建構微藻光生物反應系統並用於二氧化碳減量與微藻生物質的生產

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國 立 交 通 大 學

生 物 科 技 學 院

生 物 科 技 學 系

博士論文

建構微藻光生物反應系統並用於二氧化碳減量與微藻生物

質的生產

Establishing a Microalgae-incorporated Photobioreactor

System for CO

2

Reduction and Microalgal Biomass

Production

研 究 生: 邱聖壹

指導教授: 林志生 博士

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建構微藻光生物反應系統並用於二氧化碳減量與微藻生物質的生產

Establishing a Microalgae-incorporated Photobioreactor System for CO

2

Reduction and Microalgal Biomass Production

研 究 生:邱聖壹

Student:Sheng-Yi Chiu

指導教授:林志生

Advisor:Chih-Sheng Lin Ph.D

國 立 交 通 大 學

生 物 科 技 學 院

生 物 科 技 學 系

博 士 論 文

A Thesis

Submitted to Department of Biological Science and Technology National Chiao Tung University

In partial Fulfillment of the Requirements For the Degree of Ph.D

In

Biological Science and Technology January 2012

Hsinchu, Taiwan, Republic of China

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Acknowledgement

很感動,睽違了五年的時間,猶如又念完了一次大學的時間,跳過了一次碩班畢業, 這次的畢業取得不易,總算是有可以寫畢業致謝的一天。猶記得剛進入碩士班時,懵懵 懂懂的到現在,也撐到博士畢業得到了這頭銜,新進成員總是只能以學長尊稱,在藻類 組中辛苦的點點滴滴,從一開始的無到現在的有,確實是刻苦銘心。 回想過去研究所期間,往事依舊歷歷在目,當然要感謝的是最初進來時現在已經早 已成鳥飛去的元老學長們:建龍、俊旭和思豪學長,在當初有建龍學長嚴厲的管教之下, 才有現在對實驗嚴肅看待的我;俊旭學長則在他畢業後,實驗室電腦相關大小事都在我 身上時,才知道有俊旭學長真好(淚),學長的文件整理功力更堪稱一流,一直是讓我 佩服的五體投地的學長;思豪學長更是對我照顧有佳,也多虧了學長,讓我在博士班學 習過程中,有個很優秀的榜樣。另外,在我們藻類組最先畢業的博士班學姊筱晶,感謝 有她的協助,讓我們藻類組的戶外養殖得以順利。 當然,在我們這組中,最要感謝的就是一同陪伴我研究所生涯的千雅,沒有她,我 們藻類組也沒有辦法走到這一步,永遠的實驗室大管家,更讓我們實驗進行的更順利 了!在我們這組畢業的碩士班明達,謝謝他陪伴了我兩年運動的生涯,無聊一起去打桌 球跟游泳,跟他一起習得耐臭技能一直是我們兩一起值得驕傲的事情(誤);另外,佳 蓉雖然百勸之下還是一起加入了我們藻類組兩年,讓我感到羞愧的國文能力沒想到也有 可以驕傲的一天!在我們這一組中還要感謝一位隱藏學長棠青,猶如實驗室的媽媽般照 顧我們,也是實驗室唯一有各領域 paper 的學長,真的感謝你在我博班五年多來給予我 的協助與幫忙。 還要謝謝其他實驗室成員:從大學一直到現在的同學曜禎,雖然最後離開了我們, 但是在一起奮鬥努力的這段期間,也是有同學在一起才能互相扶持走到這一步;目前碩 二的學弟妹們,睦元、品萱以及藻類組吳克群子庭,謝謝你們在實驗室的事務上的協助 以及為實驗室犧牲奉獻的舞蹈表演,讓我在博士班最後衝刺的這段時間得以輕鬆專注面 對;最後碩一的新進成員們芳沅、琳岡與燕秋,謝謝你們陪我一起度過最後一學期,也 唯有你們才能讓實驗室變得這麼的熱鬧有趣。 在這段求學之路上,我最感謝的人就是我的指導教授林志生老師,如果沒有老師嚴 厲教導以及開放方式讓我能自由的做想做的研究方向,並在研究路途中給予我許多指

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正,導正我研究的方向才能有機會再這麼短的時間順利取得博士學位。當時碩班也是有 老師的提拔下,才能讓原本只想碩班畢業就選擇就業的我毅然決然選擇了博士班這搖 路,老師就像人生的導師ㄧ般,無論在生活上與實驗上老師都能給予悉心的指導、幫助 及鼓勵,讓我得以成長,研究得以順利進行,在此獻上最誠摯的感謝。 在此也要謝謝我的口試委員:曾慶平老師、張嘉修老師、陳俊勳老師和鍾竺均老師, 對於我的碩士論文給予寶貴的指教與建議。 而要再感謝我的摯友鎧綺,在我最後階段最失意最需要衝刺的時候,謝謝妳給我的 支持與鼓勵,讓我能夠順利走完這一段博士的階段,也唯有妳才能激起我更要努力的鬥 志,得以成就自己完成他人。 最後,我要將此論文獻給我最親愛的媽媽與姐姐以及在天上的爸爸,感謝你們一直 在背後默默的支持我、鼓勵我,讓我能夠有勇氣的持續接受一切的挑戰,沒有你們一路 的栽培以及無悔的付出就沒有今日的我,謝謝你們。希望最後選擇取得博士學位能夠讓 在天上的爸爸感到驕傲。 邱聖壹 謹誌 國立交通大學 生物科技學系博士班 中華民國一百零一年一月

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建構微藻光生物反應系統並用於二氧化碳減量與微藻生物質的生產

研究生:邱聖壹 指導教授:林志生 博士 國 立 交 通 大 學 生 物 科 技 學 院 生 物 科 技 學 系 博 士 班

摘 要

以光合生物反應器培養微藻可被用來減量廢氣中的二氧化碳(CO2),且微藻所生 產之油脂還可被轉化為生質柴油使用。在本研究中,我們篩選並分離出具有高生長潛能 之微藻細胞來減量 CO2及生產微藻生物質。此外我們也設計一氣舉式光合生物反應器用 以進行微藻的高濃度培養。 首先,我們以 CO2減量並生產生物質為目的進行微藻細胞株之篩選,分離得到具有 高生長潛能之藻株,再以起始藻細胞濃度為低濃度(i.e., 8 × 105 cells mL-1)與高濃度(i.e., 8 × 106 cells mL-1)的培養方式來進行微藻二氧化碳耐受性試驗,試驗結果顯示當微藻之起 始細胞濃度較高時,在具有二氧化碳通入培養的環境下,微藻生長速率會較為快速,由 上述結果可知微藻對於 CO2的耐受能力會因藻細胞濃度的增加而增加,因此本研究將於 微藻培養的起始階段,通入適當的 CO2 (2%),使藻細胞適應通有 CO2之環境下生長, 再配合半連續式的培養方式將微藻轉至 10%及 15% CO2環境下培養,使微藻細胞能逐 漸適應高濃度 CO2,進而克服高濃度 CO2對於微藻生長之抑制,提昇微藻對於 CO2之耐 受性。 接著,為了增加生物質生產與 CO2移除之效率,我們以半連續式之各種培養策略進 行生物質產能評估,每兩天置換四分之一培養液、每三天置換三分之一培養液及每八天 置換二分之一培養液,此半連續式培養結果顯示,以每兩天置換四分之一之培養,微藻 生物質產能可高達 0.61 g L-1 d-1,於本研究中所設計之多孔內管氣舉式光生物反應器不 但具有高生物質產能且能維持高密度培養,同時我們也評估多孔內管氣舉式光生物反應 器對於 CO2之移除效能。由結果顯示,高濃度微藻(5 g L-1)培養於通氣為 10% CO2的環 境下,CO2之移除效能大於 60%以上。 最後,我們篩選了一耐溫與耐受 CO2微藻突變株 Chlorella sp. MTF-7 實際應用於實

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場煙道廢氣養殖。研究結果顯示,我們所篩選之 Chlorella sp. MTF-7 於室內不同溫度以 中鋼煙道廢氣通氣實驗中,Chlorella sp. MTF-7 於 35o C 及 40oC 之高溫下,微藻生物質 產能則為 0.32 及 0.24 g L-1 d-1;直接引中鋼煙道廢氣進行戶外 Chlorella sp. MTF-7 養殖 六天,微藻養殖濃度可達 2.87 g L-1(起始養殖濃度為 0.75 g L-1,微藻生物質產能則為 0.52 g L-1 d-1。經由兩組間歇煙道廢氣通氣方式進行養殖,其 CO2、NO 及 SO2之移除效 能分別約為 60%、70%及 50%。由結果顯示,所篩選得之 Chlorella sp. MTF-7 可有效直 接利用煙道廢氣養殖,並能穩定生產微藻生物質與有效減除煙道廢氣中之 CO2、NO 及 SO2。 關鍵字: 煙道廢氣、二氧化碳、微藻、生物質、光生物反應器、小球藻

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Establishing a Microalgae-incorporated Photobioreactor System for CO

2

Reduction and Microalgal Biomass Production

Graduate student: Sheng-Yi Chiu Advisor: Chih-Sheng Lin Ph.D. Department of Biological Science and Technology

National Chiao Tung University

Abstract

Microalgal cultivated in photobioreactor can be used for CO2 mitigation from waste gas and microalgal lipids can be converted into biodiesel. In this study, we screened and isolated microalgal strains with high potential for CO2 reduction and microalgal biomass production. In addition, we also designed an air-lift photobioreactor for high density microalgal

cultivation.

First, the high growth potential microalgal cells were screened and isolated as a candidate for CO2 reduction and biomass production. Then, the low (i.e., 8 × 105 cells mL-1) and high (i.e., 8 × 106 cells mL-1) density of the microalgal cells inoculums for CO2 tolerance was evaluated. The results indicate that microalgal cells grew rapidly in a high-density culture with CO2 aeration. Thus, the strategy of increasing CO2 tolerance and cell density in the microalgal cultures was performed in this study. At the initiating stage of culture, the microalgal cells were grown and adapted to an enriched-CO2 (2%) environment. Then, the semicontinuous system was performed. The result shows that the microalgal cells can grow well even under the conditions of 10% and 15% CO2 aeration.

Then, for increasing biomass production and CO2 removal efficiency, the microalgal cells cultivated in the operation mode that culture broth was replaced by 1/4 (i.e., one-fourth volume of cultured broth was replaced by fresh medium at an interval of 2 days) and 1/3 (one-third broth replaced at 3 days interval) and 1/2 (one-second broth replaced at 8 days interval). The results show that the maximum biomass productivity could achieve 0.61 g L-1 d-1 in 1/4 of the culture broth recovered from the culture every 2 days. The CO2 removal efficiency was also evaluated because the high performance of biomass production and high density cultivation. The results show that > 60% of CO2 could be removed from the aerated gas which contains 10% CO2 under high density (approximate 5 g L-1) cultivation.

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CO2-tolerant mutant strain, Chlorella sp. MTF-7, were investigated. The biomass productivity of Chlorella sp. MTF-7 cultured indoors at 35 and 40oC was 0.32 and 0.24 g L-1 d-1,

respectively. The Chlorella sp. MTF-7 cultures were directly aerated with the flue gas generated from coke oven of a steel plant. The biomass concentration, productivity of

Chlorella sp. MTF-7 cultured in an outdoor 50-L photobioreactor for 6 days was 2.87 g L-1

(with an initial culture biomass concentration of 0.75 g L-1), 0.52 g L-1 d-1. By the operation with intermittent flue gas aeration in a double-set photobioreactor system, average efficiency of CO2 removal from the flue gas could reach to 60%, and NO and SO2 removal efficiency was maintained at approximately 70% and 50%, respectively. Our results demonstrate that flue gas from coke oven could be directly introduced into Chlorella sp. MTF-7 cultures to potentially produce algal biomass and efficiently capture CO2, NO and SO2 from flue gas simultaneously.

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Contents

Acknowledgement ... i

Abstract in Chinese ... iii

Abstract in English ... v

Contents ... vii

List of Tables ... xi

List of Figures ... xii

1. Introduction

... 1

1.1 General Introduction ... 1

1.2 Microalgae ... 2

1.3. Microalgal Cultivation ... 6

1.4 Open Pond System ... 7

1.5 Photobioreactor ... 9

1.6 Closed Hybrid System ... 11

1.7 Factors influencing the growth and lipid content of algae ... 13

1.8 CO2 Reduction by Microalgal Cultures ... 16

1.9 Potential of Biodiesel Produced from Microalgae ... 18

1.10 Microalgae in Other Applications ... 19

2. Research Approaches

... 23

3. Experimental Methodologies

... 26

3.1. Microalgal cultures, medium and chemicals ... 26

3.2. Experimental system with photobioreactor ... 26

3.3. Preparation of the inoculum ... 26

3.4. Experimental design of indoor batch cultivation ... 27

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3.6. Microalgal cell counting and dry weight ... 28

3.7. Measurement of growth rate ... 28

3.8. pH and light measurements ... 29

3.9. Lipid extraction and measurement ... 29

3.10. Measurement of lipid content by fluorescent spectrometry ... 29

3.11. Measurement of medium nitrate content ... 30

3.12. Determinations of CO2(g) and CO2(aq) ... 30

3.13. Photobioreactors and operation of microalgal culture ... 30

3.14. Determination of CO2 removal efficiency ... 31

3.15. Experimental system of indoor photobioreactor for on-site bioremediation experiments ... 31

3.16. Experimental system of outdoor photobioreactor ... 32

3.17. Chemical analyses ... 32

3.18. Statistics ... 32

4. Part I: Reduction of CO

2

by a high-density culture of Chlorella sp. in a

semicontinuous photobioreactor

... 33

4.1 Introduction ... 33

4.2 Results and discussion ... 34

4.2.1. Evaluation of cell density and biomass ... 34

4.2.2. Effect of CO2 on microalgal culture at different cell density ... 34

4.2.3. Effect of CO2 on cell growth in semicontinuous cultivation ... 35

4.2.4. Effect of CO2 on CO2 reduction in semicontinuous culture ... 36

4.2.5. Effect of CO2 on lipid and biomass production in semicontinuous culture ... 37

4.2.6. Performances of six-parallel photobioreactor system ... 38

5. Part II. Biomass production and CO

2

utilization of Nannochloropsis

oculata in response to CO

2

aeration

... 40

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5.1 Introduction ... 40

5.2 Results and Discussion ... 41

5.2.1. Growth of N. oculata NCTU-3 aerated with different CO2 concentration ... 41

5.2.2. Lipid content of microalga at different growth phases ... 41

5.2.3. Effect of CO2 concentration on cell growth in semicontinuous cultures 42 5.2.4. Biomass and lipid productivity in semicontinuous culture ... 43

5.2.5. Optimal CO2 concentration applied in semicontinuous cultures ... 43

5.2.6. Comparison of productive efficiencies in semicontinuous system with different culture approaches ... 44

6. Part III: The air-lift photobioreactors with flow patterning for a

high-density culture of microalgae and carbon dioxide removal

... 46

6.1 Introduction ... 46

6.2 Results and Discussion ... 47

6.2.1 Growth of Chlorella sp. NCTU-2 in the photobioreactors ... 47

6.2.2 CO2 removal of Chlorella sp. NCTU-2 in the photobioreactors ... 49

6.2.3 Biomass productivity of Chlorella sp. NCTU-2 in semicontinuous cultivation ... 49

6.2.4 CO2 removal efficiency at a variety of culture densities at different aeration rates ... 50

7. Part IV: Microalgal biomass production and on-site bioremediation of

carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using

Chlorella sp. cultures

... 52

7.1 Introduction ... 52

7.2 Results and Discussion ... 53

7.2.1. Growth parameters of Chlorella sp. MTF-7 aerated with flue gas ... 53

7.2.1.1. Indoor culture experiments ... 53

7.2.1.2. Outdoor culture experiments ... 56

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7.2.2.1. CO2 removal ... 57

7.2.2.2. NO and SO2 removal ... 57

7.2.3. Flue gas bioremediation by a gas-switching cycle operation ... 58

7.2.3.1. CO2 removal ... 58

7.2.3.2. NO and SO2 removal ... 59

8. Conclusions

... 61

8.1 Part I. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor ... 61

8.2 Part II. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration ... 61

8.3 Part III. The air-lift photobioreactors with flow patterning for a high-density culture of microalgae and carbon dioxide removal ... 62

8.4 Part IV. Microalgal biomass production and on-site bioremediation of carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp. cultures ... 63

9. Future works

... 64

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List of Tables

Table 1. Oil productivity of different energy plants. ... 76 Table 2. Advantages and disadvantages of open and closed culture systems ... 76 Table 3. Productivity of biomass grown outdoors in the various photobioreactors ... 79 Table 4. The main international companies that using microalgal cultivation for CO2

reduction and bioerergy development ... 76

Table 5. The biomass production and the specific growth rate of the low- and high-density

inoculums of Chlorella sp. growth depending on different concentrations of CO2 aeration. ... 84

Table 6. Recovery of lipid and biomass productivity of the Chlorella sp. as waste broth

in the semicontinuous photobioreactor under different concentrations of CO2

aeration compared with single and six-parallel photobioreactor. ... 84

Table 7. Daily recovery of biomass and lipid of N. oculata NCTU-3 cultured in the

semicontinuous system aerated with different CO2 concentrations ... 85

Table 8. Biomass and lipid productivity of N. oculata NCTU-3 cultured in the

semicontinuous system aerated with 2% CO2 under the treatments of one-day and three-day replacement ... 86

Table 9. Comparisons of growth potential and CO2 removal efficiency in the three types of photobioreactors. ... 87

Table 10. Performance of the different broth replacement strategies in a semicontinuous

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List of Figures

Figure 1. The typical types of photobioreactors. ... 90 Figure 2. Schematic diagram of the photobioreactor for the experiments on CO2 reduction

for batch and semicontinuous microalgal cultures ... 91

Figure 3. Schematic diagram of the different types of photobioreactor and visualization of

the liquid flow patterns. ... 92

Figure 4. Calibration curves and equations of optical density at A682 to the cell density and the biomass. ... 93

Figure 5. Effects of different concentrations of CO2 aeration on the growth of Chlorella sp.. ... 94

Figure 6. Comparisons of the total amount and efficiency of CO2 reduction in the single and the six-parallel photobioreactor of semicontinuous Chlorella sp. cultures under 2%, 5%, 10%, and 15% CO2 aeration. ... 95

Figure 7. Effect of the concentrations of CO2 aeration on the growth of N. oculata

NCTU-3. ... 96

Figure 8. Growth profiles of N. oculata NCTU-3 cultured in the semicontinuous system

aerated with 2, 5, 10, and 15% CO2.. ... 97

Figure 9. Growth profiles of N. oculata NCTU-3 cultured in the semicontinuous system

with 2% CO2 aeration and operated by one-day and three-day replacements. ... 98

Figure 10. Growth curves of Chlorella sp. NCTU-2 cultured in the three different types

of photobioreactor in batch and fed-batch culture mode.. ... 99

Figure 11. Growth profiles of Chlorella sp. NCTU-2 cultured in semicontinuous culture

mode with 5% CO2 aeration and operated by 1/4, 1/3 and 1/2 replacements in the porous centric-tube photobioreactor. ... 100

Figure 12. CO2 removal efficiency in Chlorella sp. NCTU-2 cultures operated at different aeration rates and biomass concentrations. ... 101

Figure 13. Growth profiles of Chlorella sp. (wild-type, WT) and its mutant, Chlorella sp.

MTF-7, cultured in an indoor photobioreactor aerated with continuous flue gas or CO2-enriched gas (2, 10, or 25%). ... 102

Figure 14. Growth profiles of Chlorella sp. (wild-type, WT) and its mutant, Chlorella sp.

MTF-7, cultured in an indoor photobioreactor aerated with continuous flue gas and operated at different temperatures (25, 30, 35 or 40oC). ... 103

Figure 15. The outdoor photobioreactor culture system next to the smokestack of a coke

oven at the China Steel Corporation, Kaohsiung, Taiwan. ... 104

Figure 16. A double-set of photobioreactor system for flue gas-switching cycle operation

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Figure 17. Growth profiles of Chlorella sp. MTF-7 cultured in an outdoor photobioreactor

aerated with continuous or intermittent flue gas.. ... 106

Figure 18. The efficiency of CO2, NO and SO2 removal from flue gas by Chlorella sp. MTF-7 cultures under continuous flue gas aeration. ... 107

Figure 19. The patterns of the inlet and outlet load CO2 concentrations of Chlorella sp. MTF-7 cultures aerated with intermittent flue gas at 0.05 vvm at different time intervals. The flue gas was controlled by a gas-switching cycle operation. ... 108

Figure 20. The patterns of the inlet and outlet load CO2 concentrations, pH value, and dissolved inorganic carbon (DIC) concentrations of Chlorella sp. MTF-7

cultures aerated with intermittent flue gas at 0.05 vvm. ... 109

Figure 21. The patterns of the inlet and outlet load NO (A) and SO2 (B) concentrations of Chlorella sp. MTF-7 cultures aerated with intermittent flue gas at 0.05 vvm. ... 110110

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1. Introduction

1.1 General Introduction

Global warming, which is induced by increasing concentrations of greenhouse gases in the atmosphere, is of great concern. Carbon dioxide (CO2) is the principal greenhouse gas and the concentration of atmospheric CO2 has increased rapidly since the onset of industrialization. The global atmospheric concentration of CO2 has increased from about 280 ppm of a

pre-industrial value to 387 ppm in 2010 (NOAA, 2010). Such an increasing long-lived greenhouse gas currently makes the Earth heating. And now the drastic climate change induced by increasing concentrations of greenhouse gas in the atmosphere is also of great concern. Furthermore, the drastic energy crisis is due to the shortage of oil and additionally to electricity or other natural resources. There are several means of reducing the emissions of greenhouse gases by energy production from renewable sources. This issue has received increasing attention due to the exhaustion of natural sources of fossil fuels (Favre et al., 2009).

There are various research strategies on CO2 sequestration which have been carried out, such as physical, chemical and biological methods. The example of physical method is geological storage of CO2, which it is injected into reservoirs (Packer, 2009). On the other hand, the examples chemical methods are by washing with alkaline solutions (Diao et al., 2004), multiwalled carbon nanotubes (Su et al., 2009), amine coating activated carbon (Plaza et al., 2007) for CO2 capture in order to reduce the emission of CO2. The biological method using microalgal photosynthesis is considered as an effective approach for biological CO2 fixation (de Morais and Costa, 2007a; Wang et al., 2008; González López et al., 2009; Packer, 2009; Yoo et al., 2010). By the biological approaches, CO2 can be fixed into microalgal biomass via photosynthesis. Microalgae has about 10 – 50 times higher CO2 fixation rates than terrestrial plants and can thus utilize CO2 from flue gas to produce biomass efficiently (Doucha et al., 2005, Wang et al., 2008; Brune et al., 2009). Therefore, reduction of the emission from industries or power plants by the use of microalga incorporated

photobioreactor is a potential method for directly removing CO2 from waste gas. In addition, microalgae can use CO2 efficiently and accumulate lipids, which are chemically similar to common vegetable oils and are high potential sources for biodiesel production. Compared with fossil fuels, the microalgal-based biodiesel are renewable, biodegradable, and low pollutant produced. Thus, reducing atmospheric CO2 by microalgal

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photosynthesis is considered safe and reliable for living environment. A closed

photobioreactor can be a bioscrubber for waste gas treatment and the microalgal cells cultured in the photobioreactor converting CO2 from the waste gas into biomass is energy-efficient and economical approach.

Flue gases from power plant are responsible for more than 7% of the world CO2

emissions from energy use and steel plants are the single largest source of energy-related CO2 emissions in the world. In general, the primary emission in flue gas is CO2. This CO2 is a plentiful carbon source for microalgal cultures. The direct use of the flue gas reduces the cost of pretreatment. However, the direct use of the flue gas imposes extreme conditions on the microalgae, such as the high concentration of CO2 and the presence of inhibitory compounds such as NOX and SOX. Temperature is also an inhibitory growth factor for outdoor microalgal cultivation.

Even though that the microalgae have the widen areas of characterization, the microalgae have to be improved to select more potential strains which show the potential of biomass production and remove more CO2. For on-site bioremediation of CO2, NOX and SOX from flue gas, first, we aimed to characterize the effect of CO2 on biomass production. There are lots of researches and applications on the large scale outdoor photobioreactor cultivation system for the potential in CO2 reduction. However, there are limited literatures reported about on-site bioremediation by microalgal cultures and the performance of outdoor enclosed photobioreactors still can not even achieve the values obtained at laboratory scale. Thus, we aimed to isolate thermal- and flue gas tolerant mutant strains, which can grow well in an outdoor closed photobioreactor for bioremediation under strong sunlight without the supply of cooling system in the area of subtropical region.

1.2 Microalgae

Microalgae covers all unicellular and simple multi-cellular microorganisms, including both prokaryotic microalgae and eukaryotic microalgae (Scott et al., 2010). Algae can either be autotrophic or heterotrophic; the former require only inorganic compounds such as CO2, salts and a light energy source for growth; while the latter are non-photosynthetic therefore require an external source of organic compounds as well as nutrients as an energy source. For autotrophic algae, photosynthesis is a key component of their survival, whereby they convert solar radiation and CO2 absorbed by chloroplasts into adenosine triphosphate (ATP) and O2

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the usable energy currency at cellular level, which is then used in respiration to produce energy to support growth (Falkowski and Raven, 1997).

Microalgae, just like plants, are photosynthetic microorganisms which convert sunlight, CO2 and water to biomass, potential biofuels, foods, feeds and high-value bioactives

(Borowitzka, 1999; Banerjee et al., 2002; Walter et al., 2005; Spolaore et al., 2006; Chisti, 2007). Microalgae are responsible for over 50% of primary photosynthetic productivity on earth and are budding sunlight factories for a wide range of potentially useful products, but are scarcely used commercially (Gavrilescu and Chisti, 2005; Wijffels, 2007). The large-scale cultivation of microalgae and the use of its biomass for the production of useful products were first considered seriously in Germany during World War II (Becker, 1994).

Microalgae have the potential to develop biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Microalgae produce many valuable substances such as vitamins and color pigments, essential fatty acids, amino acids, and even antibiotics and pharmaceutically-active substances, such as high-quality food, food

supplements or alternatives for synthetic substances in the cosmetics and chemical industry. Microalgae has the wide range of benefits in producing valuable chemicals or healthy foods, vitamins and as feedstock for animals on land and in aquaculture (Pulz and Gross, 2004; Spolaore et al., 2006; Raja et al., 2008), consume waste and the metallic pollutants in

wastewater (Perales-Vela et al., 2006; Jácome-Pilco et al., 2008) and produce biodiesel (Chisti, 2007; Hossain et al., 2008). Microalgae convert CO2 into biomass and use CO2 efficiently. Therefore, microalgae are cultivated at large-scale outdoor for the purpose of

industrialization.

In the reason of depleting supplies and the contribution of petroleum or natural fossil fuels to the accumulation of CO2 in the environment, continued use of these fuels is now widely recognized as unsustainable. Renewable, carbon neutral, transport fuels are necessary for environmental and economic sustainability. Biodiesel (monoalkyl esters) is nontoxic and less emissions of CO2, sulfur oxides (SOX) and nitrogen oxides (NOX), and it is biodegradable and renewable as well as environmentally safe (Ma and Hanna, 1999). Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels.

Unfortunately, biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy even a small fraction of the existing demand for transport fuels. Microalgae appear to be the only source of renewable biodiesel that is capable of meeting the global demand for transport fuels. Like plants, microalgae use sunlight to produce oils but they do so more

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efficiently than crop plants. Oil productivity of many microalgae greatly exceeds the oil productivity of the best producing oil crops (Chisti, 2007). Table 1 shows comparison of microalgae with other biodiesel feedstocks. Moreover, microalgal biomass can be used to produce biofuel by pyrolysis, direct combustion or thermal chemical liquefaction (Mata et al., 2010). The lipid fraction of microalgal biomass can be extracted and transesterified for

biodiesel production (Li et. al, 2008; Brennan and Owende, 2010; Lee et al., 2010). Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae (Rosenberg et al., 2008).

1.2.1 Chlorella

Chlorella species are encountered in all water habitats exhibiting a cosmopolitan

occurrence, having been isolated from widely differing fresh, as well as marine, water habitats. The species of the genus Chlorella have simple life cycles and nutritional requirements.

Classification is complex because Chlorella species cannot be readily discerned on the basis of morphological features, the taxonomy of Euchlorella, which comprises the most common species is, therefore, incomplete. It has indeed been proposed to use physiological and

biochemical rather than morphological criteria, for species identification. On the basis of their external morphlogy, Chlorella species could nevertheless be placed in four general groups: (1) spherical cells (ratio of the two axes equals one); (2) ellipsoidal cells (ratio of the longest axis to the shortest axis being 1.45 to 1.60); (3) spherical or ellipsoidal cells; (4) globular to subspherical cells (Richmond, 1986). In reproduction, which is exclusively asexual, each mature cell divides usually producing four or eight (and more rarely, 16) autospores, which are freed by rupture or dissolution of the parental walls.

1.2.2 Microalgal Physiology

Autotrophic organisms obtain their energy through the absorption of light energy for the reduction of CO2 by the oxidation of substrate, mainly water, with the release of O2.

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are those that cannot grow in the dark. By far, most algae belong to this category, although many require minimal quantities of organic compounds for growth, such as vitamins.

For high rates of autotrophic production, supply of CO2 and HCO3- is most important. Contrary to land plants, atmospheric CO2 cannot satisfy the C-requirements of high yielding autotrophic algal production systems. The CO2-H2CO3- HCO3-CO32- system is the most important buffer generally present in culture broth and it is the best means available to control and maintain specific pH levels that are optimal for mass-cultivated species. The

bicarbonate-carbonate buffer system can provide CO2 for photosynthesis. The buffer system reactions imply that during photosynthetic CO2 fixation, OH- accumulates in the growth solution leading to a gradual rise in pH. pH-static control via direct CO2 sparging into the culture media is the best and most convenient method for pH control and at the same time supplying CO2 for high yield in mass algal cultures. Since active photosynthesis results in an increase in pH, the opposite is true for CO2 release during respiration. The overall influence is little since as a general rule dark respiration is less than 10% of total photosynthetic

production (Grobbelaar and Soeder, 1985).

After carbon, nitrogen is the most important nutrient contributing to the biomass produced. The nitrogen content of the biomass can range from 1% to more than 10% and it not only varies between different groups but within a particular species, depending on the supply and availability. Typical responses to nitrogen limitation is discoloration of the cells and accumulation of organic carbon compound such as polysaccharides and certain oils (Becker, 1994). Nitrogen is mostly supplied as nitrate (NO3-), but often ammonia (NH4+) and urea are also used, with similar growth rates recorded. Ammonia nitrogen is often the

preferred N-source for microorganisms and the assimilation of either NO3- or NH4+ is related to the pH of the growth media. When ammonia is used as the sole source of N, the pH could drop significantly during active growth, due to the release of H+ ions. An increase in pH occurs when deciding whether to supply either nitrate or ammonia, is that the latter could be lost form the growth media due to volatilization, particularly when the pH increases.

Sulfur is generally present in small quantity in all plant cells but is probably not a limiting factor for many algae under normal conditions. Sulfur is incorporated into numerous organic compounds and sulfates are present in the vacuoles. As compared with other

macronutrients sulfur uptake and metabolism in algae have been studied only scarcely. In fact, major studies on sulfur assimilation by algae were done more than 20 years ago and present research in this field equals nearly nothing. Uptake sulfur by both Chlorella pyrenoidosa and

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Scenedesmus sp. is stimulated by light (Kylin, 1961; Tseng et al., 1971). As with N

assimilation, light could be acting by providing energy via photophosphorylation, reductant, or C skeletons. A large part of the sulfur in most algae is incorporated into protein. Two sulfur-containing amino acids, cysteine and methionine, are important in maintaining the three-dimensional configuration of proteins through sulfur bridges. Incorporation of sulfur from sulfate in the medium into normal cells of Scenedesmus was enhanced by light relatively most in the case of lipid S and least in the inorganic sulfate fraction; the effects of light were, generally, increased by the presence of CO2 and nitrogen salts.

1.3 Microalgal Cultivation

Most microalgae are strictly photosynthetic, i.e., they need light and carbon dioxide as energy and carbon sources. Photoautotrophic production is the only method which is technically and economically feasible for large-scale production of algae biomass

(Borowitzka, 1997). Two systems that have been widely constructed are based on open pond and closed photobioreactor technologies (Borowitzka, 1999). The technical viability of each system is influenced by intrinsic properties of the selected algae strain used, as well as climatic conditions and the costs of land and water (Borowitzka, 1992). Microalgae

cultivation using sunlight energy can be carried out in open ponds or closed photobioreactors, based on tubular, flat plate, or other designs. Closed systems are much more expensive than ponds, present significant operating challenges, such as gas exchange limitations, cannot be scaled up much beyond about 100 m2 for an individual growth unit. Table 2 shows the advantages and disadvantages of open and closed culture systems. Currently there are three types of industrial reactors used for algal culture: (1) open ponds, (2), photobioreactors and (3) closed and hybrid systems.

Open-pond systems are shallow ponds in which algae are cultivated. Nutrients can be provided through runoff water from nearby land areas or by channeling the water from sewage/water treatment plants. Technical and biological limitations of the open systems have given rise to the development of enclosed photobioreactors. Photobioreactors are different types of tanks or closed systems in which algae are cultivated. Microalgae cultivation using sunlight energy can be carried out in open ponds or closed photobioreactors, based on tubular, flat plate, or other designs. The closed systems have been considered to be capital intensive and are justified only when a fine chemical is to be produced. Microalgae production in closed photobioreactors is highly expensive. Closed systems are much more expensive than

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ponds. However, closed systems require much less light and agricultural land to grow algae (Chisti, 2007). As much as 25% of the biomass produced during daylight may be lost during the night due to respiration. The extent of this loss depends on the light level under which the biomass was grown, the growth temperature, and the temperature at night (Chisti, 2007). Algal cultures consist of a single or several specific strains optimized for producing the desired product. Water, necessary nutrients, and CO2 are provided in a controlled way, while oxygen has to be removed (Carlsson et al., 2007). Algae receive sunlight either directly through the transparent container walls or via light fibers or tubes that channel it from sunlight collectors. A great amount of developmental work to optimize different

photobioreactor systems for algae cultivation has been carried out (Carvalho et al., 2006, Choi et al., 2003, Hankamer et al., 2007, Janssen et al., 2003).

Photobioreactors are the preferred method for scientific researchers, and recently for some newer, innovative production designs. These systems are more expensive to build and operate; however, they allow for a very controlled environment. This means that gas levels, temperature, pH, mixing, media concentration, and light can be optimized for maximum production (Chisti 2007). Unlike open ponds, Photobioreactors can ensure a single alga species is grown without interference or competition (Campbell 2008).

1.4 Open Pond System

Open ponds are the oldest and simplest systems for mass cultivation of microalgae. The pond is designed in a raceway configuration, in which a paddlewheel circulates and mixes the algal cells and nutrients. The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid. Baffles in the channel guide the flow around the bends in order to minimize space. Algal cultures can be defined (one or more selected strains), or are made up of an undefined mixture of strains. The only practicable methods of large-scale production of microalgae are raceway ponds (Terry and Raymond 1985; Molina Grima 1999) and tubular photobioreactors (Molina Grima et al., 1999; Tredici 1999; Sánchez Mirón et al., 1999). Open architecture approaches, while possibly the cheapest of all current techniques, suffer challenges with contamination, evaporation, temperature control, CO2 utilization, and maintainability. The ponds are kept shallow because of the need to keep the algae exposed to sunlight and the limited depth to which sunlight can penetrate the pond water. The ponds are operated

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water is removed at the other end. Large-scale outdoor culture of microalgae and

cyanobacteria in open ponds, raceways, and lagoons is well established (Becker 1994). Open culture is used commercially in the USA, Japan, Australia, India, Thailand, China, Israel, and elsewhere to produce algae for food, feed, and extraction of metabolites. Open-culture

systems allow relatively inexpensive production but are subject to contamination.

Consequently, only a few algal species can be cultured in open outdoor systems. Species that grow successfully in the open include rapid growers such as Chlorella and species that require a highly selective extremophilic environment that does not favor the growth of most potential contaminants. For example, species such as Spirulina and Dunaliella thrive in highly alkaline and saline selective environments, respectively. Algae produced in quantities in open systems include Spirulina, Chlorella, Dunaliella, Haematococcus, Anabaena, and Nostoc (Chisti 2006). The size of these ponds is measured in terms of surface area, since surface area is so critical to capturing sunlight. Even at levels of productivity that would stretch the limits of an aggressive research and development program, such systems require acres of land. At such large sizes, it is more appropriate to think of these operations on the scale of a farm. Such algae farms would be based on the use of open, shallow ponds in which some source of waste CO2 could be efficiently bubbled into the ponds and captured by the algae. Careful control of pH and other physical conditions for introducing CO2 into the ponds allows for more than 90% utilization of injected CO2. Raceway ponds, usually lined with plastic or cement, are about 15 to 35 cm deep to ensure adequate exposure to sunlight. Paddlewheels provide motive force and keep the algae suspended in the water. The ponds are supplied with water and nutrients, and mature algae are continuously removed at one end. A raceway pond is made up of a closed-loop recirculation channel that is typically about 0.3 m deep. Flow is guided around bends by baffles placed in the flow channel, and raceway channels are built in

concrete or compacted earth and may be lined with white plastic. During daylight, the culture is fed continuously in front of the paddlewheel where the flow begins. Broth is harvested behind the paddlewheel on completion of the circulation loop. The paddlewheel operates all the time to prevent sedimentation. Photosynthesis is the most important biochemical process in which plants, algae, and some bacteria harness the energy of sunlight to produce food. Productivity is affected by contamination with unwanted algae and microorganisms that feed on algae. Open ponds, specifically mixed raceway ponds, are much cheaper to build and operate, can be scaled up to several hectares for individual ponds, and are the method of choice for commercial microalgae production. However, such open ponds also suffer from various limitations, including more rapid (than closed systems) biological invasions by other

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algae, algae grazers, fungi and amoeba, etc., and temperature limitations in cold or hot humid climates. Microalgae can be cultivated in coastal areas. The raceway pond system of biomass culture must be approved to achieve high and sustained growth rates and oil yields that are essential to developing an algal-based biofuel industry.

1.5 Photobioreactor

Photobioreactors are different types of tanks or closed systems in which algae are cultivated (Richmond, 2004). Photobioreactors have the ability to produce algae while performing beneficial tasks, such as scrubbing power plant flue gases or removing nutrients from wastewater (Carlsson et al., 2007). Microalgal biomass can play an important role in solving the problem between the production of food and that of biofuels in the near future. Phototropic microalgae are most commonly grown in open ponds and photobioreactors (Patil et al., 2005). Photobioreactors offer a closed culture environment, which is protected from direct fallout and so is relatively safe from invading microorganisms. This technology is relatively expensive compared to the open ponds because of the infrastructure costs. An ideal biomass production system should use the freely available sunlight. Many different designs of photobioreactor have been developed, but a tubular photobioreactor seems to be most

satisfactory for producing algal biomass on the scale needed for biofuel production (Tredici, 1999). Closed, controlled, indoor algal photobioreactors driven by artificial light are already economical for special high-value products such as pharmaceuticals, which can be combined with the production of biodiesel to reduce the cost (Patil et al., 2008).

Photobioreactors have higher efficiency and biomass concentration (2 to 5 g/L), shorter harvest time (2 to 4 weeks), and higher surface-to-volume ratio than open ponds (Lee, 2001; Wang et al., 2008). Closed systems consist of numerous designs: tubular, flat-plated,

rectangular, continued stirred reactors. The typical closed photobioreactors are demonstrated in Figure 1. Photobioreactors in general provide better control of cultivation conditions, yield higher productivity and reproducibility, reduce contamination risk, and allow greater selection of algal species used for cultivation. The photobioreactor has a photolimited central dark zone and a better lighting peripheral zone close to the surface (Chisti, 2007). CO2-enriched air is sparged into the photobioreactor creating a turbulent flow. Turbulent flow simultaneously circulates cultures between the light and dark zones and assists the mass transfer of CO2 and O2 gases. The frequency of light and dark zone cycling is depended on the intensity of

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irradiance level (Chisti, 2007). The highest cost for closed systems is the energy cost associated with the mixing mechanism (Wijffels 2008). Tubular photobioreactors consist of transparent tubes that are made of flexible plastic or glass. Tubes can be arranged vertically, horizontally, inclined, helically, or in a horizontal thin-panel design. Tubes are generally placed in parallel to each other or flat above the ground to maximize the illumination surface-to-volume ratio of the reactor. The diameter of tubes is usually small and limited (0.2-m diameter or less) to allow light penetration to the center of the tube where the linear growth rate of culture decrease with increasing unit diameter (Ogbonna and Tanaka 1997). The growth medium circulates from a reservoir to the reactor and back to the reservoir. A turbulent flow is maintained in the photobioreactor to ensure distribution of nutrients, improve gas exchange, minimize cell sedimentation, and circulate biomass for equal illumination between the light and dark zones. The tubes are generally less than 10 cm in diameter to maximize sunlight penetration. The medium broth is circulated through a pump to the tubes, where it is exposed to light for photosynthesis, and then back to a reservoir. A portion of the algae is usually harvested after it passes through the solar collection tubes, making continuous algal culture possible. In some photobioreactors, the tubes are coiled spirals to form what is known as a helical-tubular photobioreactor. The microalgal broth is circulated from a reservoir to the solar collector and back to the reservoir (Chisti 2007). Flat-plated photobioreactors are usually made of transparent material. The large illumination surface area allows high photosynthetic efficiency, low accumulation of dissolved O2

concentration, and immobilization of algae (Ugwu et al., 2008). The photobioreactors are inexpensive and easy to construct and maintain. However, the large surface area presents scale-up problems, including difficulties in controlling culture temperature and carbon dioxide diffusion rate and the tendency for algae adhering to the walls. Vertical-column

photobioreactors are compact, low-cost, and easy to operate monoseptically. Furthermore, they are very promising for large-scale cultivation of algae. It was reported that

bubble-column and airlift photobioreactors (up to 0.19 m in diameter) can attain a final biomass concentration and specific growth rate that are comparable to values typically reported for narrow tubular photobioreactors (Sánchez Mirón et al., 2002). Some bubble column photobioreactors are equipped with either draft tubes or constructed as split cylinders. In the case of draft tube photobioreactors, intermixing occurs between the riser and the

downcomer zones of the photobioreactor through the walls of the draft tube (Chiu et al., 2009b).

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1.6 Closed Hybrid System

Other system designs for algae production are possible. The Japanese, French, and German governments have invested significant R&D dollars on novel closed photobioreactor designs for algae production. The main advantage of such closed systems is that they are not as subject to contamination. When designing a photobioreactor, design parameters such as reactor dimension, flowrate, light requirements, culture condition, algae species,

reproducibility, and economic value need to be taken into consideration. Depending on the reactor dimensions, site location, and local climate, these parameters can determine the type of cultivation system needed (open versus closed). Reactor design should have good mixing properties, efficiency, and reproducibility and be easy to maintain and sterilize. An efficient photobioreactor not only improves productivity but also is used to cultivate multiple strains of algae. The performance of a photobioreactor is measured by volumetric productivity, areal productivity, and productivity per unit of illuminated surface (Riesing, 2006). Volumetric productivity is a function of biomass concentration per unit volume of bioreactor per unit of time. Areal productivity is defined as biomass concentration per unit of occupied land per unit of time. Productivity per unit of illuminated surface is measured as biomass concentration per area per unit of time. Closed photobioreactors support up to fivefold higher productivity with respect to reactor volume and consequently have a smaller “footprint” on a yield basis. Besides saving water, energy, and chemicals, closed photobioreactors have many other advantages that are increasingly making them the reactor of choice for biofuel production, as their costs are lower (Schenk et al., 2008). Closed photobioreactors permit essentially

single-species culture of microalgae for prolonged periods. Most closed photobioreactors are designed as tubular reactors, plate reactors, or bubble column photobioreactors (Pulz, 2001). Other less common designs like semihollow spheres have been reported to run successfully (Sato et al., 2006). Closed photobioreactors have been employed to overcome the

contamination and evaporation problems encountered in open ponds (Molina Grima et al., 1999). These systems are made of transparent materials and are generally placed outdoors for illumination by natural light. The cultivation vessels have a large surface-area to-volume ratio. The main problems in the large-scale cultivation of microalgae outdoors in open ponds are low productivity and contamination. To overcome these problems, a closed system consisting of polyethylenes sleeves was developed. The closed system was found to be superior to open ponds with respect to growth and production in a number of microalgae. In both closed and open systems, growth and production under continuous operation were higher than in batch

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cultivation (Cohen et al., 1991). The preferred alternative is closed photobioreactors, where the algae fluid remains in a closed environment to enable accelerated growth and better control over environmental conditions. These glass or plastic enclosures, often operated under modest pressure, can be mounted in a variety of horizontal or vertical configurations and can take many different shapes and sizes. Rigid frameworks or structures are usually used to support the photobioreactor enclosures.

In hybrid systems, both open ponds as well as closed photobioreactor system are used in combination to get better results. Open ponds are a very proficient and lucrative method of cultivating algae, but they become contaminated with superfluous species very quickly. A combination of both systems is probably the most logical choice for cost-effective cultivation of high yielding strains for biofuels. Open ponds are inoculated with a desired strain that had invariably been cultivated in a photobioreactor, whether it is as simple as a plastic bag or a high-tech fiber-optic bioreactor. Importantly, the size of the inoculums needs to be large enough for the desired species to establish in the open system before an unwanted species. Therefore, to minimize contamination issues, cleaning or flushing the ponds should be part of the aquaculture routine, and as such, open ponds can be considered as batch cultures (Schenk et al., 2008). Abundant light, which is necessary for photosynthesis, is the third requirement. This is often accomplished by situating the facility in a geographic location with abundant, uninterrupted sunshine (Brown and Zeiler 1993). This is a favored approach when cultivating in open ponds. Photobioreactors are flexible systems that can be optimized according to the biological and physiological characteristics of the algal species being cultivated, allowing one to cultivate algal species that cannot be grown in open ponds. A great proportion of light does not impose directly on the culture surface but has to cross the transparent photobioreactor walls.

In spite of their advantages, it is not expected that photobioreactor have a significant impact in the near future on any product or process that can be attained in large outdoor raceway ponds. Photobioreactors suffer from several drawbacks that need to be considered and solved. Their main limitations include: difficulty in scaling up, the high cost of building, operating and of algal biomass cultivation, overheating, bio-fouling, oxygen accumulation, and cell damage by shear stress and deterioration of material used for the photo-stage. The cost of biomass production in photobioreactors may be one order of magnitude higher than in ponds. Whereas in some cases, for some microalgae species and applications it may be low enough to be attractive for aquaculture use; in other cases, the higher cell concentration and the higher productivity achieved in photobioreactor may not balance for its higher capital

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and operating costs.

1.7 Factors influencing the growth of algae

Numerous aspects influence the growth and lipid content of algae. The reaction driving the initial conversion of sunlight into stored energy is photosynthesis. Therefore, all of the components involved in photosynthesis contribute to growth. The major factors include lighting, mixing, CO2 enrichment, O2 removal, nutrient supply, temperature, and pH (Suh and Lee, 2003a; Richmond, 2004b; Carvalho et al., 2006; Grobbelaar, 2009). It is important to note that in each category the precise conditions for optimal growth depend on the strain of algae selected for cultivation.

1.7.1 Light supply

An optimal reactor enhances light intensity/ penetration, as well as the wavelength of light and the frequency of cellular exposure to light.When selecting the light source, both the spectral quality and intensity must be considered. The spectral quality of light utilized by algae is defined by the absorption spectrum in the range of 400 to 700 nm for the chlorophyll and other photosynthetically active pigments, and the algal photosynthesis efficiency is a function of the spectral quality of the light source (Simmer et al., 1994; Suh and Lee, 2003a).

The level of light intensity is critical because at a certain level algae experience light saturation and dissipate the excess energy as heat (Mussgnug et al., 2007). Light saturation can be mitigated by the spatial dilution of light, which is the distribution of solar radiation on a greater photosynthetic surface are, and also reduces mutual shading of microalgal cells. Thus, a design principle for photobioreactor designs is to maximize the surface area to volume ratio, which can be used for comparison between reactors.

Beyond the surface area and volume, the unique geometry of a reactor influences the light distribution. For example in a tubular reactor, the light gradient is primarily determined by the diameter of the tube and the biomass density in the medium (Janssen et al., 2003). Optimal cell density is specific to each strain and needs to be maintained in order for light intensity and light penetration to remain at optimal levels (Richmond, 2004b). Light and dark cycles strongly influence the growth of algae. In both open ponds and outdoor closed reactors, natural light is subject to changes in time of day, weather, season, and geography (Pulz and Scheinbenbogen, 1998). Unfortunately, all reactors using natural light are subject to the

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absence of light during nighttime. According to Chisti (2007), biomass losses might reach as high as 25% during the night, depending on the light intensity during the day, the temperature during the day, and the temperature at night. Janssen et al. (2003) stated that the length of the light/dark cycles experienced by algae influenced photosynthetic efficiency.

1.7.2 Mixing

The level of mixing in a reactor strongly contributes to the growth of algae. When environmental conditions do not limit growth rates, mixing is the most influential factor contributing to algae growth rates (Suh and Lee, 2003a). Mixing affects growth in two

primary ways: (1) improves productivity by increasing the frequency of cell exposure to light and dark volumes of the reactor and, (2) by increasing mass transfer between the nutrients and cells (Qiang and Richmond, 1996).

Mixing and lighting are closely related, as mixing is often responsible for inducing the light and dark cycles beneficial to algae growth. Similarly, mixing offers little benefit if lighting is poor and or culture in low density (Richmond, 2004b). Ugwu et al. (2005)

demonstrated that the installation of static mixers in tubular reactors succeeded in increasing light utilization and biomass yields when the reactor was scaled up by increasing the tube diameter.

1.7.3 Carbon dioxide consumption

In addition to light and water, CO2 is necessary for photosynthesis to occur. However, an excess of CO2 can also be detrimental to photosynthesis and cell growth. CO2 concentrations from 1 to 5% (by volume) often lead to maximum growth. Despite this, laboratories routinely aerate algal cultures with 5 – 15% CO2, or even pure CO2 (Suh and Lee, 2003a).

Flue gas is a desirable source of CO2 because it reduces greenhouse gas emissions as well as the cost of algal biofuel production. Flue gas from typical coal-fired power plants contain up to 13% CO2 (Chisti, 2007). Doucha et al. (2005) studied the performance of a closed reactor utilizing flue gas as a source of CO2 versus a reactor utilizing pure CO2.

Surprisingly, productivities and photosynthetic efficiencies were very similar under conditions of pure CO2 versus flue gas. Because CO2 concentration in flue gas was relatively low, the efficiency of CO2 mass transfer was lower for flue gas than it was for pure CO2.

1.7.4 Oxygen removal

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intense sunlight and high oxygen concentration results in photooxidative damage to algal cells (Chisti, 2007).

Because of the constraint on the concentration of dissolved oxygen, tube length is limited in horizontal tubular reactors. This restriction makes it very difficult for tubular reactors to be scaled-up. In a tubular reactor designed by Molina et al. (2001), the algae culture regularly returned to an airlift zone where the accumulated oxygen from photosynthesis was stripped by air. A gas-liquid separator in the upper part of the airlift column prevented gas bubbles from recirculating into the horizontal loop of the airlift reactor. The time taken by the fluid to travel the length of the degasser must at least equal the time required by the oxygen bubbles to rise out.

1.7.5 Nutrient supply

In order to grow, algae require more than the reactants in the photosynthesis reaction. Two major nutrients are nitrogen and phosphorus, which both play a role in controlling growth rates. Other essential nutrients are carbon, hydrogen, oxygen, sulfur, calcium, magnesium, sodium, potassium, and chlorine. Nutrients needed in minute quantities include iron, boron, manganese, copper, molybdenum, vanadium, cobalt, nickel, silicon, and selenium (Suh and Lee, 2003a).

1.7.6 Temperature

Temperatures experienced by algae grown outdoors can vary as much as the extreme outdoor temperatures characteristic to the geographic region of cultivation. Although algae may be able to grow at a variety of temperatures, optimal growth is limited to a narrow range specific to each strain. Seasonal and even daily fluctuations in temperature can interfere with algae production. Temperatures can reach as high as 30°C higher than ambient temperature in a closed photobioreactor without temperature control equipment (Suh and Lee, 2003a). Evaporate cooling, water spray or shading techniques are employed frequently to inhibit temperatures of that magnitude. Whereas, a lower temperature appears to reduce the loss of biomass due to respiration during the night (Chisti, 2007).

1.7.7 pH

Each strain of algae also has a narrow optimal range of pH. The pH of the medium is linked to the concentration of CO2. Suh and Lee (2003a) mentioned that pH increases steadily in the medium as CO2 is consumed during flow downstream in a reactor. The pH affects the liquid chemistry of polar compounds and the availability of nutrients such as iron, organic

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acids, and even CO2 (Lee and Pirt, 1984). Because pH is so influential, Suh and Lee (2003a) stated that commercial pH controllers must be used in reactors to optimize growth.

1.8 CO

2

Reduction by Microalgal Cultures

Generally, microalgae can typically capture CO2 from three different sources:

atmosphere, emission from power plants and industrial processes, and from soluble carbonate (Wang et al., 2008). CO2 capture from atmosphere is probably the most basic method to sink carbon, and relies on the mass transfer from the air to the microalgae in their aquatic growth environments during photosynthesis.

There are three main CO2 mitigation strategies are normally used, (1) physical method, (2) chemical reaction-based approaches, and (3) the biological mitigation. Most of carbon capture and sequestration (CCS) discussions are about geological storage of CO2 presently. Whilst the oil and gas industry has successfully injected CO2 into reservoirs, just before date this has mainly been for increased yield of fossil hydrocarbon reserves and not for long-term storage. This is proven safe but the biggest difficulty with this approach is the added cost of separation of CO2 from the emission streams (Packer, 2009). The chemical reaction-based CO2 mitigation approaches are energy-consuming, use costly processes, and have disposal problems because both the captured CO2 and the wasted absorbents need to be disposed of. In other hand, the biological CO2 mitigation has attracted much attention in the last years since it leads to the production of biomass energy in the process of CO2 fixation through

photosynthesis (Pulz and Gross, 2004).

A number of microalgae species are able to assimilate CO2 from soluble carbonates such as sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3). Due to the high salt content and resulting high pH of the medium, it is easier to control invasive species since only a very small number of algae can grow in the extreme conditions (Colman and Rotatore, 1995; Emma et al., 2000; Wang et. al., 2008). The selection of suitable microalgae strains for CO2 bio-mitigation has significant effect on efficacy and cost competitiveness of the bio-mitigation process. The desirable characteristics for high CO2 fixation include: high growth and CO2 utilisation rates; high tolerance of trace constituents of flue gases such as SOx and NOx; possibility for valuable by-products and co-products, e.g. biodiesel and biomass for solid fuels; ease of harvesting associated with spontaneous settling or bio-flocculation characteristics; high water temperature tolerance to minimize cost of cooling exhaust flue gases; be able to

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use the strain in conjunction with wastewater treatment.

For example, de Morais and Costa (2007a) using Spirulina sp., obtained a maximum daily CO2 biofixation of 53.29% for 6% (v/v) CO2 and 45.61% for 12% (v/v) CO2 in the injected flue gas, with the highest mean fixation rate being 37.9% for 6% (v/v) CO2. With

Scenedesmus obliquus, de Morais and Costa achieved biofixation rates of 28.08% and 13.56%

for 6% (v/v) and 12% (v/v) CO2, respectively.

Chang and Yang (2003) found that certain species of Chlorella could grow in an atmosphere containing CO2 up to 40% (v/v). When comparing Botryococcus braunii,

Chlorella vulgaris and Scenedesmus sp. under flue gas conditions, Yoo et al. (2010) found Scenedesmus sp. to be the most suitable for CO2 mitigation due to high rates of biomass production (0.218 g L-1 d-1). B. braunii and Scenedesmus sp. were found to grow better using flue gas as compared to air enhanced with CO2. This is similar with an earlier study by Brown (1996) who found that microalgae can tolerant with flue gas very well.

In contrast, CO2 capture from flue gas emissions from power plants that burn fossil fuels achieves better recovery due to the higher CO2 concentration of up to 20% (Bilanovic et al., 2009), and adaptability of this process for both photobioreactor and raceway pond systems for microalgae production (Brennan and Owende, 2010). Flue gases from power plants are responsible for more than 7% of the total world CO2 emissions from energy use (Kadam, 1997). Also, industrial exhaust gases contains up to 15% CO2 (Maeda et al., 1995; Kadam, 2001), providing a CO2-rich source for microalgae cultivation and a potentially more efficient route for CO2 bio-fixation. Table 3 summarized the productivity of biomass grown outdoors in the various photobioreactors. These outdoor cultivation showed the potential of microalgal cultivation for on-site bioremediation of CO2 from flue gas.

In order to have an optimal yield, these algae need to have CO2 in large quantities in the basins or bioreactors where they grow. Thus, the photobioreactors need to be coupled with traditional electricity-producing thermal power centers that produce CO2 at an average rate of 13% of the total flue gas emissions. The CO2 is put into the photobioreactor and assimilated by the algae. Outdoor microalgal culture coupled with flue gas aeration is with economic value and potential strategy for large-scale microalgal cultivation.

GreenFuel Technologies, one of the earliest, best funded and most publicized algae companies was a startup that developed a process of growing algae using emissions from fossil fuel, mainly to produce biofuel from algae. A beta emission reduction system was

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installed at an MIT cogeneration facility in 2004 and after performing beyond expectations was moved to a larger power plant in fall 2005. Pilot units were tested at power plants in Arizona, Massachusetts and New York. Although the algal biomass produced by the process consists of proteins, lipids and carbohydrates which could be used to produce a variety of products, GreenFuel Technologies seems to be focusing on biofuel products. GreenFuel's large scale algae to biofuel process at the Arizona Public Service Redhawk power facility won the 2006 Platts Emissions Energy Project of the Year Award.

In 2007, the company had to shut down its third-generation bioreactor facility in Arizona after the plant produced more algae than the company’s equipment could handle. At the same time, the company learned that its algae harvesting system would cost twice as much as expected. Though GreenFuel Technologies finally shut down operations on May 13, 2009, GreenFuel Technologies was the frontier of industrialized process of microalgal cultivation and microalgal bioremediation. At present time, there are many microalgal industries for microalgal cultivation process and they also focus on microalgal bioenergy. Table 4 list the main international companies that using microalgal cultivation for CO2 reduction and bioerergy development.

1.9 Potential of Biodiesel Produced from Microalgae

There are four important potential benefits of algae biomass cultivation that other sources don’t have. First, algae biomass can be produced at extremely high volumes, and this biomass can yield a much higher percentage of oil than other sources. Second, algal oil has limited market competition. Third, algae can be cultivated on marginal land, fresh water, or sea water. Fourth, innovations to algae production allow it to become more productive while consuming resources that would otherwise be considered waste (Campbell, 2008). Biodiesel derived from oil crops is a potential renewable and carbon-neutral alternative to petroleum fuels. Unfortunately, biodiesel from oil crops, waste cooking oil, and animal fat cannot realistically satisfy even a small fraction of the existing demand for transport fuels.

Microalgae appear to be the only source of renewable biodiesel that is capable of meeting the global demand for transport fuels.

Like plants, microalgae use sunlight to produce oils, but they do so more efficiently than crop plants. Oil productivity of many microalgae greatly exceeds the oil productivity of the best producing oil crops. Approaches to making microalgal biodiesel economically

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