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Biochemical Engineering Journal 78 (2013) 1 10

Contents lists available at ScienceDirect

Biochemical Engineering Journal

jou rna l h om epage: www.elsev ier .com/ locate /be j

egular Article

icroalgae-based carbohydrates for biofuel production

hun-Yen Chena, Xin-Qing Zhaob, Hong-Wei Yenc, Shih-Hsin Hod, Chieh-Lun Chengd,uu-Jong Leee, Feng-Wu Baib,, Jo-Shu Changa,d,f,

University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, TaiwanSchool of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, ChinaDepartment of Chemical and Materials Engineering, Tunghai University, Taichung, TaiwanDepartment of Chemical Engineering, National Cheng Kung University, Tainan 701, TaiwanDepartment of Chemical Engineering, National Taiwan University, Taipei, TaiwanResearch Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan

r t i c l e i n f o

rticle history:eceived 23 November 2012eceived in revised form 4 March 2013ccepted 8 March 2013vailable online 15 March 2013

eywords:icroalgaeiogasrowth kinetics

a b s t r a c t

Microalgae are considered as the most promising renewable feedstock for biofuel production and biore-fineries, due to their advantages of fast growth, efficient carbon dioxide fixation, not competing for arablelands and potable water, and potentially accumulating high amounts of lipids and carbohydrates. Sincecarbohydrates in microalgae biomass are mainly cellulose in the cell wall and starch in the plastidswithout lignin and low hemicelluloses contents, they can be readily converted into fermentable sugars.However, to date there are very few studies focusing on the use of microalgae-based carbohydrates forbiofuel production, which requires more understanding and knowledge to support the technical feasi-bility of this next-generation feedstock. This review article elucidates comprehensive information on thecharacteristics and metabolism of main fermentable microalgal carbohydrates (e.g., starch and cellulose),ntegrated Processingiofuelsiorefinery

as well as the key factors and challenges that should be addressed during production and saccharifi-cation of microalgal carbohydrates. Furthermore, developments on the utilization of microalgae-basedfeedstock in producing liquid and gaseous biofuels are summarized. The objective of this article is toprovide useful knowledge and information with regard to biochemistry, bioprocess engineering, andcommercial applications to assist in the viable technology development of for biofuels generation frommicroalgae-based carbohydrates.. Introduction

The fast growth of the global population and the rise of develop-ng countries, such as China and India, have led to a rapid increasen demand for energy [1]. Currently, about 90% of energy needsome from coal, natural gas and petroleum, and sustainable energyupplies need to be developed due to the dwindling reserve ofhese fossil fuel resources [2,3]. With current consumption trends,orld oil reserves may run out by 2050 [1,4]. Moreover, the

roblems of environmental pollution and climate change are alsoainly attributed to the over-consumption of fossil fuels [5]. There-

ore, a number of countries have expressed increased interest in

Corresponding author at: National Cheng Kung University; Department ofhemical Engineering; Tainan 701; Taiwan. Tel.: +886 6 2757575x62651;ax: +886 6 2357146. Co-corresponding author at: School of Life Science and Biotechnology, Dalianniversity of Technology, Dalian 116024, China. Tel.: +86 411 8470 6308;ax: +86 411 8470 6329.

E-mail addresses: fwbai@dlut.edu.cn (F.-W. Bai), changjs@mail.ncku.edu.twJ.-S. Chang).

369-703X/$ see front matter 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bej.2013.03.006 2013 Elsevier B.V. All rights reserved.

developing alternative energy sources that are renewable, econom-ically competitive and environmentally friendly [6].

Biomass can be converted to energy by biological or thermo-chemical methods. Biological conversion includes fermentation ofdegradable components to produce energy carriers like bioethanol,biobutanol, biohydrogen and biogas, or extraction of oils forbiodiesel production. Thermo-chemical conversion includes directcombustion for heat and electricity, as well as indirect processeslike pyrolysis and gasification [7]. The most widely used biofuel isbioethanol, which is produced from sugar-based (sugar beets, sug-arcane) and starch-based (corn, wheat, barley, etc.) feedstocks [8],while technology leading to conversion of lignocellulosic materi-als (bagasse, corn stover, rice straw, switchgrass, and so on) intoethanol is under development worldwide.

The choice of biomass feedstock depends on social, environ-mental, economic and industrial factors, such as availability andcost of raw materials. However, the challenges associated with

most of the current feedstock lies in the need for arable lands andfreshwater for the cultivation of plants, and thus possible compe-tition with food production, seasonal and geographical variationsin productivity, as well as the need for herbicides [9]. Although

dx.doi.org/10.1016/j.bej.2013.03.006http://www.sciencedirect.com/science/journal/1369703Xhttp://www.elsevier.com/locate/bejhttp://crossmark.dyndns.org/dialog/?doi=10.1016/j.bej.2013.03.006&domain=pdfmailto:fwbai@dlut.edu.cnmailto:changjs@mail.ncku.edu.twdx.doi.org/10.1016/j.bej.2013.03.006

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ignocellulosic biomass is cheap and plentiful to utilize, the costf converting it into ethanol is still relatively high. Moreover,he lignin component is very difficult to ferment or degrade bio-ogically [10], and thus a more sustainable feedstock should beeveloped to overcome these barriers. Microalgae have been recog-ized as an alternative, a so-called third generation feedstock thatoes not compete for arable land or portable water, and marineicroalgae species growing in seawater can also reduce freshwa-

er consumption. In addition, the microalgae-based carbohydratesave low lignin content (high fermentable sugars), and their sac-harification is much easier, thus being a more promising andustainable biomass source for bioethanol production [1,11]. Inhis review, we provide up-to-date and comprehensive informa-ion on the metabolism, characteristics, production technologies,nd applications of microalgae-based fermentable carbohydrates.n particular, recent advances in the production and utilization ofarbohydrate-rich microalgae feedstock for biofuels are covered toromote the idea of using microalgae as a practical feedstock.

. Evolution of feedstock for biofuels production

As bulk commodities, the cost of biofuels mainly comes fromeedstock consumption. For example, the cost of feedstock con-umption is as high as 6070% for fuel ethanol produced fromtarched-based feedstocks [8]. Therefore, the availability of low-ost feedstock is always the biggest concern for biofuels productionf it is to successfully meet the global demand for energy. Due tohe issues of cost, land/freshwater requirement, food competition,nd environmental concerns, the feedstock for biofuels productionas been switched from sugar or starch-based crops (first gen-ration feedstock) to lignocellulosic materials (second generationeedstock), and then to microalgae (third generation feedstock).he advantages and limitations of these feedstocks are addressedelow.

.1. Conventional feedstocks and their limitations

Biofuels produced from sugar, grain and oleaginous crops arenown as first generation biofuels. Currently, ethanol is the mainiofuel, which is primarily produced from sugar- and starch-basedeedstocks that are easy to ferment, and thus have high biofuelonversion efficiency. However, sugarcane, the major sugar-basedeedstock, requires tropical or temperate climates, with Brazil thenly successful example of sugarcane ethanol use [12]. Starch-ased feedstocks are mostly grains, and thus are not sustainableor biofuels production, taking into account the global populationituation and increased demand for grains as the food or animaleed. For example, fuel ethanol production in the United Statesncreased from 1.6 to 13.2 billion gallons from 2000 to 2010, con-uming one third of the corn harvest of US and causing a significantncrease in global grain prices [13]. A similar situation also appliesith biodiesel, another major biofuel which is produced from ani-al fats or vegetable oils through transesterification with alcoholsatalyzed by either enzymes or chemical catalysts [14]. Much likeugarcane, palm trees, which have the highest oil yield amongleaginous plants, are limited to tropical regions such as South-ast Asia, while other vegetable oils from soybeans, peanuts andapeseeds are edible ones. Therefore, first generation feedstock hasignificant limitations and raises issues with regard to food versusuel. Therefore, alternative feedstocks are needed if biofuels are toe more widely used..2. Lignocellulosic biomass and challenges

Lignocellulosic biomass, such as agricultural and forest residues,s not food-related and abundantly available at low cost withoutering Journal 78 (2013) 1 10

competition for arable land. It was estimated that the United Statesalone produces about 1.3 billion tons of lignocellulosic biomassannually, which could be converted into 60 billion gallons ofethanol with negligible impact on the food supply and significantenvironmental benefits [15]. Therefore, lignocellulosic biomass hasbeen widely acknowledged as an ideal feedstock for the sustainableproduction of biofuels. However, due to its recalcitrance to degra-dation, lignocellulosic biomass still cannot be converted to biofuelsin an efficient and economical way [16]. The major componentsof lignocellulosic biomass are cellulose, hemicelluloses and lignin,which interact and entangle to form the lignincarbohydrate com-plex [17]. This unique crystalline structure prevents cellulases frombinding onto cellulose surfaces to liberate sugars for biofuels pro-duction, and pretreatment is thus needed to deconstruct it, whichis energy-intensive and consumes significant amounts of chemicals[18]. On the other hand, cellulases, the enzymes responsible for cat-alyzing cellulose hydrolysis, are more expensive than amylases andglucoamylases, and the cellulase dosage required for effective cel-lulose saccharification is usually very high [19]. Furthermore, thereare still problems arising from inefficient fermentation of pen-tose from the hemicellulose content in lignocellulosic feedstock,although intensive research and development to engineer variousmicroorganisms has been carried out for decades [20]. In addition,the lignin part is very difficult to ferment, thereby decreasing theoverall biomass to biofuels yield, and also leading to higher wastetreatment costs

2.3. Carbohydrate-based microalgae as innovative feedstock forbiofuels production

Although lignocellulosic biomass is much cheaper for biofuelsproduction than sugar- and starch-based feedstock, a recent out-look report predicted that large-scale production of the secondgeneration biofuels will take another five to ten years for devel-opment [21]; needless to say the ongoing controversy over landuse, even for marginal land used for the cultivation of herbaceousenergy crops. However, aquatic microalgae that can grow withhigh photosynthetic efficiency could be a solution to this problem[22]. Microalgae are photosynthetic organisms with relatively sim-ple requirements for growth when compared to other sources ofbiomass. Through the process of photosynthesis, the algae convertCO2, water and light into biomass. Some microalgae may containa large amount of carbohydrates in the cell wall (mainly in theform of cellulose and soluble polysaccharides) [23] and plastids(mainly in the form of starch) [24], which can potentially be used ascarbon sources for fermentation [11]. Moreover, some microalgaespecies are also known to accumulate lipids under stress condi-tions, and thus could also be used as the raw material for biodieselproduction [2,25].

Using microalgal feedstock has several advantages over tra-ditional feedstock. These include: (1) the high growth rate andhigh area (or volumetric) productivity; (2) the absence of ligninand low hemicellulose content, and thus only minor pretreatmentbeing needed; (3) efficient CO2 capture via photosynthesis, therebymitigating greenhouse gas emissions; (4) being able to grow onsalt or wastewater streams (saline/coastal water, brackish seawa-ter, domestic/municipal/industrial wastewater), thereby reducingfreshwater use; (5) being able to grow in areas unsuitable for agri-cultural purposes (e.g. desert and seashore lands), and thus thereis no competition with arable land for food production; (6) hav-ing a very short harvesting cycle (110 days) compared with other

feedstocks (which are harvested once or twice a year), thus provid-ing enough supplies to meet ethanol production demand; and (7)compatibility with integrated production of fuels and co-productswithin biorefineries. These advantages thus allow microalgae to be

Engineering Journal 78 (2013) 1 10 3

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Table 2Carbohydrate or starch content of green microalgal species.

Microalgal species Carbohydrate or starchcontent (%)

References

Chlorella vulgaris IAM C-534 37.0 (starch) [67]C. vulgaris CCAP 211/11B 55.0 [31]C. vulgaris P12 41.0 (starch) [86]C. vulgaris 55.0 (starch) [87]Chlamydomonas reinhardtii UTEX 90 60.0 [67]C. reinhardtii IAM C-238 55.0 (starch) [32]Chlorococum sp. 32.5 [88]Chlorococcum sp. TISTR8583 26.0 (starch) [58]S. acutiformis TISTR 8495 16.4 (starch) [89]C.-Y. Chen et al. / Biochemical

referentially selected as a clean, efficient, and sustainable feed-tock for bioethanol production [1].

. Microalgae-based carbohydrates

Carbohydrates are the major products derived from photosyn-hesis and the carbon fixation metabolism (i.e., the Calvin cycle)4]. These carbohydrates are either accumulated in the plastids aseserve materials (e.g., starch), or become the main componentf cell walls (e.g., cellulose, pectin, and sulfated polysaccha-ides). However, the composition and metabolism of carbohydratesmainly starch and cellulose) in microalgae may differ significantlyrom species to species [24,26]. It is thus of great importance toelect microalgae with high carbohydrate productivity as well asuitable sugar composition for biofuels or chemical production.or instance, microalgae that contain glucose-based carbohydratesre the most feasible feedstock for bioethanol production. How-ver, although increasing attention has been paid to the potentialf using macroalgae (e.g., seaweed) as a sugar source [27], the majorugars from brown macroalgae are glucan, mannitol, and algi-ate. The former two are relatively easily assimilated by availableicrobial ethanol producers, with a yield of 0.080.12 g ethanoler g dry biomass weight. However, the inability of industrialicrobes to metabolize alginate is a major hurdle to achieving the

ull potential of ethanol production from macroalgal biomass [27].n contrast, the carbohydrates from microalgae are very suitableor bioethanol production, although more economic cell harvestingechnology should be developed to make microalgae-based ethanolommercially viable. The carbohydrate composition and the gen-ral carbohydrate metabolism of microalgae are described in theollowing sections.

.1. Carbohydrate composition of microalgae

The cell walls of microalgae primarily consist of an inner cell wallayer and an outer cell wall layer, and can be grouped into threeypes, namely those (1) with a trilaminar outer layer, (2) with ahin outer monolayer and (3) without an outer layer [28]. The com-osition of the outer cell wall varies from species to species, butsually contains specific polysaccharides, such as pectin, agar, andlginate [28]. The inner cell wall layer of microalgae is mainly com-osed of cellulose and other materials (such as hemicellulose andlycoprotein) [28]. Table 1 shows the compositions of the cell wallsnd the storage products in different microalgal species. For someicroalgae, the glucose polymers produced via cellulose/starch are

he predominant component in the cell walls and stored products oficroalgae [29]. Microalgae are considered a promising feedstock

or bioethanol production because they have cellulose-based cell

alls, with accumulated starch as the main carbohydrate source.oth starch and most cell wall polysaccharides can be convertednto fermentable sugars for subsequent bioethanol production viaicrobial fermentation [30]. Table 2 shows the carbohydrate or

able 1omposition of microalgal cell wall and storage products.

Division Cell wall Storage products

Cyanophyta Lipopolysaccharides,peptidoglycan

Cyanophyceanstarch

Chlorophyta Cellulose, hemicellulose Starch/lipidDinophyta Absence or contain few cellulose StarchCryptophyta Periplast StarchEuglenophyta Absence Paramylum/lipidRhodophyta Agar, carrageenan, cellulose,

calcium carbonateFloridean starch

Heterokontophyta Naked or covered by scales orwith large quantities of silica

Leucosin/lipidS. obliquus CNW-N 51.8 [11]Tetraselmis sp.CS-362 26.0 [90]

starch content of different microalgae species. As indicated inTable 2, Chlorella sp. has the highest carbohydrate content. Inparticular, Chlorella vulgaris can accumulate a large amount of car-bohydrates, up to 3755% of its dry biomass. For example, after 14days cultivation in a low-nitrogen medium, the biomass concen-tration and carbohydrate content of C. vulgaris CCAP 211/11B canreach 0.52 g (dry cell weight, DCW)/L and 55%, respectively [31].Chlamydomonas reinhardtii and Scenedesmus obliquus also has thepotential to serve as a biofuels feedstock due to its high carbohy-drate content of 4560%. In a batch culture of C. reinhardtii UTEX90, the biomass concentration and starch content were found to be1.45 g DCW/L and 53%, respectively, after 3-day cultivation [32].Moreover, the biomass concentration and carbohydrate contentof S. obliquus could reach 4.96 g DCW/L and 51.8%, respectively,under 3-day nitrogen starvation [11]. Therefore, species of Chlorella,Scenedesmus, and Chlamydomonas are appropriate carbohydrate-based feedstock candidates for bioethanol production.

3.2. Carbohydrate metabolism of microalgae

The accumulation of carbohydrates in microalgae is due to CO2fixation during the photosynthetic process. Photosynthesis is abiological process utilizing ATP/NADPH to fix and convert CO2 cap-tured from the air to produce glucose and other sugars through ametabolic pathway known as the Calvin cycle [33].

The metabolic pathways of energy-rich molecules (e.g., carbo-hydrate and lipid) are closely linked. Some studies demonstratedthat there was a competition between lipid and starch synthesisbecause the major precursor for triacylglycerols (TAG) synthesisis glycerol-3-phosphate (G3P), which is produced via catabolismof glucose (glycolysis) [11,24]. Thus, to enhance biofuels produc-tion from microalgae-based carbohydrates, it is vital to understandand manipulate the related metabolisms to achieve higher microal-gal carbohydrate accumulation via strategies like increasing glucanstorage and decreasing starch degradation [34]. The starch formsaround a crystallizing nucleus and is present as an amorphousstarch grain. When a chloroplast gathers enough starch, it maybecome an amyloplast. However, the detailed changes in enzy-matic activity and metabolic flux of carbohydrate biosynthesis ofmicroalgae are poorly understood. The manipulation of the carbo-hydrate metabolisms of microalgae by genetic engineering has alsobeen proposed [34]. With the development of genetic engineeringof microalgae, and a better understanding of the biochemistry ofmicroalgae carbohydrate metabolisms, superior strains for carbo-hydrate accumulation could be developed.

Except the starch in plastids, microalgal extracellular coverings(e.g., cell wall) are another carbohydrate-rich part which could be

transformed to biofuel [1]. However, the compositions of microal-gal extracellular coverings are diverse by species [23]. Among them,cellulose is one of the main fermentable carbohydrates in most ofgreen algae [34]. Cellulose synthesis is a complicated process that

4 C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 1 10

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ncludes many enzymatic reactions. The starting substrate for cel-ulose synthesis is UDP-glucose, which is formed from the reactionf UDP and fructose catalyzed by sucrose synthase (Fig. 1) [35].Despite the understanding of main carbohydrate metabolism

n microalgae, in-depth knowledge on its regulation is still lack-ng. To meet the challenges on economic biofuels production fromicroalgae, it is important to integrate updated information ofenomic sequences, transcriptomes, proteomes, and metabolomesata at systems level. Although systems study of microalgae on car-ohydrate metabolisms is currently in its infant stage, omics studiesn microalgae have made significant progress [24]. Such a strategyill open a door for efficient carbohydrate metabolic regulation andenetic engineering of microalgae for biofuels production.

. Environmental factors affecting microalgaearbohydrate production

To enhance the economic feasibility of using algal carbohy-rates for biofuels production, the productivity is the key parameterhat needs to be improved. Unfortunately, the accumulation oficroalgal carbohydrates usually occurs when the microalgal cells

ace environmental stress (typically nutrients limitation), whichften results in poor cell growth (or lower biomass productiv-ty). Therefore, how to enhance the carbohydrate content withoutompromising the cell growth rate is crucial to the success of car-

ohydrate production from microalgae. The carbohydrate contentf microalgae could be enhanced by the use of various cultivationtrategies, such as irradiance [36], nitrogen depletion [37], tem-erature variation [38], pH shift [39], and CO2 supplement [40].Redrawn on the basis of the reference [33].

The effects of these approaches are discussed in more detail in thefollowing sections.

4.1. Irradiance

Providing appropriate irradiance is essential for the autotrophicgrowth of microalgae, since illumination can offer light energythat is further stored in the form of carbohydrates or lipids inthe microalgal biomass. The efficiency of light energy supply thusbecomes one of the major limiting factors for outdoor or large-scalemicroalgae cultivation. Therefore, the configuration of microalgaecultivation systems (such as photobioreactors) should be designedto provide uniform and sufficient irradiance to the microalgal cells.The key design factors associated with the light supply wouldbe the operation depth and agitation. The former strongly affectsthe light penetration and availability, while the latter is vital forenhancing the light distribution and uniformity. In addition, thelight intensity was also found to affect the carbohydrate accu-mulation in microalgae. Previous studies demonstrate that anincrease in the light intensity in the range of 30400 mol m2 s1

could slightly increase the accumulation of carbohydrates [41].In a recent report, a significant increase in starch content from8.5% (dry weight basis) to 40% was observed when the meanlight intensity was increased from 215 to 330 mol m2 s1 [42].However, in other cases [41], there was no obvious positive corre-

lation between light intensity and carbohydrate accumulation. Thissuggests that the accumulation of microalgal carbohydrates notonly depends on light intensity, but also on other environmentalparameters.

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.2. Nitrogen depletion

Nitrogen is an essential nutritional component for the growthf microalgae, because the formation of proteins, amino acids,hloroplast, enzymes, coenzymes, and so on is associated withitrogen assimilation [43]. A variety of nitrogen sources (such asitrate, nitrite, ammonia and urea) can be utilized by microalgae,hile different nitrogen sources may influence their biochemicalomposition. More importantly, when under nitrogen-depletiononditions many microalgal strains could transform proteins oreptides to lipids or carbohydrates as energy reserve components44]. Illman et al. [31] reported that a 55% carbohydrate content waschieved when cultivating C. vulgaris on a low-nitrogen-containingedium. DSouza and Kelly [37] also demonstrated that the culti-ation of Tetraselmis suecica under nitrogen starvation with CO2eeding could dramatically enhance the cellular carbohydrate con-ent from 10 to 57%. There are many other examples in the literaturehowing that limitation of nitrogen availability seems to be theost effective way of triggering the accumulation of carbohy-rates in microalgae [4]. However, some studies indicated thathere was a competition between lipid and carbohydrate synthe-is under stress environments (e.g., nitrogen-deficient) because theetabolic pathways associated with synthesis and degradation ofnergy-rich compounds (e.g., lipids and carbohydrates) are closelyinked [11,24,45]. Precisely, starch biosynthesis of microalgae canirectly proceed away from lipid synthesis. On the contrary, degra-ation of starch provides the metabolites for producing acetyl-CoA,hich is the precursor of fatty acid synthesis [24,46]. Thus, in someases, decreasing starch degradation by genetic modification is nec-ssary to block the synthetic pathway of lipids [34].

.3. Temperature

The effect of temperature on the accumulation of microalgal car-ohydrates are still inconclusive [38]. Some researchers observedo significant differences in biochemical composition under thetress of temperature variations in some microalgal species [47],hereas some researchers pointed out that temperature is poten-

ially able to change the biochemical composition of microalgae.herefore, the effect of temperature on carbohydrate accumula-ion in microalgae seems to be highly dependent on the microalgaltrains used.

.4. pH

pH is an important environmental condition for the metabolismf microorganisms, affecting not only cell growth rate but alsoiochemical composition [39]. In general, the adequate pH for car-ohydrate accumulation differs based on the type of microalgal

pecies used. Khalil et al. [39] indicated that the pH value signif-cantly affects the accumulation of total carbohydrates in both D.ardawil and C. ellipsoidea and their maximum carbohydrate accu-ulation was reached at pH 7.5 and 9.0, respectively. Furthermore,

able 3omparison of biomass productivity and carbohydrate productivity of microalgae strains

Microalgae strains Operationmode

Cultivationtime (days)

Biomassproduction

C. vulgaris (CCAP 211/11B) Batch 14 0.52 C. vulgaris (P12) Batch N.D. N.D. C. reinhardtii UTEX 90 Fed-batch 4 2.40 C. reinhardtii Batch 3 1.45 Tetraselmis subcordiformis Semi-batch N.D. N.D. S. obliquus CNW-N Batch 5 4.03 C. vulgaris Batch 6 1.70 C. vulgaris FSP-E Batch 5.25 7.30 ering Journal 78 (2013) 1 10 5

Taraldsvik and Myklestad. [48] reported that the extracellular car-bohydrate production of a marine diatom Skeletonema costatumwas dramatically increased from 2.1 to 17.7% when it was grownat pH 9.4.

4.5. CO2 supplementation

The addition of CO2 is required for the autotrophic growthof microalgae, and is considered to be positively related to theefficiency of photosynthesis, with the synthesis of carbohydratesas the end product. A sufficient supply of CO2 is thus one ofthe key factors influencing the accumulation of carbohydrate inmicroalgae. Some studies found that carbohydrate accumulation inmicroalgae is improved by increasing the percentage of CO2 in theinlet gas [49,50]. For example, Xia and Gao [49] pointed out thatincreasing dissolved CO2 concentration from 3 to 186 mol/L inthe cultivation of C. pyrenoidosa and C. reinhardtii could elevate thecarbohydrate content from 9.30 to 21.0% and 3.19 to 7.40% (w/w),respectively. However, many researchers believe that increasingthe CO2 concentration not only provides more carbon source forphotosynthesis to promote microalgae growth, but also induces thesynthesis of relevant proteins, which may influence the cell physiol-ogy. In some microalgal species, increases in the CO2 concentrationresult in an increase in the protein content, but a decrease or noobvious change in the carbohydrate content [51]. However, undernitrogen starvation conditions and with an adequate supply of CO2and light energy, the protein content in microalgae can be con-sumed as a nitrogen source, and the carbohydrate content mayincrease significantly during this process. Therefore, suitable addi-tion of CO2 is a key step to improving the autotrophic growth ofmicroalgal cells (i.e., improving biomass productivity and proteincontent), although it may not directly enhance carbohydrate accu-mulation in microalgae, unless appropriate stress conditions areemployed.

4.6. Comparison of the carbohydrate production performance ofdifferent microalgae

The carbohydrate production ability of microalgae varies fromspecies to species. However, as mentioned earlier, the productionof microalgal carbohydrates can be significantly improved whenappropriate cultivation conditions are applied. Table 3 summarizesthe performance of the biomass and carbohydrates production ofvarious microalgae species according to recent reports in the lit-erature, showing that the microalgal carbohydrate productivityvaries in a wide range from 0.021 to 0.687 g/L/d (Table 3). Ourrecent work showed that C. vulgaris FSP-E can achieve the high-est carbohydrates productivity of 0.687 g/L/d due to a high biomass

productivity of 1.363 g/L/d [52]. Information concerning the com-position of carbohydrates produced by microalgae is also crucial forfuture applications of microalgae feedstock. However, this infor-mation is currently very limited, as only two Chlorella strains

reported in the literature.

(g/L)Biomassproductivity (g/L/d)

Carbohydrateproductivity (g/L/d)

References

0.037 0.021 [31]0.485 0.199 (starch) [86]0.507 0.304 [61]0.484 0.257 [32]N.D. 0.255 (starch) [91]0.821 0.383 [11]0.254 0.112 [92]1.363 0.687 [52]

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C. vulgaris P12 and C. reinhardtii UTEX 90) have been shown toroduce 41% and 35% starch, respectively (Table 3).

. Saccharification of the microalgae-based carbohydrates

Saccharification is usually the rate limiting step in biofuels pro-uction using lignocellulosic materials or microalgal biomass thatontains a cellulose source. It is known that cellulose and starch arehe two major carbohydrate components in microalgal biomass.herefore, to enhance utilization of microalgae-based carbohy-rates, it is important to identify efficient methods for the cleavagef -1,4-glycosidic linkages between the hydroglucose subunits inellulose molecules, and the cleavage of -1,4-glycosidic linkages intarch. While the process of saccharification of microalgae is similaro that of lignocellulosic materials, the lack of lignin present in theicroalgal biomass simplifies the pre-treatment process [1]. Vari-us methods have been applied to produce sugars from microalgae,uch as mineral acids, alkaline, enzymes or hot compressed water53], and can be categorized into two major groups, namely enzy-atic saccharification and chemical saccharification. The details of

hose methods are described in the following subsections.

.1. Enzymatic saccharification

Enzymatic saccharification processes, involving the use of cel-ulases, amylases and glucoamylases, are widely used to hydrolyzeicroalgae to obtain sugars. For microalgae-based cellulose, which

s mainly located in the inner cell wall, lignin is absent and the hemi-ellulose content is also very low. Therefore, the lignin-degradationnzymes (e.g., laccase and lignin peroxidase) and xylanase may note necessary in the enzymatic saccharification process. In addi-ion, harsh pretreatment, such as acidic or alkaline pretreatment,r steam explosion, is also not needed, making it easier and cheapero saccharify microalgae-based cellulose when compared withignocellulosic materials. To hydrolyze microalgae-based cellu-ose, endo--1,4-d-glucanase attacks the amorphous cellulose andleaves cellulose into small fragments. The exo--1,4-d-glucanaseurther hydrolyzes the small fragments into simple sugars, such asellobiose and cellodextrin. Finally the cello-oligosaccharides areegraded to glucose by -glucosidase [54]. To hydrolyze starchnside the microalgal cells, amylase (endo-amylase) first attackshe internal -1,4-glycosidic bond of starch to produce dextrinnd glucoamylase further hydrolyzes dextrin into glucose andligosaccharides, such as maltose [55]. Enzymatic hydrolysis haseveral advantages over chemical hydrolysis (such as acid or alka-ine hydrolysis), including lower equipment costs, as hydrolysis isonducted at mild conditions, and higher glucose yields withoutugar-degradation products or toxic by-products that may affectollow-up biofuels fermentation [56]. Choi et al. [57] explored theffects of two different commercial enzymes (including amylaserom B. licheniformis and glucoamylases from Aspergillus niger) onhe bioethanol conversion efficiency of Chlamydomonas reinhardtiiiomass with a carbohydrate content of about 59.7% dry weightase. The results showed that when algal biomass was hydrolyzedt pH 4.5 and 55 C for 30 min, better sugar conversion of 0.57 gugar/g algal biomass was obtained and bioethanol production wasfficient by the separate hydrolysis and fermentation (SHF) process57]. In the other example, Chlorococum humicola was hydrolyzedy enzymes from Trichoderma reesei, obtaining a saccharificationield of 64.2% (w/w) under the conditions of 40 C, pH 4.8, and aicroalgal biomass concentration of 10 g/L [58]..2. Chemical saccharification

The chemical saccharification process is characterized by itsast reaction, but usually requires violent reaction conditionsering Journal 78 (2013) 1 10

(higher temperature, pressure, and addition of acid and alkali),resulting in the production of the inhibitors such as furfural and5-hydroxymethylfurfural, which potentially repress fermentativebiofuels production and also require costly downstream treat-ment of the waste [59]. Maintaining suitable reaction conditions(including temperature, moisture content, residence time and reac-tion agent concentration) is essential to reduce the productionof inhibitors and improve operational efficiency [60]. Due to theshort hydrolysis time required, acid/alkaline hydrolysis has beensuccessfully applied to sugar production from microalgae. In par-ticular, since the microalgae-based cellulose is not associated withlignin and is thus easier to hydrolyze when compared with ligno-celluloses, chemical hydrolysis under relatively mild conditions isof great interest (i.e., low acid or alkali concentration, low reac-tion temperature, and so on). Nguyen et al. [61] reported that byusing an extremely low acid concentration (3.0 wt.% sulfuric acid)a maximum glucose yield of 95.0% can be achieved from hydrol-ysis of algal biomass at 100 C for 30 min. Nguyen et al. [62] alsodemonstrated that a nearly 100% sugar yield of algal starch from C.reinhardtii was obtained while it was treated by 1.5% HCl. On theother hand, Harun et al. [1] used alkaline treatment (0.75% NaOH,120 C, 30 min) to produce sugar from C. infusionum with the high-est glucose yield of 0.35 g sugar/g algal biomass. It has also beenshown that bioethanol production from the sugars resulting fromthe chemical hydrolysis of microalgal biomass is quite successful[1]. These results demonstrate the advantages of using microalgae-based carbohydrates for biofuels production, as the conversion ofassimilable sugars from microalgal biomass can be achieved moreefficiently via much simpler and cheaper chemical saccharificationprocesses. In other words, the expensive and energy intensive pre-treatment and enzymatic reaction may not be necessary during thesaccharification of microalgal biomass. However, the sugar yield byacid or alkali hydrolysis may still be lower than that from enzymaticsaccharification [57].

6. Producing biofuels from microalgae-basedcarbohydrates

Microalgae possess higher lipids or carbohydrates productiv-ity, making them one of the most promising feedstock to producebiofuels [63]. The production of liquid and gaseous biofuels frommicroalgae biomass (mainly based on carbohydrates) is addressed,taking ethanol, butanol, hydrogen and methane as examples.

6.1. Liquid biofuels

Two liquid biofuels (i.e., bioethanol and biobutanol) pro-duced from microalgae-based carbohydrates are discussed here.Carbohydrates-based microalgae can be suitable feedstock forbioethanol production [64]. Although bioethanol fermentationgenerates a large amount of by-product CO2, this disadvantagecan be overcome when bioethanol fermentation is coupled withthe cultivation of carbohydrate-rich microalgae [4]. The CO2 pro-duced from bioethanol fermentation process can be fully recoveredto grow microalgae for carbohydrates accumulation. The resultingcarbohydrate-rich microalgal biomass is then used as feedstock forbioethanol production through the fermentation process. This cou-pling process can efficiently achieve the goal of CO2 mitigation andre-utilization. Moreover, the carbohydrate productivity of microal-gae is usually higher than that of lipids, since the accumulation

of the latter requires intensive stress, while carbohydrate produc-tion is readily achieved by photosynthesis through the Calvin cycle[34]. This is one of the advantages of producing bioethanol insteadof biodiesel from microalgae feedstock.

C.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 1 10 7

Fig. 2. A conceptual process of ethanol production coupled with the cultivation of starch-based microalgae. Starch-based microalgal biomass is supplemented with thep catios entate ed for

idtdgmfstimcoassursscbmbCti[wosbtft

rimary feedstocks such as corn and cassava chips, which are subjected to liquifiaccharification and fermentation (SSF) process. Ethanol is distilled from the fermthanol fermentation and the thin distillage with solid residues removed can be us

The main component in the carbohydrates of microalgal cellss starch, and some species such as Chlorella, Dunaliella, Chlamy-omonas and Scenedesmus have been reported to accumulate morehan 50% starch based on their dry cell weight [65]. Althoughifferent hydrolysis technologies have been studied for microal-al biomass [57], enzymatic hydrolysis is still the most promisingethod, and has been widely used in bioethanol fermentation

rom starch-based feedstocks. Microalgal biomass can be used as aupplement to replace part of the primary feedstocks, taking advan-age of existing technologies and facilities. A conceptual processs thus illustrated in Fig. 2. In addition to being used as supple-entary feedstock, the starch accumulated within microalgal cellsan be converted directly into bioethanol under dark and anaer-bic conditions, although bioethanol production rate and yieldre much lower [66], making this approach more scientificallyignificant in elucidating the fundamental metabolism of photo-ynthetic microalgal cells under dark conditions, rather than forse in practical applications for bioethanol production. [67] car-ied out bioethanol fermentation utilizing C. vulgaris biomass (37%tarch content) as feedstock, and obtained 65% bioethanol conver-ion when compared with the theoretically maximum bioethanolonversion from starch. Choi et al. [57] demonstrated that theioethanol yield of C. reinhardtii UTEX 90 hydrolyzed by com-ercial hydrolytic enzymes was 0.235 g bioethanol/g microalgaliomass with a separate hydrolysis and fermentation (SHF) process.hemical pretreatment (such as acid and alkaline) is an alterna-ive way to hydrolyze the carbohydrates of microalgal biomassnto fermentable sugars for bioethanol production. Nguyen et al.61] reported that when 5% (w/v dry weight basis) of algal biomassas pretreated with 3% sulfuric acid at 110 C for 30 min, 28.5 g/Lf glucose was released, representing a glucose yield of 95%. Theubsequent bioethanol yield (g bioethanol/g algae) from algae

iomass through the SHF process was 29.2%. The alkaline pre-reatment method has also been shown to be promising optionor bioethanol production. Using 0.75% (w/v) of NaOH to pre-reat microalgal biomass at 120 C for 30 min can obtain then and pre-saccharification. Fermentation is then performed by the simultaneousion broth and dehydrated as the final product. Meanwhile, CO2 produced duringmicroalgae culture.

maximum bioethanol yield of 0.26 g bioethanol/g algae [58].Although chemical saccharification usually gives a higher sugarproduction rate, the sugar-containing hydrolysates derived frommicroalgal biomass may need to be detoxified to avoid theinhibitory effect on bioethanol fermentation by the byproducts(such as furfural and 5-hydroxymethylfurfural) generated duringthe pretreatment/hydrolysis.

Biobutanol can also be produced from carbohydrate-basedmicroalgae as an alternative fuel. Butanol contains more energyand is less corrosive and water soluble [68], making this com-pound well suited for use with the existing storage and distributioninfrastructure of petroleum-based transportation fuels. Butanolalso has several advantages over ethanol as a fuel, due to its higherenergy content and lower volatility, being less hygroscopic, andmixing better with gasoline in any proportion. Biobutanol andother higher alcohols produced from biomass feedstocks are thusknown as advanced biofuels, and are expected to eventually replacebioethanol [69].

Butanol is now mainly produced by chemical synthesis usingpetroleum as the raw material [70]. Like bioethanol, tremendousresearch efforts have also been focused on biobutanol produc-tion from conventional carbon sources. However, compared withbioethanol, biobutanol fermentation is much less efficient and lessproductive, with a lower product titer and yield. This is mainly dueto the severe inhibition of biobutanol to host cells [71] and theunique metabolic pathways of the biobutanol-producing species(i.e., Clostridium spp.) that produce a significant amount of by-products, in particular acetone, bioethanol and various organicacids [72]. Moreover, the theoretical maximum biobutanol yieldis 1 mol/mol glucose (or 0.41 g/g glucose) if the fermentation canbe controlled to produce biobutanol with CO2 and H2 as the onlybyproducts, which is lower than that for bioethanol (i.e., 2 mol/mol

glucose or ca. 0.5 g/g glucose).

Biobutanol production from starch-based microalgae feedstockcould be a simpler process than that of bioethanol production,since Clostridium spp. are saccharolytic. As a result, the steps

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or starch liquefaction by amylase and saccharification of dex-rins by glucoamylase required by bioethanol fermentation withaccharomyces are no longer needed for biobutanol productionith Clostridium spp. However, there is limited research progressn biobutanol production from carbohydrates-rich microalgae,hereas butanol produced from macroalgae has been reported

73]. Efremenko et al. [74] examined the efficiency of ABE (acetone-utanol-ethanol) fermentation from various microalgal biomassesy fermentation with poly(vinyl alcohol)-immobilized C. ace-obutylicum cells. The highest biobutanol yields were obtainedhen using thermolysis pretreated Arthrospira platensis and Nan-ochloropsis sp. Ellis et al. [75] investigated the feasibility of usingicroalgae biomass cultivated with wastewater as feedstock forBE fermentation with Clostridium saccharoperbutylacetonicum N1-. They found that proper pretreatment and enzymatic hydrolysisignificantly improved the ABE yield, while adding 1% glucose alsoed to a 1.6 fold increase in total ABE production. The highest totalBE production yield and productivity obtained was 0.311 g/g and.102 g/L/h, respectively.In addition to the conversion of starch components in microal-

ae for biobutanol production, the cellulose content in microalgaean also be converted to biobutanol after appropriate hydroly-is processes. It is known that lignocellulosic materials, such asorn fiber, wheat straw [76] and wheat bran [77], can be con-erted to biobutanol with Clostridium spp. [77]. However, thereave been no reports describing biobutanol production by usingellulose originating from microalgae (mainly located in the innerell walls). Nevertheless, converting microalgal biomass that con-ains both starch and cellulose into biobutanol via fermentationith Clostridium spp. is likely to be growing trend in biobutanolroduction [78].

.2. Gaseous biofuels

In addition to liquid biofuels production from microalgae-basedarbohydrates, gaseous biofuels, such as methane and biohydrogen,ould also be produced through the aerobic/anaerobic fermenta-ion process using microalgal biomass as the carbon source. Theethane formation through the anaerobic digestion of organicaste or wastewater has been well utilized, especially for theegradation of complex materials. Hence, microalgae biomassould also be a suitable substrate for methane fermentation. Bio-ydrogen can also be produced directly through the metabolicetwork of microalgae, but the biohydrogen production efficiencys quite low [79]. In contrast, production of biohydrogen fromnaerobic fermentation using microalgae-based carbohydrates isnother attractive route, which could significant reduce the pro-uction cost of biohydrogen.When the cultivation of microalgae is associated with

ioethanol or biobutanol production from grain-based feedstocks,icroalgal residues can be processed together with grain residuess animals feed. Otherwise, the microalgal residues can be digestedor methane production to recover the remaining energy throughn anaerobic digestion process. In general, methane productionacilities can be established to the treat organic wastes producedn fermentation plants, such as bioethanol production from cas-ava chips. The same facilities can also use microalgal residues forethane production. Anaerobic digestion of algal biomass can pro-uce biogas with a high methane content (over 60%) and low sulfuroncentration to avoid corrosion problems in the power generator80]. However, the low C/N ratio of microalgal biomass may make itnsuitable for methane production. Therefore, it was proposed that

icroalgal residues and cellulosic materials (such as agricultureastes) can be co-digested to balance the C/N ratio in the optimumange of 20:1-25:1 [81]. The co-digestion of algal biomass (mainlypirulina) and waste papers can greatly enhance the biomethaneering Journal 78 (2013) 1 10

production rate. Moreover, the co-digestion of microalgal biomasswith other organic materials as well as physical pretreatment onalgal biomass was also applied to improve the digestibility of algae.

In addition to methane production from algal biomass, bio-hydrogen is another alternative biogas produced through thedigestion of algal cells. Biohydrogen has emerged as one of themost promising new energy carriers, because it is cleaner andmore efficient, particularly when it is used in fuel cells to directlygenerate electricity [82]. Kawaguchi et al. [83] reported that thestarch-containing green alga Dunaliella tertiolecta and C. reinhardtiiwere used successfully in biohydrogen fermentation, achieving bio-hydrogen yields (based on starch) of 61% and 52%, respectively.Nguyen et al. [62] used enzymatic degradation of starch with ther-mostable -amylase to enhance the biohydrogen production ofgreen alga biomass (Thermotoga neapolitana), obtaining a biohydro-gen yield of 2.5 mol H2/mol glucose through the SHF process [62].Significant amounts of biohydrogen were produced from lipid-extracted microalgal biomass residues (LMBRs) using conventionalanaerobic activated sludge fermentation technology, generating anew concept for use in biorefineries. Yang et al. [84] showed thatthe highest biohydrogen yield of 45.54 mL/g-VS was achieved fromLMBRs that underwent thermo-alkaline pretreatment at 100 C,with this being approximately three-fold higher than the yieldfrom untreated LMBRs. In addition, there has recently been growingresearch attention focused on biogas production from microalgae[85]. However, it is worth noting that the anaerobic digestion pro-cess cannot be economically competitive unless it is targeted totreat the microalgal residues remaining after the production of highgrade biofuels, such as bioethanol and biobutanol, or unless it isintegrated into a microalgae biorefinery [6].

7. Conclusions

Microalgae strains can accumulate over 50% carbohydratesintracellularly (in terms of starch and cellulose) under appropri-ate cultivation conditions. These microalgae-based carbohydrateshave the advantages of easy saccharification and requiring less pre-treatment, thereby being highly competitive against lignocellulosicmaterials as the feedstock for biofuels production and biorefiner-ies. Understanding of fundamentals underlying the carbohydratemetabolism of microalgae is a prerequisite for developing moreeffective strategies to increase the carbohydrates productivity,which should be optimized via manipulation of the key operatingfactors (such as light supply, nutrients starvation, temperature andCO2 supplementation). In addition, more economic and effectivesaccharification processes should also be developed to enhance theefficiency of biofuel conversion through the microalgae biomass.To meet the demand of the biofuels market, large scale pro-cesses for the cultivation of carbohydrates-rich microalgae shouldbe developed with appropriate photobioreactor design. Moreover,economic assessment and life cycle analyses of the microalgae-based biofuels producing system should also be conducted to assessthe commercial feasibility of converting microalgae-based carbo-hydrates into various biofuel products.

Acknowledgements

The authors gratefully acknowledge the financial supportprovided by the National Basic Research Program of China(2011CB200905) for research into the self-flocculation of microal-gal cells, as well as the support from Taiwans Ministry of Economic

Affairs (Grant no. 101-D0204-3) and Taiwans National ScienceCouncil under grant numbers NSC 100-2221-E-006-126, NSC100-2622-E-011-008-CC2, NSC 101-3113-P-110-002, NSC 101-3113-E-006-015, and NSC 101-3113-E-006-016.

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eferences

[1] R. Harun, M.K. Danquah, G.M. Forde, Microalgal biomass as a fermentationfeedstock for bioethanol production, J. Chem. Technol. Biotechnol. 85 (2010)199203.

[2] C.Y. Chen, K.L. Yeh, R. Aisyah, D.J. Lee, J.S. Chang, Cultivation, photobioreactordesign and harvesting of microalgae for biodiesel production: a critical review,Bioresour. Technol. 102 (2011) 7181.

[3] A. Demirbas, Social, economic, environmental and policy aspects of biofuels,Energy Educ. Sci. Technol. 2 (2010) 75109.

[4] S.H. Ho, C.Y. Chen, D.J. Lee, J.S. Chang, Perspectives on microalgal CO2-emissionmitigation systems a review, Biotechnol. Adv. 29 (2011) 189198.

[5] G. Sivakumar, D.R. Vail, J.F. Xu, D.M. Burner, J.O. Lay, X.M. Ge, P.J. Weathers,Bioethanol, biodiesel., Alternative liquid fuels for future generations, Eng. LifeSci. 10 (2010) 818.

[6] J.H. Mussgnug, V. Klassen, A. Schluter, O. Kruse, Microalgae as substrates for fer-mentative biogas production in a combined biorefinery concept, J. Biotechnol.150 (2010) 5156.

[7] K. Skjnes, P. Lindblad, J. Muller, BioCO2 a multidisciplinary, biologicalapproach using solar energy to capture CO2 while producing H2 and high valueproducts, Biomol. Eng. 24 (2007) 405413.

[8] F.W. Bai, W.A. Anderson, M. Moo-Young, Ethanol fermentation technologiesfrom sugar and starch feedstocks, Biotechnol. Adv. 26 (2008) 89105.

[9] Y. Sun, J.Y. Cheng, Hydrolysis of lignocellulosic materials for ethanol produc-tion: a review, Bioresour. Technol. 83 (2002) 111.

10] L.R. Lynd, Overview and evaluation of fuel ethanol from cellulosic biomass:technology, economics, the environment, and policy, Annu. Rev. Energy Envi-ron. 21 (1996) 403465.

11] S.H. Ho, C.Y. Chen, J.S. Chang, Effect of light intensity and nitrogen starvationon CO2 fixation and lipid/carbohydrate production of an indigenous microalgaScenedesmus obliquus CNW-N, Bioresour. Technol. (2012) 244252.

12] J. Goldemberg, Ethanol for a sustainable energy future, Science 315 (2007)808810.

13] R.L. Naylor, A.J. Liska, M.B. Burke, W.P. Falcon, J.C. Gaskell, S.D. Rozelle, K.G.Cassman, The ripple effect: biofuels, food security, and the environment, Envi-ronment 49 (2007) 3043.

14] D.Y.C. Leung, X. Wu, M.K.H. Leung, A review on biodiesel production usingcatalyzed transesterification, Appl. Energy 87 (2010) 10831095.

15] R.F. Service, Cellulosic ethanol biofuel researchers prepare to reap a newharvest, Science 315 (2007) 14881491.

16] M.E. Himmel, S.Y. Ding, D.K. Johnson, W.S. Adney, M.R. Nimlos, J.W. Brady,T.D. Foust, Biomass recalcitrance: engineering plants and enzymes for biofuelsproduction, Science 315 (2007) 804807.

17] S.P.S. Chundawat, G.T. Beckham, M.E. Himmel, B.E. Dale, Deconstruction of lig-nocellulosic biomass to fuels and chemicals, Annu. Rev. Cell Biomol. 2 (2011)121145.

18] P. Kumar, D.M. Barrett, M.J. Delwiche, P. Stroeve, Methods for pretreatmentof lignocellulosic biomass for efficient hydrolysis and biofuel production, Ind.Eng. Chem. Res. 48 (2009) 37133729.

19] F. Wen, N.U. Nair, H.M. Zhao, Protein engineering in designing tailored enzymesand microorganisms for biofuels production, Curr. Opin. Biotechnol. 20 (2009)412419.

20] G. Stephanopoulos, Challenges in engineering microbes for biofuels production,Science 315 (2007) 801804.

21] D. Graham-Rowe, Beyond food versus fuel, Nature 474 (2011) S6S8.22] G.C. Dismukes, D. Carrieri, N. Bennette, G.M. Ananyev, M.C. Posewitz, Aquatic

phototrophs: efficient alternatives to land-based crops for biofuels, Curr. Opin.Biotechnol. 19 (2008) 235240.

23] D.S. Domozych, M. Ciancia, J.U. Fangel, M.D. Mikkelsen, P. Ulvskov, W.G.T.Willats, The cell walls of green algae: a journey through evolution and diversity,Front. Plant Sci. 3 (2012) 82.

24] H. Rismani-Yazdi, B.Z. Haznedaroglu, K. Bibby, J. Peccia, Transcriptomesequencing and annotation of the microalgae Dunaliella tertiolecta: pathwaydescription and gene discovery for production of next-generation biofuels, BMCGenomics 12 (2011) 148.

25] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294306.26] C.D. Rangel-Yagui, E.D.G. Danesi, J.C.M. de Carvalho, S. Sato, Chlorophyll pro-

duction from Spirulina platensis: cultivation with urea addition by fed-batchprocess, Bioresour. Technol. 92 (2004) 133141.

27] A.J. Wargacki, E. Leonard, M.N. Win, D.D. Regitsky, C.N.S. Santos, P.B. Kim,S.R. Cooper, R.M. Raisner, A. Herman, A.B. Sivitz, A. Lakshmanaswamy, Y.Kashiyama, D. Baker, Y. Yoshikuni, An engineered microbial platform for directbiofuel production from brown macroalgae, Science 335 (2012) 308313.

28] T. Yamada, K. Sakaguchi, Comparative studies on Chlorella cell walls inductionof protoplast formation, Arch. Microbiol. 132 (1982) 1013.

29] F.B. Metting, Biodiversity and application of microalgae, J. Ind. Microbiol.Biotechnol. 17 (1996) 477489.

30] G.Y. Wang, X. Wang, X.H. Liu, Two-stage hydrolysis of invasive algal feedstockfor ethanol fermentation, J. Integr. Plant Biol. 53 (2011) 246252.

31] A.M. Illman, A.H. Scragg, S.W. Shales, Increase in Chlorella strains calorific val-ues when grown in low nitrogen medium, Enzyme Microb. Technol. 27 (2000)

631635.

32] M.S. Kim, J.S. Baek, Y.S. Yun, S.J. Sim, S. Park, S.C. Kim, Hydrogen productionfrom Chlamydomonas reinhardtii biomass using a two-step conversion process:anaerobic conversion and photosynthetic fermentation, Int. J. Hydrogen Energy31 (2006) 812816.

[

ering Journal 78 (2013) 1 10 9

33] A. Lehninger, D. Nelson, M. Cox, Lehninger Principles of Biochemistry, 4th ed.,W.H. Freeman, New York, 2005.

34] R. Radakovits, R.E. Jinkerson, A. Darzins, M.C. Posewitz, Genetic engineering ofalgae for enhanced biofuel production, Eukaryot. Cell 9 (2010) 486501.

35] S. Kimura, T. Kondo, Recent progress in cellulose biosynthesis, J. Plant Res. 115(2002) 297302.

36] A. Sukenik, Ecophysiological considerations in the optimization of eicosapenta-enoic acid production by Nannochloropsis-Sp (Eustigmatophyceae), Bioresour.Technol. 35 (1991) 263269.

37] F.M.L. DSouza, G.J. Kelly, Effects of a diet of a nitrogen-limited alga (Tetraselmissuecica) on growth, survival and biochemical composition of tiger prawn(Penaeus semisulcatus) larvae, Aquaculture 181 (2000) 311329.

38] M.A.C.L. De Oliveira, M.P.C. Monteiro, P.G. Robbs, S.G.F. Leite, Growth and chem-ical composition of Spirulina maxima and Spirulina platensis biomass at differenttemperatures, Aquacult. Int. 7 (1999) 261275.

39] Z.I. Khalil, M.M.S. Asker, S. El-Sayed, I.A. Kobbia, Effect of pH on growth andbiochemical responses of Dunaliella bardawil and Chlorella ellipsoidea, World J.Microbiol. Biotechnol. 26 (2010) 12251231.

40] S.D. Araujo, V.M.T. Garcia, Growth and biochemical composition of the diatomChaetoceros cf. wighamii brightwell under different temperature, salinity andcarbon dioxide levels I. Protein, carbohydrates and lipids, Aquaculture 246(2005) 405412.

41] A.P. Carvalho, C.M. Monteiro, F.X. Malcata, Simultaneous effect of irradianceand temperature on biochemical composition of the microalga Pavlova lutheri,J. Appl. Phycol. 21 (2009) 543552.

42] B.D. Fernandes, G.M. Dragone, J.A. Teixeira, A.A. Vicente, Light regime charac-terization in an airlift photobioreactor for production of microalgae with highstarch content, Appl. Biochem. Biotechnol. 161 (2010) 218226.

43] D.H. Turpin, Effects of inorganic N availability on algal photosynthesis andcarbon metabolism, J. Phycol. 27 (1991) 1420.

44] Y.X. Huo, K.M. Cho, J.G.L. Rivera, E. Monte, C.R. Shen, Y.J. Yan, J.C. Liao, Conversionof proteins into biofuels by engineering nitrogen flux, Nat. Biotechnol. 29 (2011)346351.

45] M. Siaut, S. Cuine, C. Cagnon, B. Fessler, M. Nguyen, P. Carrier, A. Beyly, F. Beisson,C. Triantaphylides, Y. Li-Beisson, G. Peltier, Oil accumulation in the model greenalga Chlamydomonas reinhardtii: characterization, variability between commonlaboratory strains and relationship with starch reserves, BMC Biotechnol. 11(2011) 7.

46] Y. Li, D. Han, G. Hu, M. Sommerfeld, Q. Hu, Inhibition of starch synthesis resultsin overproduction of lipids in Chlamydomonas reinhardtii, Biotechnol. Bioeng.107 (2010) 258268.

47] S.M. Renaud, L.V. Thinh, G. Lambrinidis, D.L. Parry, Effect of temperatureon growth, chemical composition and fatty acid composition of tropicalAustralian microalgae grown in batch cultures, Aquaculture 211 (2002)195214.

48] M. Taraldsvik, S.M. Myklestad, The effect of pH on growth rate, biochemicalcomposition and extracellular carbohydrate production of the marine diatomSkeletonema costatum, Eur. J. Pharmacol. 35 (2000) 189194.

49] J.R. Xia, K.S. Gao, Impacts of elevated CO2 concentration on biochemical compo-sition, carbonic anhydrase, and nitrate reductase activity of freshwater greenalgae, J. Integr. Plant Biol. 47 (2005) 668675.

50] M. Giordano, Interactions between C and N metabolism in Dunaliella salinacells cultured at elevated CO2 and high N concentrations, J. Integr. Plant Biol.158 (2001) 577581.

51] M.R. Brown, S.W. Jeffrey, J.K. Volkman, G.A. Dunstan, Nutritional properties ofmicroalgae for mariculture, Aquaculture 151 (1997) 315331.

52] S.H. Ho, S.W. Huang, C.Y. Chen, T. Hasunuma, A. Kondo, J.S. Chang,Characterization and optimization of carbohydrate production from anindigenous microalga Chlorella vulgaris FSP-E, Bioresour. Technol. (2012),http://dx.doi.org/10.1016/j.biortech.2012.10.100.

53] S. Van de Vyver, L. Peng, J. Geboers, H. Schepers, F. de Clippel, C.J. Gommes,B. Goderis, P.A. Jacobs, B.F. Sels, Sulfonated silica/carbon nanocomposites asnovel catalysts for hydrolysis of cellulose to glucose, Green Chem. 12 (2010)15601563.

54] R. Soni, A. Nazir, B.S. Chadha, Optimization of cellulase production by a ver-satile Aspergillus fumigatus fresenius strain (AMA) capable of efficient deinkingand enzymatic hydrolysis of Solka floc and bagasse, Ind. Crop. Prod. 31 (2010)277283.

55] M.J.E.C. van der Maarel, B. van der Veen, J.C.M. Uitdehaag, H. Leemhuis, L.Dijkhuizen, Properties and applications of starch-converting enzymes of thealpha-amylase family, J. Biotechnol. 94 (2002) 137155.

56] C. Cara, M. Moya, I. Ballesteros, M.J. Negro, A. Gonzalez, E. Ruiz, Influence ofsolid loading on enzymatic hydrolysis of steam exploded or liquid hot waterpretreated olive tree biomass, Process Biochem. 42 (2007) 10031009.

57] S.P. Choi, M.T. Nguyen, S.J. Sim, Enzymatic pretreatment of Chlamydomonasreinhardtii biomass for ethanol production, Bioresour. Technol. 101 (2010)53305336.

58] R. Harun, M.K. Danquah, Enzymatic hydrolysis of microalgal biomass forbioethanol production, Chem. Eng. J. 168 (2011) 10791084.

59] S.I. Mussatto, G. Dragone, P.M.R. Guimaraes, J.P.A. Silva, L.M. Carneiro, I.C.Roberto, A. Vicente, L. Domingues, J.A. Teixeira, Technological trends, global

market, and challenges of bio-ethanol production, Biotechnol. Adv. 28 (2010)817830.

60] K. Okuda, K. Oka, A. Onda, K. Kajiyoshi, M. Hiraoka, K. Yanagisawa, Hydro-thermal fractional pretreatment of sea algae and its enhanced enzymatichydrolysis, J. Chem. Technol. Biotechnol. 83 (2008) 836841.

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