Bioresource Technology xxx (2012) xxxxxx

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Microalgae-based biorenery 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 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
b

h i g h l i g h t s
" Components of microalgal biomass are suitable for biofuels production and bioreneries. " 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.

a r t i c l e

i n f o

a b s t r a c t
The potential for biodiesel production from microalgal lipids and for CO2 mitigation due to photoautotrophic growth of microalgae have recently been recognized. Microalgae biomass also has other valuable components, including carbohydrates, long chain fatty acids, pigments and proteins. The microalgaebased 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 considerable potential for many medical applications. This review article presents comprehensive information 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 efcient microalgae-based biorenery process. 2012 Elsevier Ltd. All rights reserved.

Article history: Available online xxxx Keywords: Microalgae Carbohydrates Lipids Biorenery Long-chain fatty acid

1. Introduction It has been estimated that about 90% of our current energy consumption 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 Laherrre, 1998; Ho et al., 2011, 2012). Moreover, even if oil reserves remain plentiful, the associated environmental pollution, including the release of CO2, 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 CO2 by photosynthetic organisms on earth has contributed signicantly 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. http://dx.doi.org/10.1016/j.biortech.2012.10.099

global carbon cycle. The CO2 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 CO2 is created. Since microalgae-based CO2 xation is much faster and more efcient than that of terrestrial plants, it has thus been considered to have the potential to serve as a commercially feasible process for mitigation of CO2 emissions (Ho et al., 2011). Most microalgae are unicellular photosynthetic microorganisms that can x the dissolved inorganic carbon and CO2 in the gaseous efuents to form chemical energy through photosynthesis. The majority of microalgae have much higher cell growth and CO2 xation rate (around 1050 times higher) than terrestrial plants. Moreover, the CO2 xation 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 benets from the microalgae CO2 xation process (Ho et al., 2011).

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

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

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 advantages 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 feasible, 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 practical biodiesel, the biomass of microalgae also contains many other valuable components, including carbohydrates, lipids, polyunsaturated fatty acid, pigments and proteins, all of which are worth developing into rened products for various applications (Lammens et al., 2012). Nevertheless, there are still several problems to be solved during the development of microalgae-based biorenery technologies. The most challenging problems for the microalgae industry include high installing and operating cost, difculty in controlling the culture 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 sustainable 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 efcient and economical feasible microalgae cultivation 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 microalgae requires different cultivation conditions and operation strategies. These would rely on more understanding on the photosynthetic metabolism and physiology of the microalgae used and more advanced engineering technology that could help design more appropriate photobioreactors and establish better environment for microalgae growth and product formation. Finally, highefciency and low-cost downstream processing, which accounts for the majority of operating cost, should be developed. For example, some innovative microalgae harvesting approaches have been proposed (Show et al., 2012; Lee et al., 2012; Cheng et al., 2011). A novel one-step transesterication method for producing microalgae-based biodiesel using wet oil-bearing microalgae biomass was also reported (Tran et al., 2012a,b). This method allows biodiesel synthesis from microalgae without dewatering and oil extraction 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 issues. Finally, life cycle analysis, energy balance and cost assessment should also be performed to justify the economic feasibility and environmental impacts. In this review, we provide up-to-date information on the formation mechanisms, production and applications of those microalgaebased components (namely, carbohydrates, lipids, pigments and proteins) (Olguin, 2012). For example, microalgal carbohydrate can serve as a carbon source to replace traditional crop carbohydrates 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 several 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 producing biofuels and bio-based chemicals based on both their natural components and rened (or fermented) products. This review aims to provide a comprehensive report on the production and application of the natural or rened 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 conversion efciency, and are able to accumulate a high carbohydrate content (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 polysaccharides. 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 storage, 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 structures and distinct biological functions. The monosaccharides of fructose, galactose, glucose, mannose and xylose, consist of microalgal polysaccharides in different ratios. Currently, algal polysaccharides represent a class of high-value compounds with many downstream applications in food, cosmetics, textiles, stabilizers, emulsiers, lubricants, thickening agents and clinical drugs (Arad and Levy-Ontman, 2010). In particular, algal polysaccharides contain 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 macrophage stimulation and modulation. As a general rule, the biological activity of sulfated polysaccharides changes with their sugar composition and degree of sulfation (Kim et al., 2012). Algal sulfated polysaccharides have been shown to exhibit a wide range of pharmacological activity, including acting as antioxidant, antitumor, anticoagulant, anti-inammatory, antiviral and immunomodulating 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 responses, 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 inammatory 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 Porphyridium sp. is to overcome extreme environmental factors, and thus it has a protective response against the oxidative stress imposed by ROS (representing antioxidant activity) (Tannin-Spitz et al., 2005). The sulfated polysaccharides derived from Porphyridium sp. have signicant potential for use in anti-inammatory 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 signicantly stimulate murine macrophages to secrete the pro-

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxxxxx Table 1 The pharmacological functions found in the microalgae. Microalgae C. vulgaris S. quadricauda Porphyridium sp. Porphyridium sp. H. lacustris G. impudium KG-03 R. reticulata C. stigmatophora P. tricornutum Polysaccharide extracts Crude polysaccharide Crude polysaccharide Crude polysaccharide Sulfated polysaccharide Water-soluble polysaccharide Sulfated polysaccharide Extracellular polysaccharide Crude polysaccharide Crude polysaccharide Pharmacological signicance Antioxidant Antioxidant Antioxidant Anti-inammatory Immunostimulating Antiviral Antioxidant Anti-inammatory/immunomodulating Anti-inammatory/immunomodulating Reference 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)

inammatory cytokine, indicating its potent immune stimulating activities (Park et al., 2011), although the detailed molecular mechanisms of macrophage activation are not currently known. Several types of microalgal sulfated polysaccharides show wide-spectrum antiviral activity because they are capable of specic interactions with viral particles or cellular surface molecules, resulting in the unique inhibition of virus-type or host cell-type independent activity (Kim et al., 2012). The sulfated polysaccharide p-KG03 of Gyrodinium impudium has specic antiviral activities, because it not only inhibits the binding site of the inuenza A virus to host cells (virus-cell interaction), but also prevents cellular internalization 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 3050% of their content by weight) under several specic cultural conditions, such as in a high C/N medium or under stress conditions. Microalgal lipids are classied into two types according to their carbon numbers, with fatty acids containing 1420 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 productivity (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 efcient microalgal lipid production, with a carbon number in the range of 1420 being suitable for biodiesel production. In contrast, polyunsaturated fatty acids (PUFAs) with more than 20 carbons are high value compounds 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, eicosapentaenoic 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 particular 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 triacylglycerol (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 CO2 into malonyl-CoA, considered the rst 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 rst step, CO2 (from HCO3-) is transferred to nitrogen by the biotin carboxylase prosthetic group of ACCase in a biotin prosthetic group attached to the e-amino group of lysine residue, which is ATP-dependent. In the second step, the activated CO2 is transferred from biotin to acetyl-CoA to form malonyl-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 rst step of TAG formation is the condensation of glycerol-3-phosphate with acyl-CoA and the formation of lysophosphatidic acid. This reaction is catalyzed by GPAT (glycerol-3-phosphate acyltransferase), which exhibits the lowest specic 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 signicantly depend on the environmental factors and medium composition. The performance of lipid production should be evaluated 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, simultaneously, to reach efcient microalgal lipid production. Lipid accumulation in microalgae usually occurs at the expense of slower cell growth, due to the stress conditions. Therefore, it is more reasonable to evaluate lipid production performance using the concept of lipid productivity, which is more important from an engineering aspect (Chen et al., 2011). The most well known factors inuencing lipid production efciency include light illumination strategy, temperature and nitrogen source availability (e.g., nitrogen starvation). These factors can affect either the lipid accumulation or cell growth rate, or both. First, the type of light source is known to affect the growth of microalgae, due mainly to the difference 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 biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

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

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 products, or from the formation of biolm on the surface of the reactor vessels (Chen et al., 2008), although mixing the cells well can reduce 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 inuenced by temperature. For instance, the lipid content of Nannochloropsis salina and Ochromonas danica increased with an increase in the culture temperature (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 1020% higher than under nitrogen sufcient conditions. However, the lipid content of cyanobacteria produced under stress conditions was

usually less than 10%, making it unsuitable for lipid production. Various nitrogen limitation strategies have been applied to foster lipid accumulation, and these can mainly be divided into two types. The rst is a two-stage nitrogen limitation method in which the microalgae cells are cultivated under nitrogen-sufcient conditions to stimulate cell growth for a set amount of time. They are then collected and switched to nitrogen-starving conditions for lipid accumulation. 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 microalgae culture will then automatically advance to the nitrogen starvation stage when the nitrogen content in the medium is depleted. The effects of the two types of nitrogen starvation on lipid production efciency 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 transesterication process. The triglycerides react with methanol in a reaction called transesterication or alcoholysis and then methyl esters of fatty acids (biodiesel) and glycerol 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 transesterication 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 saponication reaction occurs when free fatty acids are present in the

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxxxxx Table 2 DHA production by microalgae in the literature. Strain Schizochytrium sp. Crypthecodinium cohnii Schizochytrium sp. Schizochytrium limacinum Schizochytrium sp. Schizochytrium limacinum Schizochytrium sp. Aurantiochytrium limacinum Aurantiochytrium sp. Aurantiochytrium DHA productivity (g/L day) 3.3 1.15 0.51 3 2.86 3.7 2.90 DHA content (mg DHA/g biomass) 277 174 130 170 100 173 246 153 175 290 DHA (% of total fatty acids) 35.6 31.1 32.5 33.6 40 49 23.9 40 39.7 Reference 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. Strain Phaeodactylum tricornutum Navicula saprophila Rhodomonas salina Nitzschia sp. Monodus subterraneus Chlorella minutissima Phaeodactylum tricornutum Monodus subterraneus Nitzschia laevis Nitzschia laevis Monodus subterraneus EPA productivity (mg/L day) 6 0.89 0.04 0.18 4456 175 33.5 9 EPA content (mg EPA/g biomass) 13.6 38 37 22 3543 26 30 2332 EPA (% of total fatty acids) 25.8 20.1 15.4 24.7 34.2 31.3 21.4 31.531.8 22.4 11 2530 Reference 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 produced to prevent this problem, the cost of raw materials usually accounts for 6075% 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 saponication 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 foodfuel 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 microalgal 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 oilseed 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 CO2 into biomass, and may reduce the CO2 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 production of biodiesel from microalgae. Compared to terrestrial oil crops, the harvest of, and oil extraction from, microalgae is much more difcult, leading to a higher production cost for microalgae-based biodiesel. Normally, the dry microalgal biomass only accounts for 0.11% by weight of the microalgae culture. As a result, concentration 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 conventional 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). Recently, wet extraction approaches have been used to enhance the efciency and economic feasibility of lipid extraction from microalgae. 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 transesterication process. In these approaches, the lipid extraction and transesterication can be done simultaneously in one step, leading to a simplied process and reduced production costs. However, intensive dewatering of the microalgal biomass is still required to avoid the problems associated with transesterication reactions. More recently, (Tran et al., 2012a,b) demonstrated an innovative approach using wet microalgae (up to 90 wt.% water content) as the feedstock to produce biodiesel in the presence of a solvent (i.e., hexane), an excessive amount of methanol, and immobilized lipase via a one-step lipid extraction and transesterication process. They achieved a high biodiesel conversion of 97.3% when using a wet biomass of Chlorella

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

6 Table 4 The photosynthetic pigments. Pigments Chlorophylls Chlorophyll a Chlorophyll b Chlorophyll c Chlorophyll d Carotenoids a-Carotene b-Carotene Luteol Fucoxanthol Peridinin Phycobilins Phycoerythrins Phycocyanins Allophycocyanins

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

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, 576 618 650

Occurrence All higher plants and algae All higher plants and green algae Diatoms and brown algae Red algae Most plants and some algae Higher plants and most algae Green algae, red algae, and higher plants Diatoms and brown algae Dinophytes 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 1420 carbons for biodiesel production, 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 enzymes to synthesize PUFAs from more than 18 carbons, although they are essential for good functioning, conferring exibility, uidity 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 classied 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 sh oils are common sources of long-chain PUFAs, and since the PUFAs found in sh originate from digested microalgae in oceanic environments, it is reasonable to consider microalgae as a direct potential source of these substances. Among all the commercially produced microalgal PUFAs, eicosapentaenoic 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 highquality EPA and DHA, and are thus considered a potential source of this important fatty acid. Numerous strategies have been investigated for commercial production of EPA and DHA using microalgae, including screening of high EPA and DHA-yielding microalgal strains, improvement of strains by genetic manipulation, optimization of culture conditions (such as pH, temperature and the ratio of C/N), and the development of efcient cultivation systems. The productivity and yield gures 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 sh 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 rst 24 h, then shifted to 0.6 vvm for 96 h, and nally 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 substrates 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 performed 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 inammation 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 laver 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

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

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

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 oxygen 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 necrosis (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 pancreatic cancer (Busch et al., 2009). There are currently two FDA-approved photosensitizers, Photofrin and aminolevulinic acid (ALA), which induce protoporphyrin IX (PpIX). Commercial pheophorbide a is now available from Frontier Scientic, Inc. (Logan, UT, USA) (Logan, UT, USA). 4.2. Carotenoids 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 insoluble 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 ketocarotenoid, 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 (Dufoss et al., 2007). It is also a potent quencher of reactive oxygen and nitrogen species, including singlet oxygen and single- and twoelectron oxidants, as shown in several in vitro studies (Fassett and Coombes, 2011). Astaxanthin has many benets in the prevention and treatment of various conditions, such as chronic inammatory diseases, eye diseases, skin diseases, cardiovascular diseases, cancers, neurodegenerative diseases, liver diseases, metabolic syndrome, diabetes, diabetic nephropathy and gastrointestinal 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 industries. 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 salmon 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 agellated cells with an optimum growth temperature of 2225 C. Stress conditions, such as nutrient deprivation, high salinity, strong irradiance, 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 (Phafa rhodozyma); however, the

former is in limited supply and has a low astaxanthin content, while the latter has a astaxanthin content (0.42.5%) 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 market is met by synthetic astaxanthin, consumer demand for natural products provides an opportunity for the sale of Haematococcus astaxanthin. There are currently ve 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 Parrys Pharmaceuticals (Chennai, India) (Dufoss et al., 2007).

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 pyrrole rings that are linked together by single-atom bridges. Phycobilins 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 classied into three types by their phycobilin energy (absorption spectra): high-energy ones are phycoerythrins (PEs) or phycoerythrocyanins (PECs), with their main absorption at 480580 nm; intermediateenergy ones are phycocyanins (PCs), with their main absorption at 600640 nm; and low-energy ones are allophycocyanins (APCs), with their main absorption at 620660 nm. Puried phycobiliproteins are highly uorescent because there are no nearby acceptors to receive the harvested energy. The phycobiliproteins have several unique features, such as a broad absorption in a visible light spectrum, a high extinction coefcient, high uorescence quantum efciency, a large Stokes shift and very little uorescence quenching. These features make phycobiliproteins promising uorescent labeling reagents that can be employed in ow cytometry, uorescence immunoassay, uorescence microscopy, immuno-histochemistry and other biomedical research purposes ((Matamala et al., 2007; Waggoner, 2006). Blue phycobiliproteins, 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 global 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 phycobiliproteins as a uorescent 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 considered an appropriate feedstock for microbial growth and for the production 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 biorenery processes deserve further investigations. In particular, economic feasibility and life cycle assessments of such processes should be conducted to conrm the commercial viability

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxxxxx Ho, S.-H., Chen, C.-Y., Chang, J.-S., 2012. Effect of light intensity and nitrogen starvation on CO2 xation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244252. Ho, S.-H., Chen, C.-Y., Lee, D.-J., Chang, J.-S., 2011. Perspectives on microalgal CO2emission mitigation systems a review. Biotechnol. Adv. 29, 189198. 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, 14681480. 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, 621639. Huang, G.H., Chen, F., Wei, D., Zhang, X.W., Chen, G., 2010. Biodiesel production by microalgal biotechnology. Appl. Energy 87, 3846. John, R.P., Anisha, G.S., Nampoothiri, K.M., Pandey, A., 2011. Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour. Technol. 102, 186193. 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 inuenza A virus infection by marine microalgaderived sulfated polysaccharide p-KG03. Antiviral Res. 93 (2), 253259. 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, 559563. 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, 9497. 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, 168181. 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 ltration of Chlorella vulgaris. Bioresour. Technol. 108, 184189. Lembi, C.A., Waaland, J.R., 1988. Some public health aspects of microalgal products. In: Algae and Human Affairs, Cambridge University Press, pp. 181202. Lorenz, R.T., Cysewski, G.R., 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol. 18, 106167. Lu, C., Fernndez, 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, 331342. 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, 517522. Mascarelli, A., 2009. Gold rush for algae. Nature 461, 460461. Matamala, A.R., Almonacid, D.E., Figueroa, M.F., Martinez-Oyanedel, J., Bunster, M.C., 2007. A semiempirical approach to the intra-phycocyanin and interphycocyanin uorescence resonance energy-transfer pathways in phycobilisomes. J. Comput. Chem. 28, 12001207. Matsui, M.S., Muizzuddin, N., Arad, S., Marenus, K., 2003. Sulfated polysaccharides from red microalgae have antiinammatory properties in vitro and in vivo. Appl. Biochem. Biotechnol. 104, 1322. Milledge, J.J., 2011. Commercial application of microalgae other than as biofuels: a brief review. Rev. Environ. Sci. Biotechnol. 10, 3141. Mohamed, Z.A., 2008. Polysaccharides as a protective response against microcystininduced oxidative stress in Chlorella vulgaris and Scenedesmus quadricauda and their possible signicance in the aquatic ecosystem. Ecotoxicology 17, 504516. Muntendam, R., Melillo, E., Ryden, A., Kayser, O., 2009. Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts. Appl. Microbiol. Biotechnol. 84, 10031019. Olguin, E.J., 2012. Dual purpose microalgaebacteria-based systems that treat wastewater and produce biodiesel and chemical products within a biorenery. Biotechnol. Adv. 30, 10311046. 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, 10901098. Patterso, G.W., 1970. Effect of culture temperature on fatty acid composition of Chlorella sorokiniana. Lipids 5, 597600. 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-ow reactor. Bioresour. Technol. 55, 8388. 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 efcient docosahexaenoic acid production by Schizochytrium sp. Appl. Microbiol. Biotechnol. 87, 16491656. Rosa, S.M., Soria, M.A., Vlez, C.G., Galvagno, M.A., 2010. Improvement of a twostage fermentation process for docosahexaenoic acid production by Aurantiochytrium limacinum SR21 applying statistical experimental designs and data analysis. Bioresour. Technol. 101, 23672374. Schepetkin, I.A., Quinn, M.T., 2006. Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int. Immunopharmacol. 6, 317333. 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.

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

Acknowledgments The authors gratefully acknowledge the nancial support from the top university project of NCKU and by Taiwans National Science Council under grant numbers NSC 101-3113-P-110-002, NSC 101-3113-E-006-015, and NSC 101-3113-E- 006-016.

References
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, 211 215. Arad, S., Levy-Ontman, O., 2010. Red microalgal cell-wall polysaccharides: biotechnological aspects. Curr. Opin. Biotechnol. 21 (3), 358364. Behzadi, S., Farid, M.M., 2007. Review: examining the use of different feedstock for the production of biodiesel. Asia-Pacic J. Chem. Eng. 2, 480486. Boussiba, S., Vonshak, A., Cohen, Z., Avissar, Y., Richmond, A., 1987. Lipid and biomass production by the halotolerant microalga Nannochloropsis salina. Biomass 12, 3747. 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, 540542. Campbell, C.J., Laherrre, J.H., 1998. The End of Cheap Oil. Scientic American, March, pp. 7883. 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, 833840. 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 bers. Int. J. Hydrogen Energy 33, 68786885. 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, 7181. 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, 154163. Chi, Z., Liu, Y., Frear, C., Chen, S., 2009. Study of a two-stage growth of DHAproducing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl. Microbiol. Biotechnol. 81, 11411148. 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, 15371545. 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, 631640. 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 otation. Bioresour. Technol. 102, 8287. 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, 3141. Del Campo, J.A., Garca-Gonzlez, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74, 11631174. Demirbas, A., 2010. Social, economic, environmental and policy aspects of biofuels. Energy Edu. Sci. Technol. Part B Social Edu. Studies 2, 75109. Dufoss, L. 2007. Pigments from microalgae and microorganisms: sources of food colorants. In: Food Colorants: Chemical and Functional Properties, CRC Press, pp. 399426. Fassett, R.G., Coombes, J.S., 2011. Astaxanthin: a potential therapeutic agent in cardiovascular disease. Marine Drugs 9, 447465. 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, 15591564. Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21, 210216. Guzman, S., Gato, A., Lamela, M., Freire-Garabal, M., Calleja, J.M., 2003. Antiinammatory and immunomodulatory activities of polysaccharide from Chlorella stigmatophora and Phaeodactylum tricomutum. Phytother. Res. 17, 665670. Hall, D.O., Rao, K.K., 1999. Photosynthetic apparatus, 6th ed. In: Photosynthesis, The Press Syndicate of The University of Cambridge, pp. 3357.

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099

H.-W. Yen et al. / Bioresource Technology xxx (2012) xxxxxx Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 8796. 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, 126128. 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, 666672. Takaichi, S., 2011. Carotenoids in algae: distributions, biosyntheses and functions. Mar. Drugs 9, 11011118. 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, 215222. Tran, D.-T., Yeh, K.-L., Chen, C.-L., Chang, J.-S., 2012a. Enzymatic transesterication of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour. Technol. 108, 119127. Tran, D.-T., Chen, C.-L., Chang, J.-S., 2012b. Effect of solvents and oil content on direct transesterication 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 bioreneries through bioconversion of solid by-products. Bioresour. Technol. 116, 295301. Um, B.H., Kim, Y.S., 2009. Review: a chance for Korea to advance algal-biodiesel technology. J. Ind. Eng. Chem. 15, 17.

Vazhappilly, R., Chen, F., 1998. Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth. JAOCS 75, 393397. Waggoner, A., 2006. Fluorescent labels for proteomics and genomics. Curr. Opin. Chem. Biol. 10, 6266. 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, 27242730. Wen, Z.-Y., Chen, F., 2001. A perfusioncell bleeding culture strategy for enhancing the productivity of eicosapentaenoic acid by Nitzschia laevis. Appl. Microbiol. Biotechnol. 57, 316322. 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, 14471453. 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, 31033108. Xiao, F.G., Shen, L., Ji, H.F., 2011. On photoprotective mechanisms of carotenoids in light harvesting complex. Biochem. Biophys. Res. Commun. 414, 14. 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, 14311434. 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, 173185.

Please cite this article in press as: Yen, H.-W., et al. Microalgae-based biorenery From biofuels to natural products. Bioresour. Technol. (2012), http:// dx.doi.org/10.1016/j.biortech.2012.10.099