Faculty of Resource Science and Technology

Improvement of NGP medium for mass culture of vulgaris

Cassy Cassandra anak Mitaha (18141)

Bachelor of Science with Honours (Aquatic Resource Science and Management) 2010

Improvements of NGP medium for mass culture of

Cassy Cassandra anak Mitaha

A final report submitted in partial fulfillment of the Final Year Project STF 3014

Supervisor: Ass. Prof Dr. Norhadi Ismail

Aquatic Resource Science and Management Department of Aquatic Science

Faculty of Resource Science and Technology Universiti Malaysia Sarawak 3 May 2010

DECLARATION

I hereby declare that no portion of the work referred to in this dissertation has been submitted in support of an application for another degree or qualification to this university or any other institution of higher learning.

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Cassy Cassandra anak Mitaha

Aquatic Resource Science and Management

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ACKNOWLEDGEMENTS

First of all, I would like to thank God for His guidance and His strength, as well as time and wisdom that has given to me throughout all the way from the beginning until the final step in doing this Final Year Project Thesis. I am grateful to Him who has giving me the opportunity to learn and discover many challenges in different aspects, and taking this challenges in a positive manner.

I would like to express my deepest gratitude and appreciation to my supervisor, Dr.

Norhadi Ismail for his constant guidance, advice, patience and support throughout completing my thesis. The knowledge he imparted upon me, his generous assistance and concerns that contributed a lot in completing this study. Thanks also to Dr. Ruhana Hassan for her guidance and advices in writing this thesis since the beginning.

Special thanks dedicated to Ms. Iqliema Afdalia, postgraduate student in Aquatic

Botany Laboratory, who had given valuable technical advices and helped during laboratory work as well as their assistance. I also would like extend my sincere thanks to laboratory assistant, Mr. Zaidi Ibrahim and Mr. Mohd Norazlan Bujang for their cooperation and assistant.

On the other hand, I also would like to thank my family especially my parents, Mr.

Mitaha Mapang and Mdm. Selera Jiki as well as my beloved siblings, Goretty Mika, Gregory

Rapin, Geraint Jenggi and Gwendoline Nilie for their moral support, patience and encouragement for me to complete my final year project. I also would like to thank my fellow classmates, housemates and friends for their continous encouragement and support. Without their support, assistance and advices, this project would never have been completed.

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TABLE OF CONTENT

Acknowledgements I

Table of Content II

List of Abbreviations IV

List of Tables and Figures V

Abstract………………………………………………………………… 1

1.0 Introduction and Objectives………………………………………….. 2

2.0 Literature Review

2.1 Potential of as production……………………... 4

2.2 Factors affecting growth rate of algae……………………………... 7

2.3 Mixotrophic and heterotrophic algae………………………………. 8

2.4 Batch culture……………………………………………………….. 10

3.0 Materials and Methods

3.1 Laboratory work

3.1.1 Establishment of stock culture of Chlorella vulgaris…….. 11

3.1.2 Improvement of NGP medium………………………….... 11

3.2 Outdoor mass culture………………………………………………. 12

3.2.1 Growth measurement…………………………………….. 13

3.3 Harvesting of algae ………………………………………………… 14

3.4 extraction…………………………………………...... 14

4.0 Result

4.1 Improvement of NGP medium for subcultures of Chlorella vulgaris…………………………………………………. 15

II

4.2 Chlorella vulgaris mass culture……………………………………. 19

4.3 Lipid content analysis……………………………………………… 20

5.0 Discussion

5.1 Factors affecting growth of Chlorella vulgaris……………………. 21

5.2 Chlorella vulgaris mass culture under outdoor conditions…………………………………………………………. 24

5.3 Lipid content analysis of Chlorella vulgaris………………………. 26

6.0 Conclusion……………………………………………………………... 27

7.0 References……………………………………………………………… 28

8.0 Appendix

Appendix 1…………………………………………………………… 33

Appendix 2…………………………………………………………… 34

Appendix 3…………………………………………………………… 35

Appendix 4…………………………………………………………… 36

Appendix 5…………………………………………………………… 37

Appendix 6…………………………………………………………… 38

Appendix 7…………………………………………………………… 39

III

LIST OF ABBREVIATIONS

NGP Norhadi Growth Promoter

N Nitrogen

P Phosphate

K Potassium

L Liter g Gram ml Milliliter

CO2 Carbon dioxide gas

EOM Extracellular organic matter oC Degree Celcius

µm Micrometer

R & D Research and Development m Metres km Kilometers

A, B, C, D, E Treatment for algae that have different concentration of salt and Organisol

EU European Union

USA United States of America ha Hectares

SD Standard deviation wt Weight

Vt Volt

IV

LIST OF TABLES

Table 1 : The oil content of some microalgae those are potential in producing biofuel in future. (Extracted from Christi, 2007)……. 6

Table 2 : The combinations of fertilizer and growth promoter per 1 L of improved NGP medium…………………………………………. 12

Table 3 : Growth rate, K’ of Chlorella vulgaris in subcultures under different concentration of fertilizer and Organisol (growth 17 promoter).………………………………………………………...

Table 4 : The pH value of different concentration of fertilizer and Organisol before adding Chlorella vulgaris ……………………. 17

Table 5 : Summary of number of cells of Chlorella vulgaris under different concentration of fertilizer and growth promoter..……... 18

Table 6 : Summary on duration time (s) obtained by using flocculation harvesting technique at different concentration of Chitosan and 20 voltage (Volt)...…………………………………………………..

V

LIST OF FIGURES

Figure 1 : The growth curves of Chlorella vulgaris in different concentration of fertilizer and Organisol ………………………...... 16

Figure 2 : The growth curves of Chlorella vulgaris in mass culture under outdoor conditions.…………………………………………………. 19

Figure 3 : Percentage weight of dry algae obtained from different concentration of Chitosan and voltage (Volt). (Total biomass = 36).. 20

VI

Improvement of NGP medium for mass culture of Chlorella vulgaris

Cassy Cassandra anak Mitaha

Aquatic Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Chlorella vulgaris is a single-celled of green algae that are spherical in shape and do not have flagella, which can be found in freshwater. It is fast-growing green algae and has different lipid production capabilities (30–40% of dry weight) under natural conditions. In the present study, the microalgae were grown in NGP medium. It was tested to grow in different combinations amount of fertilizer and Organisol (growth promoter) as an improvement to the original NGP medium. Results showed that C. vulgaris had the highest growth rate when the NGP supplied with combination of 0.13ml L-1 growth promoter and 2.0g L-1 fertilizer. Using this nutrient combination, the algal was mass cultured outdoor in aquariums. The lipid yield produced after 26 days of cultivation was about 2.17%.

Keyword: Chlorella vulgaris, out-door conditions, NGP medium, mass culture, lipid extraction

ABSTRAK

Chlorella vulgaris ialah alga hijau yang mempunyai sel tunggal berbentuk seperti sfera dan tidak mempunyai flagella, serta boleh ditemui di air tawar. Alga ini sangat cepat bercambah dan mempunyai berbeza keupayaan menghasilkan minyak (30-40% daripada berat alga kering) dalam keadaan semulajadi. Berdasarkan kajian ini, mikroalga telah bertumbuh di dalam NGP media. Ia telah dikaji untuk bertumbuh di dalam kombinasi baja dan Organisol (penggalak tumbesaran) yang berbeza sebagai penambahbaikkan daripada NGP media yang asal. Hasil kajian menunjukkan C. vulgaris mempunyai kadar tumbesaran yang tertinggi apabila NGP disediakan dengan kombinasi 0.13ml L-1 penggalak tumbesaran dan 2.0g L-1 baja. Dengan menggunakan kombinasi nutrisi ini, alga telah dikultur di persekitaran luar secara besar-besaran di dalam akuarium. Hasil minyak yang dihasilkan selepas 26 hari pengkulturan ialah 2.17%.

Kata kunci: Chlorella vulgaris, persekitaran luar, NGP media, jisim kultur, rentapan minyak

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1.0 INTRODUCTION AND OBJECTIVES

Chlorella vulgaris is unicellular green algae that can be found in marine ecosystem, freshwater and terrestrial area. C. vulgaris (See Appendix 1) is a single-celled of green algae that are spherical in shape and do not have flagella. Their sizes are about 2-10μm in diameter and contain photosynthetic pigments including chlorophyll a and b. This algae is taxonomically placed under: Kingdom Protista, Division , Order and

Family .

Many people believed that Chlorella vulgaris could serve as a potential source of food and energy because of its photosynthetic efficiency compare to other crops such as sugar cane. It is also an attractive food source because it is high in essential nutrients such as , lipid, and .

Chlorella sp. has been the oldest commercial application of microalgae. Chlorella vulgaris is fast-growing green algae and has different lipid production capabilities (30–40% of dry weight) under natural conditions. Its heterotrophic growth mode in the presence of glucose or acetate has also been studied in the 1960s and 1970s (Pratt & Johnson, 1963;

Nichols et al., 1967; Harris & James, 1969; Podojil et al., 1978; Liang et al., 2009). C. vulgaris can be cultivated on acetate in the dark and in the light with acetate being directly converted to fatty acids (Nichols et al., 1967; Harris & James, 1969; Liang et al., 2009).

Based on study by Liang et al. (2009), C. vulgaris has the potential to reach the highest lipid productivity in large scale if shallow surface or enough light penetration is provided.

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Microalgae represented a renewable resource that can be used as a fuel and preliminary results indicated that a dried algal powder could be introduced into a diesel engine. The work is in progress to increase the algae biomass concentration and to produce emulsions with other liquid fuels (Scragg et al., 2003). Further study is also required to optimize the process in algal production as a sustainable environmental management practice

(Siranee & Pakpainb, 2007).

Despite the economic potentials showed by Chlorella vulgaris, there are limited numbers of studies done in Malaysia. In University Malaysia Sarawak (UNIMAS), the preliminary studies were carried out by Muniandy (2009) and Soojin (2009) on lipid contents of this microalga. While for this study, C. vulgaris was cultured in Norhadi Growth Promoter

(NGP) medium. The main ingredients of NGP medium were commercial fertilizer and

Organisol (growth promoter). This medium were prepared in laboratory and mass culture was conducted under out-door conditions.

The main objectives of this research are:

(i). To improve the recently developed NGP medium for mass culture of Chlorella vulgaris.

(ii). To determine the growth rate of C. vulgaris under out-door conditions.

(iii). To determine lipid yield extracted from C. vulgaris under out-door conditions.

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2.0 LITERATURE REVIEW

2.1 Potential of microalgae as biofuel production

Microalgae can exist as unicells, colonies and extended filaments that contain photosynthetic pigments. They are ubiquitously distributed throughout the biosphere and grow under the widest possible variety of conditions. They also can be cultivated under aqueous conditions ranging from freshwater up to extreme salinity. Microalgae have been found living in clouds and well known as essential components of coral reefs. This wide span of ecological requirements plays a significant role in determining the range of metabolic products they produce (Satin, 2007).

Recently, microalgae have been claimed to be a good source of that has potential to completely displace fossil diesel. Unlike other oil crops, microalgae grow extremely rapidly and many are exceedingly rich in oil. Microalgae commonly double their biomass within 24 hours. Oil content in microalgae can exceed 80% by weight of dry biomass

(Metting, 1996; Spolaore et al., 2006; Cristi, 2007).

Microalgae have much faster growth-rates than terrestrial crops. The yield of oil from algae is estimated to be between 5000 and 15.000 m3/km2/y which is around 8 to 25 times greater than the next best crop, palm oil (Kurevija & Kukulj, 2004). It can be harvested using micro screens, centrifugation or by flocculation methods.

Nowadays, many researches have been done by scientists to explore on microbial oils, which might become one of potential oil sources for biofuel production in the future.

Microbial oils are produced by some oleaginous microorganisms, such as yeast, fungi, bacteria, and microalgae (Ma et al., 2006; Li et al., 2008). It has been demonstrated that such

4 microbial oils can be used as feedstocks for biodiesel production and they are much better than other vegetable oils and animal fats.

The need for renewable liquid fuels to replace or supplement diesel is certainly required in both short term and long-term. The microalgae represented a renewable resource, which can be used as a fuel supplement in a stationary diesel engine used for the generation of electricity. The preliminary results had indicated that a dried algal powder could be introduced into a diesel engine (Scragg et al., 2003).

The production of microbial oil has many advantages due to their short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up (Li &

Wang, 1997; Li et al., 2008). Therefore, there are many works associated with microorganism-producing oils have been carried out to further research and prospects of such microbial oils used for biodiesel production are also discussed (Li et al., 2008).

Besides that, the biodiesel production currently uses around 1.4 million hectares of arable land in the EU and today there are approximately 40 plants in the EU producing up to

3,184,000 tonnes of biodiesel annually. These plants are mainly located in Germany, Italy,

Austria, France and Sweden. In the USA, the most common crop for producing biodiesel is soy while in East Asia (Malaysia and Indonesia) biodiesel is mainly produced from crude palm oil (Kurevija & Kukulj, 2004)

The usefulness of Chlorella as an experimental organism for photosynthesis has led to its selection for exploratory work on the problem of algal mass culture. It is a hardy and rapidly growing form, an algal weed. Its chloroplast takes up a large fraction of the cell, and

5 its very high rate of photosynthesis exceeds its rate of respiration by a factor of 10 to 100 times.

Apart from that, microalgae also can grow photosynthetically so that no carbon source is required for growth. Any carbon dioxide released on combustion will have been previously fixed, so that the energy supply will be carbon dioxide neutral (Scragg et al., 2003). In addition, microalgae also have fast proliferation rates, wide tolerance to extreme environments, potential for intensive cultures and lesser land area requirement. Once the are extracted from the harvest algae, potential use for the microalgae residue include fodder for livestock, food and chemicals, colorants, perfumes, and , which leads to greater economically feasibility of the project (Kurevija & Kukulj, 2004).

The advantages of biological sources of energy are they are renewable, biodegradable, produce fewer emissions and do not contribute to the increase in carbon dioxide in the atmosphere (Cook & Beyea, 2000; Scragg et al., 2003). The oil productivity from microalgae can contribute to greater production compare to other oil crops that applied for biodiesel producing before. For example, microalgae contributed to 136900 L/ha of oil yield compare to soybean, canola, jatropha and oil palm (Christi, 2007; Rao, 2008).

Table 1: The oil content of some microalgae those are potential in producing biofuel in future (Adopted from Christi, 2007).

Scientific name Oil content (% dry wt)

Botryococcus braunii 25–75

Chlorella sp. 28–32

Crypthecodinium cohnii 20

Cylindrotheca sp. 16–37

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Dunaliella primolecta 23

Isochrysis sp. 25–33

Monallanthus salina 20

Nannochloris sp. 20–35

Nannochloropsis sp. 31–68

Neochloris oleoabundans 35–54

Nitzschia sp. 45–47

Phaeodactylum tricornutum 20–30

2.2 Factors affecting growth rate of algae

Temperature and light intensity are factors that can affect the growth rate of algae in culture system. According to study done by Babel et al. (2001), the algae could grow well in optimum temperature between 28oC – 35oC in laboratory culture. The algae growth rate was inhibited below and above the optimum temperature or strong solar radiation. While under the positive temperature, the cells grow rapidly and have high negative charge which is difficult to neutralize. Chlorella sp. is negatively charged and causes the low resistance (Burlew,

1964).

Under out-door cultures, Chlorella sp. was difficult to grow in glass bottles without a cover net due to extremely high radiation and temperature. The high radiation and temperature inhibited the growth of algae, and then lead to high extracellular organic matter

(EOM) attachment on cells of Chlorella sp. in natural environment (Babel et al., 2001).

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Hoek et al. (1995), indicated the cell wall of Chlorella sp. consists of sporopollenin-like structure.

Optimum temperatures of algae are generally between 28oC and 35oC, but the thermophilic strains of Chlorella sp. grow best at about 40oC. However, the optimum temperatures may vary with light intensity and concentration of certain nutrients. According to Fogg & Thake (1987), the difference between low and high temperature strains are depended on largely on the differential effect of temperature changes on photosynthesis and respiration.

2.3 Mixotrophic and heterotrophic algae

The algae can be defined as mixotrophics because of their dual modes of nutrition and phototrophy. A mixotrophic culture might be used as an alternative way to conventional photoautotrophic mass culture system production of algae. The possibility of using mixotrophic culture to achieve high cell densities was investigated using fed-batch culture in a

3.7-1 fermentor (Chen & Zhang, 1997).

According to Chen (1996), heterotrophic culture may provide a cost-effective and large-scale alternative method of cultivation for some microalgae to utilize organic carbon substances. Microalgae required light energy to undergo photosynthesis and support their growth. The uses of algae have been recognized for decades and mainly produced as nutrient supplements, agriculture and aquaculture. Due to their high value of chemical and pharmaceutical compounds, the heterotrophic culture may be used for future commercial production of microalgae products as well as biofuel to replace other energy sources.

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According to Burlew (1976), the systematic research into photoautotrophic mass culture of microalgae has begun in the early 1950s in Washington, USA. In heterotrophic cultures, the optimal growth and productions conditions can be easily maintained. The contaminations or predation organisms also can be eliminated by sterilization of the medium and aseptic operation (Chen, 1996).

The mode of growth eliminates the requirement for the light and increased the cell concentration of algae as well as productivity on large scale. In the late 1970s in Japan and

Taiwan, two Chlorella sp. were cultivated heterotrophically in stainless steel tanks by using glucose and acetic acid as carbon and energy sources (Kawaguchi, 1980; Chen, 1996).

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2.4 Batch culture

Batch culture is one of the simplest forms of culture where the initial lag phase is minimal growth. Typically, the experience shows that the lag is short if the inoculum is large and the parent culture has been acclimated to similar conditions. If the concentrations of nutrients are high and the incidents irradiance relatively low, there is also the potential for the culture to shade itself and become light limited as cell density becomes very high (Macintyre

& Cullen, 2005).

It is very difficult to define a period of acclimated growth in batch cultures because the progressive accumulation of biomass changes the availability of nutrients and light. The reduction in mean irradiance available to the cells as the culture starts to shade itself causes a response of increased pigmentation. Batch cultures covered multiple growth phases and physiological state representative of nutrient-replete growth at the experimental irradiance

(Macintyre & Cullen, 2005).

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3.0 MATERIALS AND METHODS

3.1 Laboratory work

3.1.1 Establishment of stock culture of Chlorella vulgaris

The original stock of Chlorella vulgaris was obtained from National Institute of

Environmental Studies in Japan and maintained in Zarrouk medium in Aquatic Botany

Laboratory. At first, the stock culture was a mixing of diluted 2ml growth promoter, 2 teaspoons of fertilizer and 200ml of C. vulgaris. The stock culture was later subcultured in

NGP medium containing fertilizer Kwik Bloom 67Q (N:P:K = 18:36:18) and Organisol

(growth promoter). This stock culture was observed and the algal cells counted to determine any growth in the medium.

3.1.2 Improvement of NGP medium

The improvement of the NGP medium was carried out by manipulation of the amount of fertilizer and Organisol (See Appendix 2) in the medium. Each treatment had different combinations of fertilizer and Organisol, while the control consisted only 0.25ml of Organisol and without fertilizer addition (See Table 2). The combinations of fertilizer and Organisol

(growth promoter) added in NGP medium are as following:

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Table 2: The combinations of fertilizer and Organisol (growth promoter) per 1 L of improved NGP medium.

TREATMENTS

Control A B C D E

ORGANISOL (ml) 0.25 0.25 0.25 0.5 0.3 0.13 FERTILIZER (g) 0 2 4 2 2 2

50ml of Chlorella vulgaris stock culture was later added into the medium of each treatment. Then, the cultures were placed on the rack under 12L: 12D light conditions. The light intensity used was 21Klux (Digital Lightmeter, TECPEL 530).

The density (cells/ml) of Chlorella vulgaris in each treatment was determined using a haemacytometer. Treatment that produced the best growth rate has been chosen for mass culture in aquarium.

3.2 Outdoor mass culture

Chlorella vulgaris was further cultured in large volume of 20 L of culture in aquariums. There were 4 replicates of aquariums (See Appendix 3) which contained culture of

C. vulgaris by using best treatment which was Treatment E (0.13ml L-1 Organisol and 2.0g L-

1 fertilizer) from previous experiment as medium. Each of aquariums was seeded with 350ml of stock culture of C. vulgaris.

The initial pH has been chosen from best treatment (Treatment E). The pH 5.53 was standardized for all aquariums. Each of the aquariums was provided with continuously

12 aeration that supplied by Hi-Blow Diaphragm Air Pump (Model HAP-60). Natural light intensity at the corridor was 55 Klux and temperature was 26.5oC.

3.2.1 Growth measurement

Algal cell densities in the aquariums were determined every two days by using a haemacytometer. The growth of cells Chlorella vulgaris was observed under compound microscope. Before pipette out the culture from conical flasks, Bunsen burner was switch on to avoid the contamination. A drop of well mixed algae sample was filling both chambers of haemacytometer by using pasteur pipette. The observation, growth pattern and data collected have been recorded. The cell density of C. vulgaris culture also determined. The number of cells in 1 ml of culture was obtained by using the formula given:

If all the cells in individual blocks are counted:

N1 and N2 = cell density at time 1 (t1) and time 2 (t2) respectively.

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3.3 Harvesting of algae

Chlorella vulgaris cultured in the aquariums were harvested on the 4th week. The algae were harvested by using flocculation method where Chitosan was used as the electrolytes. The algae were harvested by using different concentration of Chitosan (1.0g,

1.5g, 2.0g, 2.5g and 3.0g) with different level of voltage (2Vt, 4Vt, 6Vt, 8Vt, 10Vt). Then, algal biomass collected was dried in the oven at temperature about 60oC for three days.

3.4 Lipid extraction analysis

The extraction of the lipid was conducted by using Soxhlet method using n-hexane.

Lipid extraction was conducted for 24 hours. After that, the lipid together with n-hexane was placed in rotation vapour (50% rotation) until lipid was completely separated from solvents.

The lipid was further dried in the oven at temperature of 50oC for 6 days. The lipid yield produced at the end of experiment was determined by the formula as following:

m

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4.0 RESULTS

4.1 Improvement of NGP medium studies in the laboratory

The growth curve of Chlorella vulgaris in the laboratory under different treatments is

n n F . D n , m (K’) n treatment E (see Table 3). Therefore, treatment E was used for mass outdoor cultivation of this algae.

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