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Bioresource Technology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Bioresource Technology
Bioresource Technology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Bioresource Technology

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

journal homepage: www.elsevi er.com/locate/biortech Microalgae-based biorefinery – From biofuels to natural

Microalgae-based biorefinery – From biofuels to natural products

Hong-Wei Yen a , I.-Chen Hu b , c , Chun-Yen Chen d , Shih-Hsin Ho e , Duu-Jong Lee f , Jo-Shu Chang d , e , g ,

a Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan

b Far East Bio-Tec Co. Ltd., Taipei, Taiwan

c Far East Microalgae Ind Co. Ltd., Ping-Tung, Taiwan

d University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan

e Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan

f Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan

g Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan


" Components of microalgal biomass are suitable for biofuels production and biorefineries.

" Characterization and application of microalgae-based lipids, carbohydrates, pigments and proteins are elucidated.

" Critical comments were made on the role of microalgae in fermentation, food, and pharmaceutical industries.

article info

Article history:

Available online xxxx


Microalgae Carbohydrates Lipids Biorefinery Long-chain fatty acid


The potential for biodiesel production from microalgal lipids and for CO 2 mitigation due to photoautotro- phic growth of microalgae have recently been recognized. Microalgae biomass also has other valuable components, including carbohydrates, long chain fatty acids, pigments and proteins. The microalgae- based carbohydrates consist mainly of cellulose and starch without lignin; thus they can be ready carbon source for the fermentation industry. Some microalgae can produce long chain fatty acids (such as DHA and EPA) as valuable health food supplements. In addition, microalgal pigments and proteins have con- siderable potential for many medical applications. This review article presents comprehensive informa- tion on the current state of these commercial applications, as well as the utilization and characteristics of the microalgal components, in addition to the key factors and challenges that should be addressed during the production of these materials, and thus provides a useful report that can aid the development of an efficient microalgae-based biorefinery process.

2012 Elsevier Ltd. All rights reserved.

1. Introduction

It has been estimated that about 90% of our current energy con- sumption is provided by coal, natural gas and petroleum, with less than 10% coming from renewable energy sources ( Chen et al., 2011; Demirbas, 2010 ). Based on current consumption habits, it is very likely that there will be no more oil reserves after 2050 ( Campbell and Laherrère, 1998; Ho et al., 2011, 2012 ). Moreover, even if oil reserves remain plentiful, the associated environmental pollution, including the release of CO 2 , is widely seen as presenting a serious threat to the current global order, particularly through its effects on climate change ( Ho et al., 2011 ). Fixation of CO 2 by pho- tosynthetic organisms on earth has contributed significantly to the

Corresponding author at: University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan. Tel.: +886 6 2757575x62651; fax:

+886 6 2357146. E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).

0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.

global carbon cycle. The CO 2 produced from natural or human activities can be consumed by plants and algae, converting it to biomass and other metabolic products through photosynthesis and Calvin cycle. Phototrophic growth of microalgae can transport atmospheric carbon into a cycle in which no additional CO 2 is cre- ated. Since microalgae-based CO 2 fixation is much faster and more efficient than that of terrestrial plants, it has thus been considered to have the potential to serve as a commercially feasible process for mitigation of CO 2 emissions ( Ho et al., 2011 ). Most microalgae are unicellular photosynthetic microorganisms that can fix the dis- solved inorganic carbon and CO 2 in the gaseous effluents to form chemical energy through photosynthesis. The majority of microal- gae have much higher cell growth and CO 2 fixation rate (around 10–50 times higher) than terrestrial plants. Moreover, the CO 2 fix- ation is accompanied by production of microalgae biomass, which could be converted to a variety of biofuels, pigments, cosmetic, nutritious food and animal feed, representing additional benefits from the microalgae CO 2 fixation process ( Ho et al., 2011 ).

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorefinery – From biofuels to natural products. Bioresour. Technol. (2012) , http://

2 H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Microalgal biomass cultivation is


H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx

Microalgal biomass cultivation is regarded as a potential way to overcome our current reliance on fossil fuels for a number reasons, such as the high area yields compared with other crops, high oil contents in some strains, low water consumption rates, and the possibility of producing microalgae on infertile lands. These advan- tages have attracted several oil companies, such as Exxon, BP, Chevron, Shell and Neste Oil, to invest in this area ( Mascarelli, 2009 ). Moreover, a recent study indicated that the production of microalgal biofuels is relatively close to being economically feasi- ble, given expected developments in both market conditions and production technology ( Stephens et al., 2010 ). In addition to the microalgal lipids that could potentially be converted to practi- cal biodiesel, the biomass of microalgae also contains many other valuable components, including carbohydrates, lipids, polyunsatu- rated fatty acid, pigments and proteins, all of which are worth developing into refined products for various applications ( Lammens et al., 2012 ). Nevertheless, there are still several problems to be solved dur- ing the development of microalgae-based biorefinery technologies. The most challenging problems for the microalgae industry include high installing and operating cost, difficulty in controlling the cul- ture conditions, contamination bacteria or alien algae, unstable light supply and weather. Several strategies have been proposed to cope with those challenges. First, it is of great importance to isolate good microalgae/cyanobacteria strains that are rich in the target products, tolerate high (or low) temperature, and easily become predominant in the culture environment. Attaining good microalgae strains is the key factor leading to stable and sustain- able microalgae cultivation, which is extremely important in industrial applications of microalgae. Next, identifying preferable culture conditions for improving target-product production as well as designing efficient and economical feasible microalgae cultiva- tion system (or photobioreactors) are also critical to improve the productivity of microalgal biomass or target products ( Chen et al., 2011 ). In particular accumulation of different components (such as lipids, carbohydrates, proteins and pigments) in microal- gae requires different cultivation conditions and operation strategies. These would rely on more understanding on the photo- synthetic metabolism and physiology of the microalgae used and more advanced engineering technology that could help design more appropriate photobioreactors and establish better environ- ment for microalgae growth and product formation. Finally, high- efficiency and low-cost downstream processing, which accounts for the majority of operating cost, should be developed. For exam- ple, some innovative microalgae harvesting approaches have been proposed ( Show et al., 2012; Lee et al., 2012; Cheng et al., 2011 ). A novel one-step transesterification method for producing microal- gae-based biodiesel using wet oil-bearing microalgae biomass was also reported ( Tran et al., 2012a,b ). This method allows biodie- sel synthesis from microalgae without dewatering and oil extrac- tion steps. In addition, appropriate treatment of the wastes produced from microalgae systems as well as recycling of water used during microalgae cultivation processes are also critical is- sues. Finally, life cycle analysis, energy balance and cost assess- ment should also be performed to justify the economic feasibility and environmental impacts. In this review, we provide up-to-date information on the forma- tion mechanisms, production and applications of those microalgae- based components (namely, carbohydrates, lipids, pigments and proteins) ( Olguin, 2012 ). For example, microalgal carbohydrate can serve as a carbon source to replace traditional crop carbohy- drates in the fermentation industry, while lipids extracted from the microalgal biomass could be used as a potential feedstock for biodiesel production. In addition, some long chain fatty acids (such as DHA and EPA) are important health food supplements, and sev- eral proteins ( Aikawa et al., 2012 ) and pigments found in microalgae

have been applied in the pharmaceutical industry in the treatment of diseases. Therefore, microalgae could play important roles in pro- ducing biofuels and bio-based chemicals based on both their natural components and refined (or fermented) products. This review aims to provide a comprehensive report on the production and applica- tion of the natural or refined products of microalgae in order to aid the greater the utilization of microalgal biomass (also known as a third generation feedstock) for different purposes.

2. Carbohydrates from microalgae

Carbohydrates are one of the most important sources of energy and biological nutrients. Algae have a relatively high photo conver- sion efficiency, and are able to accumulate a high carbohydrate con- tent (potentially higher than 50% of its dry weight) ( Ho et al., 2012 ). In general, the algal carbohydrates are mainly composed of starch, glucose, cellulose/hemicelluloses, and various kinds of polysaccha- rides. Of these, algal starch/glucose is conventionally used for biofuel production, especially for bioethanol ( John et al., 2011 ) and hydrogen ( Chochois et al., 2009 ), while polysaccharides have various important biological functions in algae cells, mainly as stor- age, protection and structural molecules ( Arad and Levy-Ontman, 2010 ). Recently, algal polysaccharides (e.g. seaweed) have come to be regarded as new bioactive materials, due to their novel struc- tures and distinct biological functions. The monosaccharides of fructose, galactose, glucose, mannose and xylose, consist of microalgal polysaccharides in different ratios. Currently, algal poly- saccharides represent a class of high-value compounds with many downstream applications in food, cosmetics, textiles, stabilizers, emulsifiers, lubricants, thickening agents and clinical drugs ( Arad and Levy-Ontman, 2010 ). In particular, algal polysaccharides con- tain sulfate esters called sulfated polysaccharides (e.g., fucoidan, carrageenans and agarans), and are gaining wide attention due to their unique medical applications. Although the pharmaceutical mechanism of algal polysaccharides is still under investigation, the basic mechanism of their therapeutic effects is based on macro- phage stimulation and modulation. As a general rule, the biological activity of sulfated polysaccharides changes with their sugar com- position and degree of sulfation ( Kim et al., 2012 ). Algal sulfated polysaccharides have been shown to exhibit a wide range of phar- macological activity, including acting as antioxidant, antitumor, anticoagulant, anti-inflammatory, antiviral and immunomodulat- ing agents ( Table 1 ) ( Chen et al., 2010; Guzman et al., 2003; Kim et al., 2012; Matsui et al., 2003; Mohamed, 2008; Park et al., 2011; Tannin-Spitz et al., 2005 ). Many microalgal polysaccharides can modulate the immune system via activating the functions of macrophages and inducing the production of reactive oxygen species (ROS), nitric oxide (NO) and various kinds of cytokines/chemokines ( Schepetkin and Quinn, 2006 ). Macrophages are able to regulate various innate re- sponses, as well as secrete connecting cytokines and chemokines, such as interleukin (IL)-6, IL-8, IL- b and the tumor necrosis factor (TNF-a ), which are the signaling molecules for the immune system and inflammatory reactions ( Park et al., 2011 ). For example, Tannin-Spitz and his colleagues mentioned that the main function of the cell-wall sulfated polysaccharide from the red microalga Por- phyridium sp. is to overcome extreme environmental factors, and thus it has a protective response against the oxidative stress im- posed by ROS (representing antioxidant activity) ( Tannin-Spitz et al., 2005 ). The sulfated polysaccharides derived from Porphyridi- um sp. have significant potential for use in anti-inflammatory skin treatments because of their ability to inhibit the migration and adhesion of polymorphonuclear leukocytes (PMNs) ( Matsui et al., 2003 ). The sulfated polysaccharides of Haematococcus lacustris significantly stimulate murine macrophages to secrete the pro-

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorefinery – From biofuels to natural products. Bioresour. Technol. (2012) , http://

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Table 1 The pharmacological functions

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx

Table 1 The pharmacological functions found in the microalgae.



Polysaccharide extracts

Pharmacological significance




Crude polysaccharide


Mohamed (2008) Mohamed (2008) Tannin-Spitz et al. (2005) Matsui et al. (2003) Park et al. (2011) Kim et al. (2012) and Lee et al. (2009) Chen et al. (2010) Guzman et al. (2003) Guzman et al. 2003)



Crude polysaccharide Crude polysaccharide Sulfated polysaccharide


Porphyridium sp.


Porphyridium sp.




Water-soluble polysaccharide



impudium KG-03

Sulfated polysaccharide




Extracellular polysaccharide




Crude polysaccharide




Crude polysaccharide


inflammatory cytokine, indicating its potent immune stimulating activities ( Park et al., 2011 ), although the detailed molecular mech- anisms of macrophage activation are not currently known. Several types of microalgal sulfated polysaccharides show wide-spectrum antiviral activity because they are capable of specific interactions with viral particles or cellular surface molecules, resulting in the unique inhibition of virus-type or host cell-type independent activ- ity ( Kim et al., 2012 ). The sulfated polysaccharide p-KG03 of Gyrodinium impudium has specific antiviral activities, because it not only inhibits the binding site of the influenza A virus to host cells (virus-cell interaction), but also prevents cellular internaliza- tion of the virus (virus-cell fusion) ( Kim et al., 2012 ). Therefore, microalgal polysaccharides have attracted considerable attention as sources of biologically active molecules, in particular as natural therapeutic agents, cosmetic additives and functional food ingredients.

3. Lipids from microalgae

Many lipids are accumulated by algae (normally accounting for 30–50% of their content by weight) under several specific cultural conditions, such as in a high C/N medium or under stress condi- tions. Microalgal lipids are classified into two types according to their carbon numbers, with fatty acids containing 14–20 carbons used for biodiesel production, and polyunsaturated fatty acids (with over 20 carbons) used as health food supplements. Two parameters are generally considered for the evaluation of lipid accumulation for biofuel production: one is the lipid content (% lipid per dry weight of biomass), and the other is the lipid produc- tivity (amount of lipid produced per liter of working volume per day). Both the lipid content and biomass production rate should be considered simultaneously to ensure efficient microalgal lipid production, with a carbon number in the range of 14–20 being suitable for biodiesel production. In contrast, polyunsaturated fatty acids (PUFAs) with more than 20 carbons are high value com- pounds for use in the health food market, as they are essential nutrients that cannot be synthesized by higher eukaryotes. Among all of the commercially produced microalgal PUFAs, eicosapentae- noic acid (EPA, 20:5, x -3) and docosahexaenoic acid (DHA, 22:6, x -3) are reported to have levels of bioactivity, and thus are of par- ticular interest. Studies have shown that microalgae may contain large quantities of high-quality EPA and DHA, and thus they are considered a good potential source of these valuable fatty acids ( Spolaore et al., 2006; Vazhappilly and Chen, 1998 ).

3.1. Lipid biosynthesis metabolism of microalgae

Microalgae are able to produce a wide variety of biofuels. The main energy-rich compounds stored in microalgae are triacylglyc- erol (TAG) and starch, which can be used to produce biodiesel and bioethanol, respectively. Recently, more attention has been paid to producing lipids from microalgae for biodiesel synthesis. There are

three major steps in the synthesis pathway of TAG ( Fig. 1 ): (1) the conversion of acetyl-CoA to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACCase); (2) the elongation of the carbon chain of fatty acids; and (3) TAG formation ( Huang et al., 2010 ). The conversion of acetyl-CoA and CO 2 into malonyl-CoA, con- sidered the first phase in fatty acid synthesis, occurs in chloroplasts ( Hu et al., 2008 ). This reaction has two steps, and is catalyzed by a single enzyme complex. In the first step, CO 2 (from HCO 3 - ) is trans- ferred to nitrogen by the biotin carboxylase prosthetic group of AC- Case in a biotin prosthetic group attached to the e -amino group of lysine residue, which is ATP-dependent. In the second step, the activated CO 2 is transferred from biotin to acetyl-CoA to form mal- onyl-CoA, which is catalyzed by carboxyltransferase. The fatty acid elongation in the fatty acid synthesis cycle condenses malonyl-CoA molecules and acetyl-CoA; after several repeated reaction steps, saturated C16 and C18-ACP are formed, and then the ACP-thioesterase cleaves the acyl chain and liberates the fatty acid ( Courchesne et al., 2009 ). The first step of TAG formation is the condensation of glycerol-3-phosphate with acyl-CoA and the for- mation of lysophosphatidic acid. This reaction is catalyzed by GPAT (glycerol-3-phosphate acyltransferase), which exhibits the lowest specific activity of the TAG synthesis pathway, and is suggested to be the rate-limiting step ( Courchesne et al., 2009 ). After this, phosphatidic acid, diacylglycerol and TAGs are synthesized by a series of catalytic reactions.

3.2. Strategies to enhance microalgal lipid production

In both lab-scale and pilot-scale microalgae cultivation systems, growth characteristics and microalgae composition are known to significantly depend on the environmental factors and medium composition. The performance of lipid production should be eval- uated in two ways: one is the lipid contents (% lipid per dry weight of biomass) and the other is the lipid productivity (amount of lipid produced per liter of working volume per day). Both lipid content and biomass production rates should be considered, simulta- neously, to reach efficient microalgal lipid production. Lipid accu- mulation in microalgae usually occurs at the expense of slower cell growth, due to the stress conditions. Therefore, it is more rea- sonable to evaluate lipid production performance using the con- cept of lipid productivity, which is more important from an engineering aspect ( Chen et al., 2011 ). The most well known factors influencing lipid production efficiency include light illumi- nation strategy, temperature and nitrogen source availability (e.g., nitrogen starvation). These factors can affect either the lipid accu- mulation or cell growth rate, or both. First, the type of light source is known to affect the growth of microalgae, due mainly to the dif- ference in the coverage of the wavelength range. Light intensity is also known to be a critical factor in microalgae growth. In order to obtain maximum productivity, the saturation light intensity needs to be distributed throughout the entire photobioreactor; however, this is impractical in practice, since the light distribution inside the

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorefinery – From biofuels to natural products. Bioresour. Technol. (2012) , http://

4 H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Fig. 1. Microalgae metabolic


H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx

Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Fig. 1. Microalgae metabolic pathways for different

Fig. 1. Microalgae metabolic pathways for different kinds of biofuel production.

photobioreactor will decrease rapidly due to the light shading effects arising from the increased concentration of cells and prod- ucts, or from the formation of biofilm on the surface of the reactor vessels ( Chen et al., 2008 ), although mixing the cells well can re- duce the impacts of these. In addition, some reports investigated the effect of light intensity on the lipid contents of microalgae. Under a high light intensity the total polar lipid content decreases, while the contents of neutral storage lipids (TAGs) increases ( Hu et al., 2008 ). The culture temperature can also affect the degree of saturation, and an increase in the content of saturated fatty acids was observed when the temperature was increased ( Hu et al., 2008 ). The lipid content is also sometimes influenced by tempera- ture. For instance, the lipid content of Nannochloropsis salina and Ochromonas danica increased with an increase in the culture tem- perature ( Boussiba et al., 1987 ). Nevertheless, other studies showed that there was almost no difference in the lipid contents of Chlorella Sorokiniana when it was grown at various temperatures ( Patterso, 1970 ). Lipid accumulation in microalgae usually occurs when they are cultivated under stress conditions. The major physical stimuli are temperature and light intensity, while the major chemical stimuli are pH, salinity, mineral salts and nitrogen source ( Hu et al., 2008 ). Among these stress conditions, nitrogen limitation is the most widely used and reliable strategy for increasing the lipid content of microalgae. Hu and his colleagues collected the data for the lipid content of various classes of microalgae and cyanobacteria under normal growth and stress conditions ( Hu et al., 2008 ). Their results clearly showed that the lipid content of green microalgae, diatoms and some other species under stress conditions was 10–20% higher than under nitrogen sufficient conditions. However, the lipid content of cyanobacteria produced under stress conditions was

usually less than 10%, making it unsuitable for lipid production. Var- ious nitrogen limitation strategies have been applied to foster lipid accumulation, and these can mainly be divided into two types. The first is a two-stage nitrogen limitation method in which the micro- algae cells are cultivated under nitrogen-sufficient conditions to stimulate cell growth for a set amount of time. They are then col- lected and switched to nitrogen-starving conditions for lipid accu- mulation. The second method is a one-stage approach in which the initial nitrogen concentration is adjusted to a desirable level to control the time required to reach nitrogen starvation. The microal- gae culture will then automatically advance to the nitrogen starva- tion stage when the nitrogen content in the medium is depleted. The effects of the two types of nitrogen starvation on lipid produc- tion efficiency seem to be microalgal species-dependent.

3.3. Producing biodiesel from microalgal lipids

Plant or microalgal oil, which contains triglycerides, is used to make biodiesel via the transesterification process. The triglycerides react with methanol in a reaction called transesterification or alco- holysis and then methyl esters of fatty acids (biodiesel) and glyc- erol are produced. Each mole of triglyceride requires 3 mol of alcohol to produce 1 mol of glycerol and 3 mol of methyl esters. In industrial processes, 6 mol of methanol are used for each mole of triglyceride to ensure that the reaction is driven in the direction of methyl esters. Because the alkali-catalyzed transesterification is about 4000 times faster than the acid-catalyzed reaction, alkalis such as NaOH and KOH are commonly used as the catalyst. However, there is a major problem when using an alkali-catalyzed process. A saponifi- cation reaction occurs when free fatty acids are present in the

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H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Table 2 DHA production by

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx

Table 2 DHA production by microalgae in the literature.



DHA productivity (g/L day)

DHA content (mg DHA/g biomass)

DHA (% of total fatty acids)


Schizochytrium sp. Crypthecodinium cohnii Schizochytrium sp. Schizochytrium limacinum Schizochytrium sp. Schizochytrium limacinum Schizochytrium sp. Aurantiochytrium limacinum Aurantiochytrium sp. Aurantiochytrium




Yaguchi et al. (1997) Swaaf et al. (2003) Wu et al. (2005) Chi et al. (2007) Ganuza et al. (2008) Chi et al. (2009) Ren et al. (2010) Rosa et al. (2010) Hong et al. (2011) Yang et al. (2012)
























Table 3 EPA production by microalgae in the literature.



EPA productivity (mg/L day)

EPA content (mg EPA/g biomass)

EPA (% of total fatty acids)


Phaeodactylum tricornutum Navicula saprophila Rhodomonas salina Nitzschia sp. Monodus subterraneus Chlorella minutissima Phaeodactylum tricornutum Monodus subterraneus Nitzschia laevis Nitzschia laevis Monodus subterraneus



Reis et al. (1996) Kitano et al. (1997) Kitano et al. (1997) Kitano et al. (1997) Vazhappilly and Chen (1998) Vazhappilly and Chen (1998) Vazhappilly and Chen (1998) Lu et al. (2001) Wen and Chen (2001) Wen et al. (2002) Lu et al. (2002)


























triglyceride feed. Since high-quality feedstock needs to be pro- duced to prevent this problem, the cost of raw materials usually accounts for 60–75% of the total cost of the biodiesel fuel ( Huang et al., 2010 ). Acid catalysts, which can be used for feedstock with high free fatty acid content, could overcome the saponification problems that arise in an alkali-catalyzed process. Nevertheless, reactions catalyzed by acid catalysts have lower reaction rates and yields than those catalyzed by an alkali process. Therefore, a two-stage biodiesel production, which converts free fatty acids to methyl esters under an acid-catalyzed process in the primary stage followed by an alkali-catalyzed process, has been examined in a number of studies ( Behzadi and Farid, 2007 ). Biodiesel is now produced mainly from vegetable oils; however, these are mostly derived from food crops, raising the issue of food- fuel competition. In addition, because of the increasing demand for these oil crops, their market price has increased, thus raising the cost of producing biodiesel. Alternative feedstocks thus need to be developed to solve this problem. Among the various candidates for this, microalgae appear to be one of the most promising ones ( Trzcinski et al., 2012 ), due to their high lipid content, fast growth rate and large lipid production capacity. The lipid content in micro- algal biomass can range from 16% to 77%, with levels of over 30% commonly seen in a variety of microalgal species. The advantages of using microalgae as a source of biodiesel feedstock are as follows ( Um and Kim, 2009 ):

1. The growth of microalgae is extremely fast compared with terrestrial plants, and the biomass can be doubled within 24 h.

2. The oil content of the microalgal biomass can reach over 50% of the dry cell weight.

3. The oil yield by cultivated area is larger than that of the oil- seed crops.

4. Microalgae are aquatic microorganisms, and thus do not compete for the land needed for agriculture crops.

5. The production of microalgae does not compete with human food production.

6. Microalgae are able to grow under conditions that are not suitable for conventional crops.

7. Microalgae can convert CO 2 into biomass, and may reduce the CO 2 concentration in the atmosphere.

8. The biofuels produced from microalgae do not contain sulfur, and are non-toxic and highly biodegradable.

However, there are several critical issues regarding the produc- tion of biodiesel from microalgae. Compared to terrestrial oil crops, the harvest of, and oil extraction from, microalgae is much more dif- ficult, leading to a higher production cost for microalgae-based biodiesel. Normally, the dry microalgal biomass only accounts for 0.1–1% by weight of the microalgae culture. As a result, concentra- tion of the microalgal biomass and the dewatering processing are both costly and energy intensive. In addition, since microalgae are small in size, the microalgal lipid cannot be easily extracted by con- ventional mechanical methods used for obtaining oil from oil crops, such as palm trees or soybeans, raising another technical hurdle for converting microalgal lipid into biodiesel ( Chen et al., 2011 ). Re- cently, wet extraction approaches have been used to enhance the efficiency and economic feasibility of lipid extraction from microal- gae. In this way, FAME yields of 84% from microalgae can be achieved under a water content of 50% (w/w) using methanol as the co-solvent ( Wahlen et al., 2011 ). Another approach is using dry microalgae powder as the starting material to make biodiesel with a direct or in situ transesterification process. In these ap- proaches, the lipid extraction and transesterification can be done simultaneously in one step, leading to a simplified process and reduced production costs. However, intensive dewatering of the microalgal biomass is still required to avoid the problems associ- ated with transesterification reactions. More recently, ( Tran et al., 2012a,b) demonstrated an innovative approach using wet microal- gae (up to 90 wt.% water content) as the feedstock to produce bio- diesel in the presence of a solvent (i.e., hexane), an excessive amount of methanol, and immobilized lipase via a one-step lipid extraction and transesterification process. They achieved a high biodiesel conversion of 97.3% when using a wet biomass of Chlorella

6 H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx Table 4 The photosynthetic


H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx

Table 4 The photosynthetic pigments.


Characteristic absorption maxima (nm)



(in organic solvents) 420, 660 435, 643 445, 625 450, 690 (in organic solvents) 420, 440, 470 425, 450, 480 425, 445, 475 425, 450, 475 375, 495 (in water) 490, 546, 576


Chlorophyll a

All higher plants and algae All higher plants and green algae Diatoms and brown algae Red algae

Chlorophyll b

Chlorophyll c

Chlorophyll d



a -Carotene

Most plants and some algae Higher plants and most algae Green algae, red algae, and higher plants Diatoms and brown algae Dinophytes

b -Carotene







Red algae and some cyanobacteria Cyanobacteria and some red algae Cyanobacteria and red algae





vulgaris microalgal biomass (lipid content = 63% per dry weight) in the presence of 71% water content ( Tran et al., 2012a,b ).

3.4. Polyunsaturated fatty acids

Besides using the fatty acids of 14–20 carbons for biodiesel pro- duction, the polyunsaturated fatty acids (PUFAs) with more than 20 carbons are high value compounds in the health food market. Higher forms of plants and most animals lack the required en- zymes to synthesize PUFAs from more than 18 carbons, although they are essential for good functioning, conferring flexibility, fluid- ity and selective permeability properties to membranes. PUFAs consist of three or more double bonds on a fatty acid skeletal chain containing 18 or more carbons, and they are further classified into x -6 and x -3 forms, depending on the position of the last double bond proximal to the methyl end of the fatty acid. Fish and fish oils are common sources of long-chain PUFAs, and since the PUFAs found in fish originate from digested microalgae in oceanic envi- ronments, it is reasonable to consider microalgae as a direct poten- tial source of these substances. Among all the commercially produced microalgal PUFAs, eico- sapentaenoic acid (EPA, 20:5, x -3) and docosahexaenoic acid (DHA, 22:6, x -3) are reported to have bioactivities of particular interest. Moreover, microalgae contain large quantities of high- quality EPA and DHA, and are thus considered a potential source of this important fatty acid. Numerous strategies have been inves- tigated for commercial production of EPA and DHA using microal- gae, including screening of high EPA and DHA-yielding microalgal strains, improvement of strains by genetic manipulation, optimiza- tion of culture conditions (such as pH, temperature and the ratio of C/N), and the development of efficient cultivation systems. The productivity and yield figures shown in Tables 2 and 3 reveal that the production of EPA and DHA by algae is a potential replacement for the conventional extraction of EPA and DHA from fish meats. The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1500-L bioreactor using fed-batch fermentation. The aeration rate was controlled at a 0.4 volume of air per volume of liquid per min (vvm) for the first 24 h, then shifted to 0.6 vvm for 96 h, and finally switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L) and a high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained with a constant aeration rate ( Ren et al., 2010 ). In order to achieve lipid accumulation in a microorganism, it is necessary to grow it in a medium with excess carbon sub- strates and limited amounts of other nutrients, usually nitrogen.

However, different nutritional conditions are likely to be required for the stages of growth and DHA accumulation. Therefore, a two-stage cultivation process (growth and production) was per- formed for DHA production by Aurantiochytrium limacinum SR21, which produced 154 mg DHA/L/h ( Rosa et al., 2010 ).

4. Pigments and Proteins from microalgae

The colorful appearance of algae is derived from their pigments,

which absorb visible light and initiate photosynthesis reactions. The three major classes of photosynthetic pigments that appear in algae are chlorophylls, carotenoids and phycobilins ( Table 4 ) ( Hall and Rao, 1999 ). Chlorophyll a is the primary pigment in all photosynthetic organisms, and it absorbs the most energy from the wavelengths of violet-blue and orange-red light, and then serves as a primary electron donor in the electron transport chain. The other accessory pigments include chlorophyll b (also c and d in different algae), carotenoids (such as beta-carotene in most algae) and phycobilins (in cyanobacteria and red algae) ( Hall and Rao,

1999 ).

4.1. Chlorophylls

Chlorophylls are greenish pigments which contain a porphyrin (tetrapyrrole ring), a central magnesium atom, side chains and a phytol tail. Chlorophyll is situated in the chloroplast lamellae in

a form where the porphyrin head binds to the protein layers, and

a phytol tail extends into the lipid layers. Green algae have the highest chlorophyll content among all algae, and this well-commercialized green alga belongs to the Chlorella species. Chlorella is produced worldwide, with an annual production of 2,000 metric tons of dry powder, over 50% of which is manufactured in Taiwan ( Milledge, 2011 ). Pheophorbide a is a chlorophyll derivative obtained from removing the phytol and the central magnesium atom. A high dose of pheophorbide a will cause an allergic reaction in humans, including inflammation and

a red rash on the skin. As a result, the pheophorbide content in food

containing algae (such as Chlorella , Spirulina and Nori (a purple la-

ver product)) is limited by food safety and hygiene regulations. These levels have been set by the Department of Health in Taiwan (<0.8 mg/g for Chlorella and < 1 mg/g for Spirulina ) and Japan (0.8 mg/g) for more than 20 years ( Lembi and Waaland, 1988 ), with both species being widely consumed in these countries for health reasons. The photosensitizer characteristics of pheophorbide mean that it has been applied in photodynamic therapy (PDT) since the

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1990s ( Busch et al., 2009 ). Three key components of modern PDT applications are photosensitizers, light sources and tissue oxygen. The process occurs as follows: a light source with the appropriate wavelength excites the photosensitizer to produce a reactive oxy- gen species, and then the reactive singlet oxygen rapidly reacts with any nearby molecules. These destructive reactions will kill the target, such as malignant cancers, through apoptosis or necro- sis ( Chen et al., 2002 ). Pheophorbide a- based PDT has been utilized in treating human uterine sarcoma line MES-SA, human colon adenocarcinoma, human hepatoma (Hep3B cells) and rat pancre- atic cancer ( Busch et al., 2009 ). There are currently two FDA-approved photosensitizers, Photofrin and aminolevulinic acid (ALA), which induce protoporphyrin IX (PpIX). Commercial pheo- phorbide a is now available from Frontier Scientific, Inc. (Logan, UT, USA) (Logan, UT, USA).

4.2. Carotenoids

former is in limited supply and has a low astaxanthin content, while the latter has a astaxanthin content (0.4–2.5%) much lower than that seen in microalgae ( Muntendam et al., 2009 ). The annual worldwide aquaculture market for astaxanthin was estimated at US$200 million in 2004, with estimations of the global astaxanthin market rising to US$257 million in 2009 (Del Campo et al., 2007). Synthetic astaxanthin is valued at US$2500/kg, while the natural product is sold for over US$7000/kg. Although 95% of mar- ket is met by synthetic astaxanthin, consumer demand for natural products provides an opportunity for the sale of Haematococcus asta- xanthin. There are currently five major producers of Haematococcus astaxanthin: Algatechnologies, Ltd. (Kibbutz Ketura, Israel), BioReal Inc. (Kihei, Hawaii, USA, a subsidiary of Fuji Chemical Industry, Toyama, Japan), Cyanotech Corporation (Kailua-Kona, Hawaii, USA), Mera Pharmaceuticals (Kailua-Kona, Hawaii, USA) and Parry’s Pharmaceuticals (Chennai, India) (Dufossé et al., 2007).

Carotenoids are yellow, orange or red pigments found in most photosynthetic organisms. Carotenoids belong to the group of tetraterpenoids with a 40-carbon skeletal formation built up from isoprene subunits. Two small six-carbon rings are connected to the carbon skeleton. The known structures of carotenoids mentioned in this review are shown in ( Takaichi, 2011 ). Carotenoids are insol- uble in water and are usually attached to membranes within cells, being situated in the chloroplast in most algae or photosynthetic lamellae of cyanobacteria. Carotenoids serve as photo-protectors against the photo-oxidative damage resulting from excess energy captured by light-harvesting antenna ( Xiao et al., 2011 ). Astaxanthin is an oxygenated carotenoid, also a kind of ketoca- rotenoid, known for its powerful antioxidant activity. The reported antioxidant activity of astaxanthin is 10 times higher than that of b -carotene and more than 500 times that of a -tocopherol ( Dufossé et al., 2007 ). It is also a potent quencher of reactive oxygen and nitrogen species, including singlet oxygen and single- and two- electron oxidants, as shown in several in vitro studies ( Fassett and Coombes, 2011 ). Astaxanthin has many benefits in the preven- tion and treatment of various conditions, such as chronic inflam- matory diseases, eye diseases, skin diseases, cardiovascular diseases, cancers, neurodegenerative diseases, liver diseases, met- abolic syndrome, diabetes, diabetic nephropathy and gastrointesti- nal diseases. This powerful antioxidant activity means that there have been a wide range of applications of astaxanthin in the food, dietary supplements (or nutraceuticals), cosmetics and food indus- tries. The United States Food and Drug Administration (US FDA) approved astaxanthin as a food additive for use in the aquaculture industry in 1987, and in 1999 astaxanthin was further approved for use as a dietary supplement ( Guerin et al., 2003 ). Natural sources of astaxanthin are microalgae, yeast, shrimp, krill and plankton. Astaxanthin also provides the red color of sal- mon meat and the feathers of some birds after these animals have ingested it. Haematococcus pluvialis is a freshwater green alga that can synthesize and accumulate astaxanthin under oxidative stress. Its life cycle consists of four cell stages: vegetative cell growth, encystment, maturation and germination ( Collins et al., 2011; Kobayashi et al., 1997 ). The vegetative cells are motile flagellated cells with an optimum growth temperature of 22–25 C. Stress conditions, such as nutrient deprivation, high salinity, strong irra- diance, high temperature or combinations of these stresses, trigger carotenogenesis and cell differentiation from the vegetative cells stage to cyst cells. The cyst cells are nonmotile aplanospores with thick walls. Astaxanthin is contained in aplanospore ( Lorenz et al., 2000 ), and its content can reach up to 4% of the total cellular dry weight ( Collins et al., 2011; Kobayashi et al., 1997 ). Other natural sources of astaxanthin are crustacean exoskeletons and yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) ; however, the

4.3. Phycobilins/phycobiliproteins from algae

The chemical structures of phycobilins are related to those of chlorophylls. Each phycobilin is a linear tetrapyrrole with four pyr- role rings that are linked together by single-atom bridges. Phycobi- lins are covalently attached to polypeptides to form water-soluble phycobiliproteins. The phycobilins further give phycobiliproteins a distinct absorption spectra, ranging from 460 to 670 nm, in which chlorophyll a has a low absorption. Phycobiliproteins are classified into three types by their phycobilin energy (absorption spectra):

high-energy ones are phycoerythrins (PEs) or phycoerythrocyanins (PECs), with their main absorption at 480–580 nm; intermediate- energy ones are phycocyanins (PCs), with their main absorption at 600–640 nm; and low-energy ones are allophycocyanins (APCs), with their main absorption at 620–660 nm. Purified phycobiliproteins are highly fluorescent because there are no nearby acceptors to receive the harvested energy. The phyco- biliproteins have several unique features, such as a broad absorp- tion in a visible light spectrum, a high extinction coefficient, high fluorescence quantum efficiency, a large Stokes shift and very little fluorescence quenching. These features make phycobiliproteins promising fluorescent labeling reagents that can be employed in flow cytometry, fluorescence immunoassay, fluorescence micros- copy, immuno-histochemistry and other biomedical research purposes (( Matamala et al., 2007; Waggoner, 2006 ). Blue phycobili- proteins, APC, can be produced by Spirulina sp., and red ones, B-PE and R-PE, are produced by red microalgae, such as Porphyridium sp., Rhodella sp. and Bangia sp., on a kilogram scale per year. The glo- bal market was estimated to be approximately US$50 million in 1997, with prices varying from US $3/mg to US $25/mg ( Milledge, 2011). The leading manufacturers and suppliers of phycobilipro- teins as a fluorescent labeling reagent are Far East Bio-Tec Co. Ltd. (Taiwan, also known as FEBICO with the product brand name of Flogen ) and ProZyme, Inc. (Canada).

5. Conclusions

The components of microalgae are valuable, with a wide range of applications. The carbohydrates present in microalgae are consid- ered an appropriate feedstock for microbial growth and for the pro- duction of various fermentation products. The high lipid content in algal biomass makes it promising for biodiesel production, while the related long-chain fatty acids, pigments and proteins have their own nutraceutical and pharmaceutical applications. Therefore, microalgal biorefinery processes deserve further investigations. In particular, economic feasibility and life cycle assessments of such processes should be conducted to confirm the commercial viability

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of converting the components of microalgae into biofuels and other valuable products.


The authors gratefully acknowledge the financial support from the top university project of NCKU and by Taiwan’s National Sci- ence Council under grant numbers NSC 101-3113-P-110-002, NSC 101-3113-E-006-015, and NSC 101-3113-E- 006-016.


Aikawa, S., Izumi, Y., Matsuda, F., Hasunuma, T., Chang, Jo-Shu, Kondo, A., 2012. Synergistic enhancement of glycogen production in Arthrospira platensis by optimization of light intensity and nitrate supply. Bioresour. Technol. 108, 211–


Arad, S., Levy-Ontman, O., 2010. Red microalgal cell-wall polysaccharides:

biotechnological aspects. Curr. Opin. Biotechnol. 21 (3), 358–364. Behzadi, S., Farid, M.M., 2007. Review: examining the use of different feedstock for the production of biodiesel. Asia-Pacific J. Chem. Eng. 2, 480–486. Boussiba, S., Vonshak, A., Cohen, Z., Avissar, Y., Richmond, A., 1987. Lipid and biomass production by the halotolerant microalga Nannochloropsis salina . Biomass 12, 37–47. Busch, T.M., Cengel, K.A., Finlay, J.C., 2009. Pheophorbide a as a photosensitizer in photodynamic therapy: in vivo considerations. Cancer Biol. Therapy 8, 540–542. Campbell, C.J., Laherrère, J.H., 1998. The End of Cheap Oil. Scientific American, March, pp. 78–83. Chen, B., You, W., Huang, J., Yu, Y., Chen, W., 2010. Isolation and antioxidant property of the extracellular polysaccharide from Rhodella reticulata . World J. Microbiol. Biotechnol. 26, 833–840. Chen, C.Y., Saratale, G.D., Lee, C.M., Chen, P.C., Chang, J.S., 2008. Phototrophic hydrogen production in photobioreactors coupled with solar-energy-excited optical fibers. Int. J. Hydrogen Energy 33, 6878–6885. Chen, C.Y., Yeh, K.L., Aisyah, R., Lee, D.J., Chang, J.S., 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production:

a critical review. Bioresour. Technol. 102, 71–81. Chen, J., Keltner, L., Christophersen, J., Zheng, F., Krouse, M., Singhal, A., Wang, S.S., 2002. New technology for deep light distribution in tissue for phototherapy. Cancer J. 8, 154–163. Chi, Z., Liu, Y., Frear, C., Chen, S., 2009. Study of a two-stage growth of DHA- producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl. Microbiol. Biotechnol. 81, 1141–1148. Chi, Z., Pyle, D., Wen, Z., Frear, C., Chen, S., 2007. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Proc. Biochem. 42, 1537–1545. Chochois, V., Dauvillee, D., Beyly, A., Tolleter, D., Cuine, S., Timpano, H., Ball, S., Cournac, L., Peltier, G., 2009. Hydrogen production in chlamydomonas:

photosystem II-dependent and -independent pathways differ in their requirement for starch metabolism. Plant Physiol. 151, 631–640. Cheng, Y.-L., Juang, Y.-C., Tsai, P.-W., Ho, S.-H., Chen, C.-Y., Chang, J.S., Chen, W.-M., Liu, J.M., Lee, D.J., 2011. Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation. Bioresour. Technol. 102, 82–87. Collins, A.M., Jones, H.D., Han, D., Hu, Q., Beechem, T.E., Timlin, J.A., 2011. Carotenoid distribution in living cells of Haematococcus pluvialis (Chlorophyceae). PLoS One 6, e24302. Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41. Del Campo, J.A., García-González, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74, 1163–1174. Demirbas, A., 2010. Social, economic, environmental and policy aspects of biofuels. Energy Edu. Sci. Technol. Part B Social Edu. Studies 2, 75–109. Dufossé, L. 2007. Pigments from microalgae and microorganisms: sources of food colorants. In: Food Colorants: Chemical and Functional Properties, CRC Press, pp. 399–426. Fassett, R.G., Coombes, J.S., 2011. Astaxanthin: a potential therapeutic agent in cardiovascular disease. Marine Drugs 9, 447–465. Ganuza, E., Anderson, A.J., Ratledge, C., 2008. High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system. Biotechnol. Lett. 30, 1559–1564.

Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin :

applications for human health and nutrition. Trends Biotechnol. 21, 210–216. Guzman, S., Gato, A., Lamela, M., Freire-Garabal, M., Calleja, J.M., 2003. Anti- inflammatory and immunomodulatory activities of polysaccharide from Chlorella stigmatophora and Phaeodactylum tricomutum . Phytother. Res. 17,


Hall, D.O., Rao, K.K., 1999. Photosynthetic apparatus, 6th ed. In: Photosynthesis, The Press Syndicate of The University of Cambridge, pp. 33–57.

Ho, S.-H., Chen, C.-Y., Chang, J.-S., 2012. Effect of light intensity and nitrogen starvation on CO 2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244–252.

Ho, S.-H., Chen, C.-Y., Lee, D.-J., Chang, J.-S., 2011. Perspectives on microalgal CO 2 - emission mitigation systems – a review. Biotechnol. Adv. 29, 189–198. Hong, W.-K., Rairakhwada, D., Seo, P.-S., Park, S.-Y., Hur, B.-K., Kim, C.H., Seo, J.-W.,

2011. Production of lipids containing high levels of docosahexaenoic acid by a

newly isolated microalga, Aurantiochytrium sp. KRS101. Appl. Biochem.

Biotechnol. 164, 1468–1480. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A.,

2008. Microalgal triacylglycerols as feedstocks for biofuel production:

perspectives and advances. Plant J. 54, 621–639. Huang, G.H., Chen, F., Wei, D., Zhang, X.W., Chen, G., 2010. Biodiesel production by microalgal biotechnology. Appl. Energy 87, 38–46. John, R.P., Anisha, G.S., Nampoothiri, K.M., Pandey, A., 2011. Micro and macroalgal

biomass: a renewable source for bioethanol. Bioresour. Technol. 102, 186–193. Kim, M., Yim, J.H., Kim, S.-Y., Kim, H.S., Lee, W.G., Kim, S.J., Kang, P.-S., Lee, C.-K.,

2012. In vitro inhibition of influenza A virus infection by marine microalga-

derived sulfated polysaccharide p-KG03. Antiviral Res. 93 (2), 253–259. Kitano, M., Matsukawa, R., Karube, I., 1997. Changes in eicosapentaenoic acid content of Navicula saprophila , Rhodomonas salina and Nitzschia sp. under mixotrophic conditions. J. Appl. Phycol. 9, 559–563.

Kobayashi, M., Kurimura, Y., Kakizono, T., Nishio, N., Tsuji, Y., 1997. Morphological changes in the life cycle of the green alga Haematococcus pluvialis . J. Ferment. Bioeng. 84, 94–97. Lammens, T.M., Franssen, M.C.R., Scott, E.L., Sanders, J.P.M., 2012. Availability of protein-derived amino acids as feedstock for the production of bio-based chemicals. Biomass Bioenergy 44, 168–181. Lee, C.-K., Kim, H.S., Nam, J.R., Lee, M.-J., Yim, J.-H., Lee, H.K., De Clercq, E., 2009. Anti-picornavirus activity and other antiviral activity of sulfated exopolysaccharide from the marine microalga Gyrodinium impudicum Strain KG03. Antiviral Res. 82, A40. Lee, D.-J., Liao, G.-Y., Chang, Y.-R., Chang, J.-S., 2012. Coagulation-membrane filtration of Chlorella vulgaris . Bioresour. Technol. 108, 184–189. Lembi, C.A., Waaland, J.R., 1988. Some public health aspects of microalgal products. In: Algae and Human Affairs, Cambridge University Press, pp. 181–202. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol. 18, 106–167. Lu, C., Fernández, F.G.A., Guerrero, E.C., Hall, D.O., Grima, E.M., 2002. Overall assessment of Monodus subterraneus cultivation and EPA production in outdoor helical and bubble column reactors. J. Appl. Phycol. 14, 331–342. Lu, C., Rao, K., Hall , D., Vonshak, A., 2001. Production of eicosapentaenoic acid (EPA) in Monodus subterraneus grown in a helical tubular photobioreactor as affected by cell density and light intensity. J. Appl. Phycol. 13, 517–522. Mascarelli, A., 2009. Gold rush for algae. Nature 461, 460–461. Matamala, A.R., Almonacid, D.E., Figueroa, M.F., Martinez-Oyanedel, J., Bunster, M.C.,

2007. A semiempirical approach to the intra-phycocyanin and inter-

phycocyanin fluorescence resonance energy-transfer pathways in phycobilisomes. J. Comput. Chem. 28, 1200–1207. Matsui, M.S., Muizzuddin, N., Arad, S., Marenus, K., 2003. Sulfated polysaccharides from red microalgae have antiinflammatory properties in vitro and in vivo. Appl. Biochem. Biotechnol. 104, 13–22. Milledge, J.J., 2011. Commercial application of microalgae other than as biofuels: a brief review. Rev. Environ. Sci. Biotechnol. 10, 31–41. Mohamed, Z.A., 2008. Polysaccharides as a protective response against microcystin- induced oxidative stress in Chlorella vulgaris and Scenedesmus quadricauda and their possible significance in the aquatic ecosystem. Ecotoxicology 17, 504–516. Muntendam, R., Melillo, E., Ryden, A., Kayser, O., 2009. Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts. Appl. Microbiol. Biotechnol. 84, 1003–1019. Olguin, E.J., 2012. Dual purpose microalgae–bacteria-based systems that treat wastewater and produce biodiesel and chemical products within a biorefinery. Biotechnol. Adv. 30, 1031–1046.

Park, J.K., Kim, Z.H., Lee, C.G., Synytsya, A., Jo, H.S., Kim, S.O., Park, J.W., Park, Y.I.,

2011. Characterization and immunostimulating activity of a water-soluble

polysaccharide isolated from Haematococcus lacustris . Biotechnol. Bioproc. Eng. 16, 1090–1098. Patterso, G.W., 1970. Effect of culture temperature on fatty acid composition of Chlorella sorokiniana . Lipids 5, 597–600. Reis, A., Gouveia, L., Veloso, V., Fernandes, H.L., Empis, J.A., Novai, J.M., 1996. Eicosapentaenoic acid-rich biomass production by the microalga Phaeodactylum tricornutum in a continuous-flow reactor. Bioresour. Technol. 55, 83–88. Ren, L.-J., Ji, X.-J., Huang, H., Qu, L., Feng, Y., Tong, Q.-Q., Ouyang, P.-K., 2010. Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. Appl. Microbiol. Biotechnol. 87, 1649–1656. Rosa, S.M., Soria, M.A., Vélez, C.G., Galvagno, M.A., 2010. Improvement of a two- stage fermentation process for docosahexaenoic acid production by

Aurantiochytrium limacinum SR21 applying statistical experimental designs and data analysis. Bioresour. Technol. 101, 2367–2374. Schepetkin, I.A., Quinn, M.T., 2006. Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int. Immunopharmacol. 6,


Show, K.-Y., Lee, D.-J., Chang, J.-S., 2012. Algal biomass dehydration. Bioresour. Technol. http://dx.doi.org/10.1016/j.biortech.2012.08.021 .

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx 9 Spolaore, P., Joannis-Cassan, C.,

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxx–xxx


Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. Stephens, E., Ross, I.L., King, Z., Mussgnug, J.H., Kruse, O., Posten, C., Borowitzka, M.A., Hankamer, B., 2010. An economic and technical evaluation of microalgal biofuels. Nat. Biotechnol. 28, 126–128.

Swaaf, M.E.D., Sijtsma, L., Pronk, J.T., 2003. High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii . Biotechnol. Bioeng. 81, 666–672. Takaichi, S., 2011. Carotenoids in algae: distributions, biosyntheses and functions. Mar. Drugs 9, 1101–1118. Tannin-Spitz, T., Bergman, M., van-Moppes, D., Grossman, S., Arad, S., 2005. Antioxidant activity of the polysaccharide of the red microalga Porphyridium sp. J. Appl. Phycol. 17, 215–222. Tran, D.-T., Yeh, K.-L., Chen, C.-L., Chang, J.-S., 2012a. Enzymatic transesterification of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour. Technol. 108, 119–127. Tran, D.-T., Chen, C.-L., Chang, J.-S., 2012b. Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized lipase as the biocatalyst. Bioresour. Technol. http://dx.doi.org/10.1016/j.biortech.2012.

09.101 . Trzcinski, A.P., Hernandez, E., Webb, C., 2012. A novel process for enhancing oil production in algae biorefineries through bioconversion of solid by-products. Bioresour. Technol. 116, 295–301. Um, B.H., Kim, Y.S., 2009. Review: a chance for Korea to advance algal-biodiesel technology. J. Ind. Eng. Chem. 15, 1–7.

Vazhappilly, R., Chen, F., 1998. Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth. JAOCS 75,


Waggoner, A., 2006. Fluorescent labels for proteomics and genomics. Curr. Opin. Chem. Biol. 10, 62–66. Wahlen, B.D., Willis, R.M., Seefeldt, L.C., 2011. Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresour. Technol. 102, 2724–2730. Wen, Z.-Y., Chen, F., 2001. A perfusion–cell bleeding culture strategy for enhancing the productivity of eicosapentaenoic acid by Nitzschia laevis . Appl. Microbiol. Biotechnol. 57, 316–322. Wen, Z.-Y., Jiang, Y., Chen, F., 2002. High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development. Proc. Biochem. 37, 1447–1453. Wu, S.-T., Yu, S.-T., Lin, L.-P., 2005. Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31. Proc. Biochem. 40, 3103–3108. Xiao, F.G., Shen, L., Ji, H.F., 2011. On photoprotective mechanisms of carotenoids in light harvesting complex. Biochem. Biophys. Res. Commun. 414, 1–4. Yaguchi, T., Tanaka, S., Yokochi, T., Nakahara, T., Higashihara, T., 1997. Production of high yields of docosahexaenoic acid by Schizochytrium sp . strain SR21. J. Am. Oil Chem. Soc. 74, 1431–1434. Yang, H.-L., Lu, C.-K., Chen, S.-F., Chen, Y.-M., Chen, Y.-M., 2012. Isolation and characterization of Taiwanese heterotrophic microalgae: screening of strains for docosahexaenoic acid (DHA) production. Mar. Biotechnol. 12, 173–185.