Improvement of NGP medium for mass culture of Chlorella vulgaris

Cassy Cassandra anak Mitaha (18141)

Bachelor of Science with Honours

(Aquatic Resource Science and Management)

2010

Faculty of Resource Science and Technology

Improvements of NGP medium for mass culture of Chlorella vulgaris

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.

________________________________________

Cassy Cassandra anak Mitaha

Aquatic Resource Science and Management

Department of Aquatic Science

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

I

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.

II

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 microalgae as biofuel 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 Lipid extraction………………………………………….................. 14

4.0 Result

4.1 Improvement of NGP medium for subcultures of

Chlorella vulgaris………………………………………………….

15

III

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

IV

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

V

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

promoter).………………………………………………………...

17

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

voltage (Volt)...…………………………………………………..

20

VI

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

1

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

2

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 Chlorophyta, Order Chlorellales and

Family Chlorellaceae.

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

protein, lipid, carbohydrates and vitamin.

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.

3

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.

4

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 biodiesel 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

5

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

6

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

lipids are extracted from the harvest algae, potential use for the microalgae residue include

fodder for livestock, food and chemicals, colorants, perfumes, and vitamins, 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

7

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 – 35

oC 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).

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

8

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 35

oC, 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.

9

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).

10

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).

11

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:

12

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

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

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

13

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.

14

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

15

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.