophyll contents of Chlorella can be

ranked as 15ppm > (10ppm = 20ppm) > 5ppm (L.S.D = 0.1463). In Spirulina

platensis, GA3 at 15ppm concentration had the highest effect on chlorophyll content

as shown in fig 8 with an average value of 156.9 mg/g. This represents about 2-fold

increase in chlorophyll contents yield over the control (78.4 mg/g).The time courses

of phytohormones effects on chlorophyll contents of Chlorella sorokiniana in

response to the phytohormones is shown in fig 9. As is evidenced in fig 9, IAA did

have a higher effect and recorded an average value of 552.75 mg/g in chlorophyll

content over the control 129.6 mg/g. In contrast, kinetin increased in chlorophyll

content in the first 2 days, but declined on the day 4-6 and maintained a gradual and

steady increase thereafter resulting in an 8-day average of 140.85 mg/g below the

control (153.95 mg/g).

. In Spirulina platensis, as shown in fig 10, the effect of GA3 was all-time higher than

IAA even after the first six days.

xlvi


acid.

Fig 7. Effect of phytohormones on chlorophyll content of Chlorella sorokiniana after 8 days cultivation

0

100

200

300

400

500

600

700



Day 8

Ch

loro

ph

yll c

on

ten

t (

mg

/g)

xlvii


Fig 8. Effect of phytohormones on chlorophyll content of Spirulina platensis after 8 days cultivation.

0

20

40

60

80

100

120

140

160

180

Control 5ppm 10ppm 15ppm 20ppm 5ppm 10ppm 15ppm 20ppm


Day 8

Ch

loro

ph

yll

co

nte

nt

(mg

/g)

xlviii


acid

Fig 9 :Effect of different phytohormones on chlorophyll content of Chlorella sorokiniana at 15ppm

concentration

71.25

125.5

225.2 221.1238.6

71.25

254.8

378.25

552.75

594.2

71.25

149.61 155.8140.7 141.65

71.25

253.3 258.8

104.2

140.9

71.25

137.7

100

129.6153.95

0

100

200

300

400

500

600

700


Days

Ch

loro

ph

yll

co

nte

nt

(mg

/g)

GA3 15ppm

IAA 15ppm

IBA 15ppm

Kinetin 15ppm

C Control

xlix


Fig 10 :Effects of different phytohormones on chlorophyll content of Spirulina platensis at 15ppm

concentration

67.5

54.79

100.7 101

156.92

67.5

79.49

42.11

35.2429.25

67.5 67.8

78.57

95.3588.98

0

20

40

60

80

100

120

140

160

180


Days

Ch

loro

ph

yll c

on

ten

t (m

g/g

)

GA3 15ppm

IAA 15ppm

C Control

l

3.1.5 Effect of phytohormones on protein content of Chlorella sorokiniana and

Spirulina platensis after 8 days cultivation

The effect of phytohormones on protein content of Chlorella sorokiniana and

Spirulina platensis are shown in figs 11 and 12 respectively. Treatments of Chlorella

with (GA10ppm = GA20ppm), Kinetin15ppm, and (IBA15ppm = IAA15ppm) gave protein

contents of 46.64% 45.83% and 45.81% respectively. These compare with the control

which had 43.38% protein content after 8 days cultivation. The effectiveness of

phytohormones in increasing protein content of Chlorella sorokiniana can be ranked

as GA3 > Kinetin > (IBA =IAA). In the case of Spirulina platensis, treatments with

IAA15ppm, and (GA15ppm = IAA20ppm) resulted in biomass with 55.64% and 54.07%

protein respectively, against the control which had 51.18%. Data analysis using

Duncan multiple range tests, showed that in each phytohormone, only 15ppm gave

higher protein content in the two microalgae.

li


acid.

0

10

20

30

40

50

60



Pro

tein

co

nte

nt

(%)

day 8

Fig 11: Effect of Phytohormones on protein conent of Chlorella sorokiniana

after 8 days cultivation

lii


0

10

20

30

40

50

60

70

Control 5ppm 10ppm 15ppm 20ppm 5ppm 10ppm 15ppm 20ppm


Pro

tein

co

nte

nt

(%)

Day 8

Fig12:Effect of phytohormones on protein content of Spirulina platensis after

8 days cultivation

C…

liii

3.2.1 Effect of combined phytohormones on the dry cell weight of Chlorella

sorokiniana.

The effect of combinations of phytohormones on the dry cell weight of

Chlorella sorokiniana is shown in fig 13. The combination of phytohormones IBA

(12.5ppm) + GA (2.5ppm) exhibited the highest effect with an average dry cell weight of

3.364 g/l after 8 days of cultivation. This compares with the control (without

phytohormones) which recorded 0.394 g/l dry cell weight after 8 days of cultivation.

Treatments IBA (10ppm) + GA (5ppm) and IAA (12.5ppm) + Kinetin (2.5ppm) gave an 8 and 4

fold increase in dry cell weight respectively relative to the control. The effectiveness

of the combined phyothormones on dry cell weight can be ranked as IBA (12.5ppm) +

GA (2.5ppm) > IBA (10ppm) + GA (5ppm) > IBA (7.5ppm) + GA (7.5ppm) > IAA (12.5ppm) +

Kinetin (2.5ppm) > (IAA (12.5ppm) + GA (2.5ppm) = IBA (5ppm) +GA (10ppm)) > IAA (10ppm) +

GA (5ppm) > IAA (10ppm) + Kinetin (5ppm) > IAA (7.5ppm) + GA (7.5ppm) > (IAA (7.5ppm) +

Kinetin (7.5ppm) = IBA (2.5ppm) + GA (12.5ppm) = IAA (5ppm) + GA (10ppm)) > (IAA (2.5ppm) +

GA (12.5ppm) =IAA (5ppm) + Kinetin (10ppm)) > IAA (2.5ppm) +Kinetin (12.5ppm). L.S.D =

0.2174. Statistical analysis using Duncan’s multiple range tests showed that all the

phytohormones combinations gave significantly higher biomass concentrations than

the control, although, none of the combined phytohormones exhibited a higher effect

than single treatment of IAA at 10ppm concentration (fig 3).

liv

Fig 13. Effect of combined phytohormones on dry cell weight of Chlorella

sorokiniana after 8 days cultivation.

0

1

2

3

4

5

6

Co

ntr

ol

5p

pm

10

pp

m

5p

pm

10

pp

m

5p

pm

10

pp

m

5p

pm

10

pp

m

IAA

10

+G

A5

IAA

10

+K

inti

n5

IAA

12

.5+

GA

2.5

IAA

12

.5+

Kin

eti

n2

.5

IAA

2.5

+G

A1

2.5

IAA

2.5

+K

ine

tin

12

.5

IAA

5+

GA

10

IAA

5+

Kin

eti

n1

0

IAA

7.5

+G

A7

.5

IAA

7.5

+K

ine

tin

7.5

IBA

10

+G

A5

IBA

12

.5+

GA

2.5

IBA

2.5

+G

A1

2.5

IBA

5+

GA

10

IBA

7.5

+G

A7

.5

IAA IAA IBA IBAGA3GA3KinetinKinetin Day 8

Dry

we

igh

t (g

/L)

…

lv

3.2.2 Effect of combined phytohormones on the chlorophyll contents of Chlorella

sorokiniana.

The effect of combinations of phytohormones on the chlorophyll contents of

Chlorella sorokiniana is shown in fig 14. The chlorophyll contents of Chlorella

sorokiniana followed the same trend as the biomass productivity. IBA (12.5ppm) + GA

(2.5ppm) and IAA (7.5ppm) + kinetin (7.5ppm) treatments resulted in significantly higher

chlorophyll content than the control (p< 0.05). As in the case of cell biomass, none of

the combined phytohormones gave higher chlorophyll content than the single

treatment of IAA at 10ppm concentration (fig 3).

lvi

Fig 14: Effect of combined phytohormones on chlorophyll content of Chlorella sorokiniana after 8

days cultivation

0

100

200

300

400

500

600

Co

ntr

ol

5pp

m

10

pp

m

5pp

m

10

pp

m

5pp

m

10

pp

m

5pp

m

10

pp

m

IAA

10+

GA

5

IAA

10

+K

inetin

5

IAA

12

.5+

GA

2.5

IAA

12

.5+

Kin

etin

2.5

IAA

2.5

+G

A1

2.5

IAA

2.5

+K

ine

tin1

2.5

IAA

5+

GA

10

IAA

5+

Kin

etin

10

IAA

7.5

+G

A7.5

IAA

7.5

+K

inetin

7.5

IBA

10+

GA

5

IBA

12

.5+

GA

2.5

IBA

2.5

+G

A1

2.5

IBA

5+

GA

10

IBA

7.5

+G

A7.5

IAA IAA IBA IBA GA3 GA3 KinetinKinetin Day 8

Ch

loro

ph

yll c

on

ten

t (m

g/g

)

lvii

CHAPTER FOUR

DISCUSSION

The mercantile potential for microalgae represents a largely untapped resource. Many

biotechnological pathways are currenlty under consideration for effective utilization

of these potentials for cost-effective biomass production. The present study,

considered the effects of phytohormones on growth and productivity of Chlorella

sorokiniana and Spirulina platensis. Results obtained from the measurment of cell

size of Chlorella sorokiniana showed that all the treatments had marked increases in

cell size of the organism compared to the control (Fig 3). The best performing

phytohormone was GA3 at 20 ppm concentration. This is in line with the report of

Gonai et al., (2004). The harvesting of unicellular microalgae is an important cost

factor for established production processes with photoautotrophic microalgae in

conventional open ponds or photobioreactors due to the low densities of these cells.

The discovery of phytohormones and the optimum concentration that lead to increases

in cell size of Chlorella sorokiniana is very significant as it will lead to reduced cost

in downstream processing of the microalgal biomass.

In contrast to the above findings, there was a negative correlation between cell size

increment in Chlorella sorokiniana and biomass productivity in this study. GA3 had

the least effect in biomass productivity. The best performing phytohormone was IAA

(10ppm). Hu et al., (2010) reported that naphthalene acetic acid NAA (5ppm) had a 2.3 times

increase in biomass productivity over a 10 days cultivation period. In this study, IAA

(10ppm) had more than a 5 fold increase in biomass productivity over 8 days cultivation

period. A high density culture is the apparent prime focus of most microalgal

biotechnological processes. Desirous also is to achieve the highest product

lviii

concentration of the desired quality in the shortest possible time which will translate

into reduced cost in the downstream processing of the microalgal biomass.

Auxins are known to stimulate growth (thallus branching, rhizogenesis, polarization)

and development (induction of cell division, formation of reproductive structures),

(Tarakhovskaya et al., 2007). Higher biomass productivity exhibited by IAA

treatment could be due to longer exponential phase resulting in net increase in

biomass productivity over the 8 days cultivation period. Czerpak et al., (1999),

reported that auxins suppress oxidation and degeneration of chlorophylls and

carotenoids thus delaying senescence. Fig 5 shows that IAA did not have higher effect

in the first 3 days.

This could be as a result of a longer acclimation phase required by the algal cells.

In Spirulina platensis, the same trend was observed as in Chlorella sorokiniana.

Chlorophyll content yield followed the same trend as in biomass production; IAA

recorded the highest extractable chlorophyll content per gram of the biomass used.

0

0.5

1

1.5

2

2.5

3

3.5

4


Dry

weig

ht

(g/L

)

Days

Fig 5 :Effect of different phytohormones on dry weight of Chlorella sorokiniana at 5ppm concentration

GA3 5ppm

IAA 5ppm

IBA 5ppm

Kinetin 5ppm

C Control

lix

This is in agreement with Czerpak et al., (1999) who reported that IAA delays

senescence by suppressing degradation of chlorophyll and carotenoids. The discovery

of a phytohormone that simultaneously leads to higher biomass production and high

pigment production is a useful contribution to advancing biotechnological

applications of microalgae in cosmetic and food industries.

In contrast to the above, IAA did not exhibit the same effect in Spirulina platensis.

The differential response observed in this study, could be as a result of longer period

required by algal cells to acclimatize to IAA treatment. Moreover, in different species

of microalgae, the variety, content and activity of phytohormone receptor proteins

within cells differ (Li et al., 2007).

As shown in Fig 11 and Fig 12 for Chlorella sorokiniana and Spirulina platensis

respectively, the protein contents of these microalgae were not enhanced significantly,

relative to the control, by the phytohormones treatments. Hunt et al., (2010) reported

that increase in protein content, lipids, and carbohydrate in algae are generally

observed in algal cells in response to stress induced by temperature, depletion of

nutrients such as nitrogen and phosphorus from growth medium. In this study, all the

experiments were conducted in static batch cultures maintaining optimum growth

conditions.

The combination of phytohormones IBA (12.5 ppm) + GA (2.5 ppm) recorded the highest

biomass production (3.364 g/l) and showed about 8-fold increase over control (0.394

g/l) on day 8 (Fig 13). Combinations IBA (1O ppm) + GA (5 ppm), IBA (7.5 ppm) + GA (7.5

ppm) and IAA (12.5 ppm) +Kinetin (2.5 ppm), recorded 8-fold, 5-fold, and 4-fold increase

respectively over the control on day 8. In single treatments, IBA and GA3 did not

perform better than IAA at all the concentrations experimented on in this study. The

combination of IBA (12.5ppm) and GA (2.5ppm) exhibited synergistic effect on Chlorella

lx

sorokiniana in terms of biomass and chlorophyll productivity and protein yield.

Hence, the combinational phytohormones identified here that simultaneously lead to

higher biomass, chlorophyll and protein productivity is attractive for cosmetic and

pharmaceutical industries. Hu et al., (2010) reported that NAA in all combinations

with GA3, IBA, and zeatin showed only marginal increase in average productivity

between 0 – 5 days of cultivation over the control. Contrary to this finding, IAA (12.5

ppm) + kinetin (2.5 ppm) and IAA (12.5 ppm) + GA (2.5 ppm) showed about 3-fold and 2- fold

increase in average biomass productivity over the control between 0 – 5 days of

cultivation. This could be as a result of long exponential phase of IAA. However,

none of the IAA combinations with GA3 had above 3-fold increase in biomass

productivity over the control on day 8. This is in agreement with Bradley and Cheney

(1990) who suggested that auxins be combined with cytokines to enhance the growth

of cultured sea weeds. The combination of IBA (12.5ppm) + GA (2.5ppm) recorded 4-fold

increase in chlorophyll content yield (425.0 mg/g) over the control (107.97 mg/g) on

day 8 (Fig 14). The results in Fig 14, shows that IAA combinations did not result in

any significant synergistic effect over the entire growth period which in agreement

with Vance (1987) who combined three phytohormones, namely IAA, GA and kinetin

with Chlorella pyrenoidosa.

lxi

CONCLUSION

Experiments in this study were conducted in static batch cultures which indicated that

the phytohormones such as auxins, gibberelins, and cytokinins individually and in

combination stimulated microalgal growth and doubled the biomass productivity

compared to the untreated cells.

The discovery of a phytohormone (IAA) that simultaneously leads to higher biomass

production and high pigment production is a useful contribution to advancing

biotechnological applications of microalgae in cosmetic and food industries.

A high density culture is the apparent prime focus of most microalgal

biotechnological processes. Desirous also is to achieve the highest product

concentration of the desired quality in the shortest possible time which will translate

into reduced cost in the downstream processing of the microalgal biomass. This was

achieved in this study with IAA at 10 ppm concentration on day 8 of the cultivation

period.

The combinational phytohormones identified here (IBA (12.5ppm) + GA (2.5ppm) ) that

simultaneously lead to higher biomass, chlorophyll and protein productivity is

attractive for cosmetic and pharmaceutical industries

lxii

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APPENDICES

ANOVA for the effect of phytohormones on cell size of Chlorella sorokiniana

after 8 days cultivation Variate: cellsize Source of variation d.f. s.s. m.s. v.r. F pr. Phytohormone 3 742.04 247.35 10.73 <.001 Phtohormone conc 4 3226.60 806.65 35.01 <.001 Days 3 36041.50 12013.83 521.35 <.001 Residual 229 5277.00 23.04 Total 239 45287.15 Grand mean 54.22 Phytohormone GA3 IAA IBA kinetin 54.98 55.82 51.25 54.84 Phytoconc. 5ppm 10ppm 15ppm 20ppm control 50.32 53.43 57.54 59.35 50.47 Days 2 4 6 8 38.62 47.22 60.39 70.66 *** Least significant differences of means (5% level) *** Table Phytohor Phytoconc Days rep. 60 48 60 d.f. 229 229 229 l.s.d. 1.727 1.931 1.727

lxvii

ANOVA for the effect of phytohormones on dry cell weight of Chlorella

sorokiniana after 8 days cultivation

Variate: Dry cell weight Source of variation d.f. s.s. m.s. v.r. F pr. Phytohormone 3 60.3531 20.1177 48.90 <.001 Phytohormone conc 4 11.5004 2.8751 6.99 <.001 Days 3 43.0811 14.3604 34.91 <.001 Residual 229 94.2080 0.4114 Total 239 209.1427 ***** Tables of means ***** Variate: Dry cell weight Grand mean 0.791 Phytohormones GA3 IAA IBA Kinetin 0.294 1.572 0.884 0.416 Phytohormone conc 5ppm 10ppm 15ppm 20ppm control 0.742 0.915 1.016 0.894 0.390 Day 2 4 6 8 0.251 0.608 0.897 1.409 *** Least significant differences of means (5% level) *** Table Phytohormone Phyto conc Day rep. 60 48 60 d.f. 229 229 229 l.s.d. 0.2307 0.2580 0.2307

lxviii

ANOVA for the effect of phytohormones on chlorophyll content of Chlorella

sorokiniana after 8 days cultivation Variate: Chlorophyll content Source of variation d.f. s.s. m.s. v.r. F pr. Phytohormone 3 24.1712 8.0571 48.71 <.001 Phytohormone conc 4 8.9576 2.2394 13.54 <.001 Days 3 30.7155 10.2385 61.90 <.001 Residual 229 37.8775 0.1654 Total 239 101.7218 Variate: Chlorophyll content Grand mean 0.784 Phytohormone GA3 IAA IBA Kinetin 0.573 0.900 1.244 0.418 Phytohormone conc 5ppm 10ppm 15ppm 20ppm control 0.654 0.880 1.053 0.839 0.493 Days 2 4 6 8 0.330 0.667 0.815 1.323 *** Least significant differences of means (5% level) *** Table Phytohormone Phytohormone conc Day rep. 60 48 60 d.f. 229 229 229 l.s.d. 0.1463 0.1636 0.1463

lxix

ANOVA for the effect of phytohormones on protein content of Chlorella

sorokiniana after 8 days cultivation Variate: Portein content Source of variation d.f. s.s. m.s. v.r. F pr. Phytohormone 3 411.08 137.03 11.54 <.001 Phytohormone conc 4 85.65 21.41 1.80 0.129 Days 3 558.74 186.25 15.69 <.001 Residual 229 2718.27 11.87 Total 239 3773.74 ***** Tables of means ***** Variate: Protein content Grand mean 43.93 Phytohormone GA3 IAA IBA Kinetin 45.68 42.57 42.77 44.71 Phytohormone conc 5ppm 10ppm 15ppm 20ppm control 43.79 42.98 44.85 43.99 44.04 Day 2 4 6 8 41.29 44.94 44.85 44.63 *** Least significant differences of means (5% level) *** Table Phytohormone Phytohormone conc Day rep. 60 48 60 d.f. 229 229 229 l.s.d. 1.239 1.386 1.239

lxx

ANOVA for the effect of phytohormones on protein content of

Chlorella sorokiniana after 8 days cultivation Variate: Cell number Source of variation d.f. s.s. m.s. v.r. F pr. C1 3 243.737 81.246 32.58 <.001 C2 4 116.197 29.049 11.65 <.001 C3 3 46.800 15.600 6.26 <.001 Residual 229 571.098 2.494 Total 239 977.833 ***** Tables of means ***** Variate: C8 Grand mean 3.18 C1 1 2 3 4 2.58 4.80 3.22 2.14 C2 1 2 3 4 5 3.20 3.39 3.77 3.70 1.86 C3 1 2 3 4 3.54 2.52 3.05 3.62 *** Standard errors of differences of means *** Table C1 C2 C3 rep. 60 48 60 d.f. 229 229 229 s.e.d. 0.288 0.322 0.288 *** Least significant differences of means (5% level) *** Table C1 C2 C3 rep. 60 48 60 d.f. 229 229 229 l.s.d. 0.568 0.635 0.568

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