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CHAPTER 17 Production of Biodiesel from Algal Biomass: Current Perspectives and Future Yi-Feng Chen, Qingyu Wu* School of Life Sciences, Tsinghua University, Beijing 100084, People’s Republic of China *Corresponding author: E-mail: [email protected] 1 INTRODUCTION Biodiesel is defined as monoalkyl esters of plant oils or animal fats through transesterification reactions. These neutral lipids serving as energy stores in plants and animals bear a common structure of triple esters where usually three long-chain fatty acids are coupled to a glycerol. Transesterification action turns the triple esters to single ones by displacing glycerol with small alcohols (e.g., methanol). Biodiesel was initially adopted for the compression-ignition (diesel) engine invented about one century ago by a German engineer Rudolf Diesel. His first engine was run with peanut oils, and subsequent experiments confirmed that biodiesel was better than raw oils. However, biodiesel was not as popular in the world as diesel engines, replaced by petroleum-derived diesel with cheap and abundant supplies shortly after the 1920s. Recently, the rise in petroleum price and the need to reduce greenhouse gas emission reactivated interest in biodiesel. Commercial application of biodiesel started in the 1990s in the United States and European countries and now has expanded into many regions of the world including China. Despite its acceptance worldwide and various sources of feedstocks of vegetable oils or ani- mal fats, the current global yield of biodiesel only accounts for less than one per cent of the whole diesel market. One of the main obstacles is the supply of biodiesel feedstocks currently in usage is nonsustainable or limited to small scales. For example, soybean, a major form of feedstocks for the United States biodiesel market, is produced from arable land and also con- sumed as food. The feedstock competition for food and biofuel should therefore restrict the development of large-scale industrial biodiesel from soybean. Additional sources of feedstocks coming from animal fats or used oils of restaurants obviously have problems in 399 Biofuels: Alternative Feedstocks and Conversion Processes # 2011 Elsevier Inc. All rights reserved.

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Page 1: Biofuels || Production of Biodiesel from Algal Biomass

Biofuels: Alternative Feedstocks and Conversion P

C H A P T E R

17

Production of Biodieselfrom Algal Biomass: Current

Perspectives and FutureYi-Feng Chen, Qingyu Wu*

School of Life Sciences, Tsinghua University, Beijing 100084, People’s Republic of China

*Corresponding author: E-mail: [email protected]

1 INTRODUCTION

Biodiesel is defined asmonoalkyl esters of plant oils or animal fats through transesterificationreactions. These neutral lipids serving as energy stores in plants and animals bear a commonstructure of triple esters where usually three long-chain fatty acids are coupled to a glycerol.Transesterification action turns the triple esters to single ones by displacing glycerol with smallalcohols (e.g., methanol). Biodiesel was initially adopted for the compression-ignition (diesel)engine invented about one century ago by a German engineer Rudolf Diesel. His first enginewas runwith peanut oils, and subsequent experiments confirmed that biodieselwas better thanraw oils. However, biodiesel was not as popular in the world as diesel engines, replaced bypetroleum-derived diesel with cheap and abundant supplies shortly after the 1920s. Recently,the rise in petroleum price and the need to reduce greenhouse gas emission reactivated interestin biodiesel. Commercial application of biodiesel started in the 1990s in the United States andEuropean countries and now has expanded into many regions of the world including China.

Despite its acceptanceworldwide and various sources of feedstocks of vegetable oils or ani-mal fats, the current global yield of biodiesel only accounts for less than one per cent of thewhole diesel market. One of the main obstacles is the supply of biodiesel feedstocks currentlyin usage is nonsustainable or limited to small scales. For example, soybean, a major form offeedstocks for the United States biodiesel market, is produced from arable land and also con-sumed as food. The feedstock competition for food and biofuel should therefore restrictthe development of large-scale industrial biodiesel from soybean. Additional sources offeedstocks coming from animal fats or used oils of restaurants obviously have problems in

399rocesses # 2011 Elsevier Inc. All rights reserved.

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400 17. PRODUCTION OF BIODIESEL FROM ALGAL BIOMASS: CURRENT PERSPECTIVES AND FUTURE

limited and unstable supply. To these problems, more and more people through the explora-tion of the field believe the eventual answersmight be found frommicroalgae due to ultrahighbiomass yields and high contents of oil in some algal species. A nationwide research activitynamed the Aquatic Species Programwas initiated in 1978 by the Department of Energy of theUnited States and lasted for 18 years (Sheehan et al., 1998). One of the main tasks of the Pro-gram was to collect and identify microalgal strains with high contents of lipids and developthe cultivation systems, all of which was expected to lead to biodiesel production at commer-cial levels. Though the project was terminated in 1996 because of the budget difficulty, plentyof lessons and knowledge have been generated. Some of them are introduced here: (1) morethan 3000microalgal strainswere collected and 10%of them turned to bemost promising in oilcontents and other traits; (2) the feasibility of mass cultivation of these promising strains inopen ponds was confirmed and thus the culture technology was established; (3) methodsto analyze algal lipids were partially established, for instance the dyer Nile red used forin vivo monitoring of lipid droplets in algal cells; (4) genetic improvement through key genesof lipid metabolism pathways was preliminarily proved effective in eukaryotic microalgae.All these successful efforts inspired continual exploration at the field, and nowadays obviousprogresses have been made in both insights into algal metabolism pathways especiallysurrounding the model system Chlamydomonas and development of genetic tools.

The advantages of algal biomass as feedstock of biodiesel have been discussed in severalreview articles and proceedings, exemplified byChisti (2007), Rosenberg et al. (2008), Hu et al.(2008) and a latest report of the DOE of the U.S. on the National Algal Biofuels TechnologyRoadmap (Ferrell and Sarisky-Reed et al., 2010). We intend to provide a brief summary ofthese advantages of algal feedstock below. For more details, please refer to these articles.

(1) High biomass productivity. Microalgae have been demonstrated to possess higherefficiency in photosynthesis and adaptation to stressful conditions. Many microalgalspecies exist in single cells or simple clusters of a few cells. These simple cellularorganizations usually enablemicroalgae to grow fast or accumulatemore lipid or starch atexcessive energy. Microalgae possess multiple metabolic pathways and areinterchangeable under different nutrient and sunlight situations. Algal bioreactors mightbe built up vertically or in a multiple-layer manner to utilize the space to maximum. Onthe other hand, microalgae of some fast-growing species could be harvested continuouslyat a daily basis. All this greatly increase algal biomass productivity per area annually.

(2) High oil contents or yields. Oleaginousmicroalgae contain storage oils at triacylglycerides(TAGs) more than 50% of dry cell weight. It has been known that autotrophic microalgaecould accumulate more neutral lipids up on stresses such as nitrogen deficiency. Moreinterestingly, some heterotrophic species such as Chlorella protothecoides that are able togrow fast meanwhile accumulate over 50% of neutral lipids, and eventually producemuch higher yields of oil.

(3) Less usage of arable land and freshwater. Becausemicroalgae will not be cultured directlyin soil, theoretically any kind of lands including many nonarable lands could be exploitedfor algal mass cultivation. Also, algal bioreactors could be arranged in an industrialmanner, which is far advanced to crops of conventional agriculture. Consequently,efficiency of the actual land usage is projectioned to be improved drastically. Microalgaecan utilize various wastewaters, seawater, and other forms of produced water which

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4011 INTRODUCTION

cannot be introduced into the agricultural system. These features of microalgal cultureobviously reduce competition for limited land and freshwater resources.

(4) In contrast to crops of conventional agriculture, microalgae of some species may utilizehigh doses of CO2 present in flue gases, indicating they have the potential to reducegreenhouse gas emission. It is also interest for researchers to investigate if the high-doseCO2 adaptation could further improve photosynthesis and thus biomass.

(5) Algal cultivation for biomass productionmight be compatiblewith biorefinery to producea variety of fuels and value-added coproducts. In this way, all biomass components mightbe fully utilized.

Among these advantages of microalgae, microalgal metabolic pathways are particularlyimportant, mainly because these pathways directly determine algal biomass formation andlipid accumulation, and the lipid yield is the most important trait for liquid transportationfuel production. We will preliminarily introduce microalgal metabolic pathways and theirrelevance to biomass and lipid production. More specific descriptions of their metabolicproperties will be placed in the next section.

There are threemetabolic pathways present inmicroalgae—autotrophy, heterotrophy, andmixtrotrophy. Autotrophic algae uptake light, CO2 under simple inorganic media; their mainstorage products include starch and/or lipids. Under optimal light and temperature, someautotrophic algal species are able to rapidly grow at a rate of 0.2 g(DW)/l/day and eventualbiomass can be formed at 2 g (DW)/l (Gouveia and Oliveira, 2009). For most of autotrophicalgae, their rapid growth usually alleviates lipid accumulation and vice versa. Stresstreatments like deficit in nutrients (N, P, or Si) result in lipid accumulation but meanwhilereduce growth rates. In the presence of organic carbon sources such as glucose or acetate,some species from Chlorella, Chlamydomonas, and so on can thrive with increased growth ratesfor example of 24 g(DW)/l/day in comparison with autotrophic conditions, and eventualmaximal biomass yield could be over 100 g(DW)/l of the best strains (Wu and Shi, 2006).Among these species, a few of them can accumulate higher contents of TAG, usually over50% (Miao and Wu, 2006), whose final lipid yields are thus extremely higher than those ofautotrophic partners. When both light and organic carbons are present, some microalgaemay additively make use of inorganic and organic carbons, through amixotrophic (or namedphotoheterotrophic) pathway. The consequences of this pathway in biomass production arehowever hard to predict, maybe beyond the heterotrophy plus autotrophy (Lee, 2004), orbetween within the ranges of heterotrophy and autotrophy in most of cases. Less is knownabout lipid accumulation under mixotrophic conditions. These three metabolic pathwaysmight be switched from one to another in certain algal species after acclimation of manygenerations; for example, in C. protothecoides, autotrophy can be reversibly changed to hetero-trophy monitored by availability of glucose or light (Miao and Wu, 2006).

In this chapter, we will focus on the development of biodiesel technology based on hetero-trophic microalgae since its significant progresses during the last several years in comparisonto the autotrophic microalgae-based biodiesel technology. Additional progresses in regard tolipid analysis methods, improvement of algal biodiesel quality, updated transesterificationreactions, and integration of biodiesel production with environmental remediation throughalgae (e.g., wastewater-rich nutrients or flue gas high-dose CO2) are also introduced to try togive readers a balanced view of the current status of the field.

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2 TWO APPROACHES LEADING TO LIPID ACCUMULATIONIN MICROALGAE

2.1 Photoautotrophic Microalgae Accumulate Lipids upon Stresses Butat Expense of Slow Growth

At present, researchers of algal biodiesel make use of two strategies to produce biodiesel,which are inherently associated with two fundamental physiological processes ofmicroalgae—photosynthesis or aerobic respiration. Photosynthesis is the most obvious fea-ture of all microalgae that distinguishes themselves from other microorganisms (e.g., bacteriaand fungi). The energy driving photosynthesis is provided by sunlight, so fair amounts ofefforts are being devoted to improve light absorbance and thus increase biomass. For this pur-pose, many suggestions had been made from lowering chlorophyll contents to adapt to highlight, tubular photobioreactors to increase area to volume ratios, and so on, which washighlighted in Chisti (2007). There are a plenty of publications on the theme of photoautotro-phic biomass. Due to the limitation of space, details as to structure, function, and regulation ofmicroalgal photosynthetic apparatus are not mentioned, but its general characterizations arepointed out in this review. All feedstocks used in biofuel production are derived, directly orindirectly, from photosynthesis, which enables algae and plants occupy a unique position inresearch and program of biofuel technology. On the other hand, photosynthesis is a cheapprocess in nature, whichmight be an inherent driving power for researchers to try to integrateinto industrial infrastructures.

Glucose, sucrose, and starch are the main carbohydrate products of algal photosynthesis.Under normal conditions, autotrophic algal cells accumulate few amounts of lipids, usuallyless than 20% of dry cell weight in eukaryotic microalgae or even less than 10% of dry cellweight in prokaryotic cyanobacteria (Hu et al., 2008). The underlying mechanism might beinterpreted by the negative relationship between photosynthetic starch accumulation andlipid synthesis (Li et al., 2010). Li et al. (2010) observed that triacylglycerol (TAG) wasoverproduced by 10-fold in a Chlamydomonas starchless mutant with inactivation of ADP-glucose pyrophosphorylase. This work suggests a strategy to increase lipid production bydirecting more photosynthetic carbon partitioning to lipid.

On the other hand, many stress conditions ranging from nutrition deficiency (N, P, or Si) toosmotic or temperature changes induce neutral lipid accumulation within cells frequentlyover 50% of dry cell weight while alleviate cellular growth rates (Rodolfi et al., 2009;Zhekisheva et al., 2002). Naturally oleaginous autotrophic algae, albeit with higher contentsof lipids, show slow growth, which might be the native indicator of the inherent conflictbetween rapid growth and lipid accumulation. This conflict might generally exist in all kindsof photosynthetic organisms from algae to higher plants.

A technology of two-phase cultivation was developed to increase lipid accumulation inautotrophic microalgae with starvation. It composes of algal growth under sufficient nutrientsfirst for biomass formation then under nitrogen deficiency for a couple of days for the conver-sion to lipids. There are several shortages of the technologyneeded to overcome. First, the oper-ation to switch low-cell-density algae in the nitrogen concentrations from high to low is noteasy at large scale, but the reverse operation is easy to achieve by feeding certain nitrogen-containing chemicals. Second, the two-phase cultivation is discontinuous for algae harvesting

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4032 TWO APPROACHES LEADING TO LIPID ACCUMULATION IN MICROALGAE

and lipid production, since the first phase is dedicated to build up aproper algal biomass that isconverted in the subsequent phase to lipid under starvation. This design should limit lipidyields if compared with lipid yield at a daily basis. Third, the whole cultivation process lastsfor a longer period for one time production, given the lower biomass formation by autotrophicalgae,which limits the technology to be scaled up for commercial application.As an example, alatest attemptwas aimed to improve some of these disadvantages by Rodolfi et al. (2009). From30 microalgal strains, Nannochloropsis sp. F&M-M24 was characterized with 60% lipid contentafter nitrogen starvation. Further experiments were performed in a 110-liter photobioreactor.Under nitrogen starvation, lipid accumulated by the species at 0.204 g (DW)/l/day with 60%final lipid content, the growth rate was 0.3 g(DW)/l/day.

2.2 Heterotrophic Microalgae Accumulate Lipids and Grow Fast,But Consume more Glucose or Other Organic Carbons

A few of microalgal species, exemplified by C. protothecoides, has ability to grow withorganic carbon sources. These strains, after acclimation of many generations, were conferredwith expanded capacities in organic carbon utilization at high efficiency in well-controlledfermenters. Under optimized conditions, their biomass formation and high cell densitiesare comparable even to those of bacteria or yeasts which are typical industrialmicroorganisms widely employed for food and drug production at large scales. Both bacteriaand yeasts have been recently adopted for biofuel production of substances such asbioethanol and long-chain alcohols (Peralta-Yahya and Keasling, 2010) based on matureindustrial technologies. The same situation might apply to heterotrophic microalgaeaccording to their apparent similarities to other microorganisms of aerobic fermentation.Investigations during the past several years from our group and other laboratories overthe world have confirmed that heterotrophic microalgae may fit well into the infrastructureof mature fermentation industry to express the high potential in biomass productivity as wellas lipid productivity (Alabi et al., 2009; Li et al., 2007; Rosenberg et al., 2008).

Heterotrophic microalgae display another advantage, that is, there is a coordinate growthwith lipid accumulation under normal growth conditions. Lipid content in C. protothecoides inthe presence of glucose was about 4-fold of that under the light and without glucose (Miaoand Wu, 2006). This phenotype is impressively different from the situation in autotrophicmicroalgae where fast growth and lipid accumulation usually conflict. As a consequence,the coordination of these two fundamental characters confers heterotrophic microalgae withextremely high efficiency in lipid accumulation for biodiesel production. The underlyingmechanism was preliminarily exploited in heterotrophic C. protothecoides through metabolicflux analysis (Xiong et al., 2010b). A metabolic network composed of the glycolysis, the pen-tose phosphate pathway, and the tricarboxylic acid cycle was revealed, which might beinvolved in biomass formation and lipid accumulation. It was confirmed that excessiveNADPH requirements for lipid biosynthesis, indicated by increased relative activity of thepentose phosphate pathway to the glycolysis under heterotrophic conditions interfered withnitrogen limitation. The nitrogen limitation experiment further revealed that although thestress altered the growth rate and cellular oil content and thus absolute fluxes, relative globalflux distribution in the species remained stable, suggesting that the heterotrophic alga

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404 17. PRODUCTION OF BIODIESEL FROM ALGAL BIOMASS: CURRENT PERSPECTIVES AND FUTURE

possesses high capacity of metabolic buffering up on variable environments, which mightensure the alga with stable and high metabolic output for neutral lipid accumulation. Theseresults may also arouse additional exploration into the autotrophic partner in regard togrowth and lipid biosynthesis under nitrogen limitation in the near future.

A series of explorations were made to improve the cultivation of heterotrophic microalgae.One attempt was made to modify the preliminary fed-batch culture by accuratelymaintaining glucose feeding rate, pH, temperature, dissolved oxygen through computer.The supplement of glucose was limited no more than 24 g/l (Xiong and Wu, 2008). It wastested with heterotrophic C. protothecoides in a 5-liter fermenter. By using this strategy, thealgal biomass increased from 16.8 in preliminary fed-batch culture to 51.2 g/l in the modifiedculture within about 1 week of fermentation. Other important issues relevant to the cultiva-tion of heterotrophic algae will be addressed in subsequent separate sections, including costreduction by displacing glucose with cheap or even waste organic carbon sources as well asmass cultivation in a 11,000-liter fermenter to examine the scalability of the technology.

Asmentioned earlier, somemicroalgal species are able to run amixotrophic pathwaywhenfaced by both sunlight and organic carbon nutrients of the surroundings. At present, little isknown about characterizations andmechanisms of the pathway in microalgae. Apparently, afew of microalgal species might attain benefits of both autotrophy under light and heterotro-phy under organic carbons, an additive phenotype undermixotrophic conditions. Xiong et al.(2010a) successfully observed the similar results in the PFM (the photosynthesis-fermentationmodel) system they developed. In the PFM, the same Chlorella species first grows under lightto develop functional photosynthetic apparatus to capture solar energy then to be switched toorganic carbons toperformheterotrophic growth.Algal biomass and lipid accumulation in thePFM systemwas further improved in comparison to sole heterotrophic growth,which impliesan approach based on the new metabolic mechanism might developed, which provides anovel strategy for the cost reduction in addition to selection of cheap alternatives of glucoseor the scaleup in mass cultivation.

3 EFFORTS TO FURTHER REDUCE COSTS OF MASS CULTUREOF HETEROTROPHIC MICROALGAE IN SEARCH FOR

CHEAP SUBSTITUTES OF GLUCOSE

Because glucose supplement accounts for most of the medium cost of algal cultivation(it was estimated up to 80%), the cost reduction by seeking alternatives of cheap organic car-bon sources becomes the priority task of the technological improvement, in addition to otherapproaches like the scaleup for cost dilution. The price of starch from products such as corn ishalf of that of glucose, and themedium cost could be reduced to 40%when glucose is replacedwith starch. Corn powder hydrolysate has been used to replace glucose for developing acheap medium for heterotrophic C. protothecoides (Xu and Wu, 2006). To further reduce themedium cost, many additional options of organic carbon sources were screened and testedfor feasibility instead of glucose and starch, including juices from sugar cane (Cheng et al.,2009a) or sweet sorghum (Gao et al., 2010). Even for a single carbon source like starch, thereis still some room to reduce cost by selecting cheaper commercial starch products of varioussources, exemplified by an extensive survey from Jerusalem artichoke tube (Cheng et al., 2009b)

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4054 THE SCALEUP OF HETEROTROPHIC MICROALGAL BIOMASS PRODUCTION

to cassava starch (Lu et al., 2010; Wei et al., 2009). However, all these organic carbons are thefeedstocks widely used for food and chemical industries as well as for other biofuels likeethanol, which should arouse a competition between algal biodiesel and other purposes.Sugar degraded from lignocellulosic feedstock might provide potentially much cheaperand sustainable source and an approval-of-concept work has showed the application ofsemicellulose as a substrate for engineered Escherichia coli for liquid biofuel production(Steen et al., 2010). Yet, the realistic application awaits the breakthrough in the enzymaticdegradation of cellulose at commercial scales.

Industrial ormunicipal wastewaterswith abundant organic carbon contentsmight be idealalternatives of glucose to support heterotrophic microalgae, but so far few evidence has beenraised to approve the speculation. The municipal wastewater is usually introduced from thesurroundings to a centered facility for the treatment and thus with massive and stable dailysupplies. The rich nutrients in these wastewaters include nitrogen, phosphate, vitamins, andtrace elements, in addition to organic carbon substances, variable depending on differentsources. These wastewaters generate serious environmental concerns and must be treatedbefore released out. Potentially, growingmicroalgae in suchwastewaters may reach the effectof “one stone hits two birds.” Inhibitors and toxic chemicals present in them threaten growthand even survival of manymicroalgae. So screening and acclimation become an essential stepto acquire adequate microalgal species.

Waste molasses as the byproduct in sugar refinery is a kind of industrial wastewaters.It contains nearly 50% of the total sugar content and other nutrients necessary for micro-organism growth. The baker’s yeast could grow in waste molasses (Sirianuntapiboon andPrasertsong, 2008), implying that it might be suitable for algal growth too. A report fromYan et al. (2011) indicated that the biomass and lipid accumulation in C. protothecoides grownin treated waste molasses were comparable to those grown in glucose, which confirmed thefeasibility of the innovation along the direction.

4 THE SCALEUP OF HETEROTROPHIC MICROALGALBIOMASS PRODUCTION

Although so far no commercial oil and biodiesel have been reported from heterotrophicmicroalgae, there was a piece of news from U.S. Department of Energy that Solazyme, Inc.will build up a demonstration facility at the size of 12 metric tons of dry feedstock per dayby using heterotrophic microalgae for biodiesel production. They aim to collect, from thefacility, the data necessary to complete design of the first commercial plant.

Li et al. (2007) compared biomass yield and lipid accumulation in heterotrophic C.protothecoides when grown in fermenters with volumes of 5, 750, up to 11,000 liters, respec-tively. Within about 8 days of fed-batch culture, the algal biomass achieved 15.5, 12.8, and14.2 g(DW)/l, respectively in the 5, 750, and 11,000 l fermenters. The lipid contents reached46.1, 48.7, and 44.3% of dry cell weight accordingly. With up to 98.15% of the conversion rateof transesterification, the biodiesel production rates were 7.02, 6.12, and 6.24 g/l, respectively,which confirmed quite well the stability of the technology based on heterotrophic microalgaeduring the scaleup.

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406 17. PRODUCTION OF BIODIESEL FROM ALGAL BIOMASS: CURRENT PERSPECTIVES AND FUTURE

A theoretic prediction for the commercial scale and cost reduction was made based on thedemands of the European diesel market (Wijffels and Barbosa, 2010). If the annual need fornearly 0.4 billion m3 of transportation fuels was replaced by algal biodiesel, and given thealgal biodiesel productivity was 40,000 l per hectare annually based on a 3% photosyntheticefficiency and 50% oil content, 9.25million hectare of landwould therefore be required for thebiodiesel supply. It was proposed that the biodiesel production scale needs to leap at leastthree orders of magnitude and the cost has to be decreased by a factor of 10 (Wijffels andBarbosa, 2010). The prediction was based on the autotrophic microalgae. When the heterotro-phic microalgae with higher efficiency in biomass and oil production produced underconcentrated chemical energy supplies and in well-controlled fermentation facility and otheradvantages were considered into the framework of the prediction, less than 9.25 million hect-are of land would be expected to achieve the similar goal.

5 PROGRESSES IN LIPID ANALYSIS

General requirements in lipid analysis include high-throughput screening, on-site and invivo monitoring, small samples, less time, and of course accuracy. During the past severalyears, a lot of efforts have been made to boost the improvements in lipid analysis. Onlyseveral cases were adopted herein as snapshots of the area due to the limited space ofthe article.

The Soxhlet method was proved efficient for total lipid extraction and gravimetric quanti-fication frommicroalgae. It is frequently adopted as a standard to evaluate other newly devel-oped methods. However, it has two obvious shortages. The conventional organic solventssuch as n-hexane are costly and also not environmentally friendly. It takes considerabletime-consuming steps and also consumes samples at large amounts, for example with mini-mum requirement of at least 100-ml cultures.Wawrik andHarriman (2010) developed a rapidand colorimetric method for the quantification of algal lipid from 1 ml of cultures. Algal lipidsare saponified to fatty acids and then mixed with a copper reagent and a color developerdiethyldithiocarbamate. The formed yellow product is then colorimetrically measured. Fattyacidswith chain lengths of C10:0 to C16:0 fell into the ranges of linear responses, belowC10 orbeyond C16 caused underestimation. But the method could be formatted in microcentrifugetubes and an analysis of 30 samples could be finished in less than 2 hwhichwas confirmed bymonitoring dynamic total lipid contents in Phaeodactylum tricornutum and Chlorella vulgaris,indicating the method might be suitable for fast monitoring or screening of species in termsof lipid contents.

Su et al. (2008) proposed a rapid method for nondestructive detection of chlorophyll a andlipid contents of microalgae by using RGBmodel of two linear correlation functions based onthe brightness values of the three primary colors (red, green, and blue). The prediction resultsfrom the model were agreeable with experimental results and the reliability of the model wasalso verified in a photosyntheticmicroalgaNannochloropsis oculata. It is curious that if this sim-ple approach could be tested in a broad ranges of algal species, for example to estimate lipidcontents in yellow heterotrophic algal cells.

Under optimized conditions, the dyer Nile red specifically binds to nonpolarmacromolecules like neutral lipids. By using this feature, the Nile red staining method

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4076 THE CONVERSION OF ALGAL BIOMASS TO BIODIESEL

was established to determine neutral lipids in a broad spectrum of organisms includingmicroalgae in about half a century ago. The method is fast, sensitive, and requires just afew of in vivo algal cells, and therefore has the potential for the high-throughput screening.However, the staining signals are affected by the thickness of algal cell walls that variesamong species, which limits its application for the large-scale screen. Chen et al. (2009)introduced DMSO (dimethyl sulfoxide) with mild heating to speed up the penetration ofthe dyer throughout algal cell walls. They claimed this modification can buffer the differencesof staining signals caused by differences in cell wall thickness. With such, the modifiedmethod could be fitted into 96-well plates on a fluorescent spectrometer that serves for quan-tification or screening of large-scale microalgal samples. They also showed that microwavepretreatment within less than 1 min could further improve themeasurement of in vivo neutrallipids stained with Nile red (Chen et al., 2010).

Gao et al. (2008) have applied time-domain nuclear magnetic resonance (TD-NMR) toquantify lipid contents in C. protothecoides. The method was found simple, quick, and lessexpensive, but still with desired accuracy when compared with ordinary NMR. In themethod, spin-echo NMR pulse sequence is employed to separate the lipid hydrogen nucleisignal from other hydrogen nuclei signals, and after calibration lipid contents in microalgaecan be measured accurately.

As a nondestructive and rapid analytic technique, the near-infrared spectroscopy hasmany advantages in comparison to standard and traditional techniques such as the chro-matographic method. Near-infrared reflectance spectroscopy (NIRS) has been used to deter-mine the oil content and fatty acid composition in intact seeds (Kim et al., 2007). To ourknowledge, a similar approach is being transplanted to analyze lipid levels and categoryin several microalgal species by Dr. Al Darzins’s group and others.

6 THE CONVERSION OF ALGAL BIOMASS TO BIODIESEL

There are two general strategies for the conversion of algal biomass to biodiesel—lipidextracted from algae is transesterified to biodiesel or the whole algal biomass is decomposedvia physical processing to bio-oil which in turn is refined to biodiesel. The past several yearshave seen obvious progresses in both directions(Farrell et al., 2010).

The conversion of lipid extracts is the typical mode of biodiesel production from algae,especially from highly dense heterotrophic microalgae that offer high yields of oil and turnthe extraction to easy and low cost processing. The transesterification reactions are applied toconvert algal triacylglycerols to FAMEs (fatty acid methyl esters), a displacement process ofglycerol by mono-alcohols (e.g., methanol or ethanol). The technique is well developed tomaturity and used as a standard in the conversion of vegetable oils into biodiesel. Thereactions could be completed chemically with inorganic catalysts, or biochemically withthe enzyme lipase (Farrell et al., 2010).

The transesterification reaction could be achieved under bases or acids. The base-catalyzedroute is preferred though the acid-catalyzed one is an attractive alternative in certainsituations, for example less sensitivity to water presence or reduction of saponification andemulsification (Wahlen et al., 2008). However, in general, acid catalysis possesses lower activ-ity over that of base catalysis. As a result, higher temperatures and longer reaction times are

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408 17. PRODUCTION OF BIODIESEL FROM ALGAL BIOMASS: CURRENT PERSPECTIVES AND FUTURE

required in these alkaline or acid-catalyzed processes. It means the chemical transesteri-fication is energy intensive, and also requires the separation of glycerol and alkaline catalystfrom biodiesel as well as the treatment of alkaline wastewater. Several variants of thetransesterification reaction were developed to address these problems, including fast heatingaided bymicrowave (Refaat and El Sheltawy, 2008), or improvedmixing and heating by ultra-sonic treatments (Kalva et al., 2008). Ultrasound-aided transesterification can also run inlinewhich is superior to batch reaction in traditional methods. Particularly as the core of the tech-nique, various catalysts of new types and catalysis modes are being developed. For example,heteropolyacids (HPA, e.g., H3PW12O40/Nb2O5) have been shown to lower the requiredtemperatures and decrease the reaction times (Alsalme et al., 2008; Cao et al., 2008). MildLewis acid catalysts such as AlCl3 or ZnCl2 are extremely efficient, in the presence of acosolvent such as tetrahydrofuran, to convert triacylglycerols into fatty acid methyl estersup to 98% (Soriano et al., 2009). The third example is catalysts derived from the titanium com-pound (e.g., HTiNbO3) or vanadate metal compounds (e.g., TiVO4). These hydrophobiccatalysts are insensitive to or reducing free fatty acid concentration, and used to achieveFAME and glycerol yields over 90% under moderate temperature and pressure (e.g., 200 �Cand35bar;Cozzolino et al., 2006).Due to their insolubility toall other substancesof the reactionsystem as well as their stability, they may offer an ideal option toward commercial transes-terification reaction. Cheaper alternatives of this type might be identified from MgO, CaO,or Al2O3.

Lipases as biocatalysts are characterized with extremely specificity of transesterificationreaction under quite mild conditions. The enzymes are also more attractive in environmentalcompatibility than classic or other inorganic catalysts. However, currently high prices andshort lifetime of the enzymes limit their application at large scales. Biochemical engineeringis required to improve their stability and lower the cost. Alternatively, by offering the inter-face for heterogeneous catalysis, immobilized lipases could reach high conversion rate up to98%, which was demonstrated in microalgal oils (Xiong and Wu, 2008). The immobilizationmay also extend the lifetime of lipases and therefore improve the economy of enzymatictransesterification reaction.

By using supercritical alcohols (methanol or ethanol), oil extraction and transesterificationreaction might be simplified to one-step conversion from whole wet biomass to biodiesel, inwhich the alcohols may function as both extractor of lipids and stimulator of transesteri-fication reaction. The new processing has been confirmed in vegetable oils (Demirbas, 2006,2009). The advantages of the approach are immediately foreseeable such as (1) convenient—all processing being done at one spot, (2) selective—multiple extractions of differentcomponents at high purity and concentration from algal biomass by selecting supercriticalfluids, (3) “green”processingdue to only alcohols beingused and catalyst-free, (4) highqualityand stability of biodiesel and other value-added compounds attainable at modest conditions(e.g., less than 50 �C), (5) efficient—whole native algae being employed without dewateringand oil extraction. Yet, the supercritical manipulation with methanol or ethanol may stilldecompose algal biomass that may potentially reduce biodiesel yield (Hawash et al., 2009;Vieitez et al., 2009). Further study is required to detail the operating conditions in themicroalgal system (Patil et al., 2010).

When algal biomass contains lower amounts of lipids or when the secondary conversionfrom the remnants of oleaginous microalgae should be performed after lipid extraction, the

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4097 THE QUALITY AND ECONOMIC ANALYSIS OF ALGAL BIODIESEL

physical processing such as pyrolysis or hydrothermal liquefaction might be suitable to turnthese kinds of algal biomasses to biodiesel. Pyrolysis is used to decompose condensed bio-mass by extremely fast heating (e.g., a couple of seconds) in the absence of oxygen. For exam-ple, heating to 350-500 �C is completed within less than 2 s in fast pyrolysis in which finelyground particles of biomass should be provided. This created an advantageous situation formicroalgal biomass because microalgae exist as single cells with micrometer-scale sizes (e.g.,10-30 mm in diameter), and therefore the grinding processing is not necessary for microalgalparticles. Oxygen contents in different sources of biomass might affect the quality of bio-oil,the product of pyrolysis. Our previous observation showed that bio-oil from heterotrophicmicroalgae contained lower oxygen over that from autotrophic microalgae of the same spe-cies (Miao and Wu, 2004), implying the metabolic pathways might alternate oxygen contentsin algal biomass and presenting a practical biological approach to monitor the quality ofbiofuels. The bio-oil may directly enter the biorefinery stream for production of useablebiodiesel. The research in this direction is still in its infancy.

Wet biomass is suitable to supply to a hydrothermal liquefaction processingwherewater isheld in a liquid state above 100 �C under pressure (called subcritical water). Biocrude, themain product of the processing, contains smaller molecules of high energy density that mightbe comparable to fossil diesel. The biocrudemay contain additional valuable compounds andneed further upgrade. Except a couple of old reports that indicated bio-oil yields of 37-64%could be generated at 300 �C and 10 MPa from Botryococcus braunii (Sawayama et al., 1995) orDunaliella tertiolecta (Minowa et al., 1995), quite few reports on the topic could be identifiedfrom the recent literature. Since the processing may mimic the natural geological processesassociated with petroleum formation and it is believed microalgae like diatom might havea major contribution to the evolution of fossil fuels, more researches are interestingly awaitedin the near future.

7 THE QUALITY AND ECONOMIC ANALYSIS OF ALGAL BIODIESEL

Based on the ASTM biodiesel standard (e.g., ASTM International, 2009a,b), Miao and Wu(2006) examined properties of biodiesel from microalgal oil. The parameters range from den-sity, viscosity, flash point, cold filter plugging point, solidifying point, to heating value. Mostof the parameters comply with the ASTM standard. The properties of algal biodiesel werealso comparable to those of fossil diesel, indicating that algal biodiesel is probably able toblend with fossil diesel.

Three culture modes—autotrophic algae in raceway open pond or closed photobioreactor,and heterotrophic algae in fermenter—were compared in terms of economic parameters frombiomass production and harvest, oil extraction, capital, labor, to operational costs based onconditions in British Columbia (Alabi et al., 2009). Total costs for these three modes couldbe evaluated by using a thermodynamic model into which local light and temperature valuesaswell as various cost parameterswere integrated. The results showed that the base case costsfor the three biomass production systems were $14.44 (raceway), $24.60 (photobioreactor),and $2.58 (fermenter) per liter of algal oil, respectively. As a reference, the cost for per literof canola oil was $0.88. All these biological oils currently cannot compete with petroleumfor fuel production. An immediate conclusion from this analysis could be heterotrophic

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410 17. PRODUCTION OF BIODIESEL FROM ALGAL BIOMASS: CURRENT PERSPECTIVES AND FUTURE

fermentation appears to be the most promising approach for biofuel production, while auto-trophic algae in photobioreactor are unaffordable economically. Further analysis indicatedthat the cost of fermentation was mainly composed of the power and organic carbon sub-strate. As addressed before, the two critical issues could be solved through cheap alternativesof current organic carbons and through large-scale production. The cost for oil conversion tobiodiesel and the comparison between autotrophic and heterotrophicmodes are not yet avail-able, but a case study in vegetable oil showed that the conversion costs were $0.26 or$0.51 gal�1 when supercritical or traditional alkaline transesterification was employed(Anitescu et al., 2008). The supercritical transesterification showed lower cost over the con-ventional one because it could be finished in one step using alcohols. Taken together, theseprecommercial analyses favor the heterotrophic fermentation routemight lead to commercialproduction of algal biodiesel.

8 CONCLUDING REMARK AND FUTURE PERSPECTIVES

Through the examination of major aspects of algal biodiesel production from upstreamalgal culture to downstream oil extraction and conversion, this review has seen significantprogresses in algal biodiesel production, particularly from heterotrophic microalgae, weremade during the past several years. Along with continual efforts imposed into the field,by focusing on lowering the costs of the whole process through technical improvement, com-prehensive utilization of algal biomass components, and scaleup in production, it is hopedthat the innovative algal biodiesel technologywill probably find its application in commercialplants at large scale.

Specific tasks aiming to further yield algal biodiesel in quantity and quality are proposedand might be the focus of the next phase explorations in the field, in addition to generalconcerns on algal biofuels addressed by Wijffels and Barbosa (2010) and many others.

(a) In-depth insights are required about lipid metabolism in either autotrophic orheterotrophic microalgae. Particularly, the knowledge of lipid droplet biology rangingfrom dynamic structure, storage or degradation, to regulation will directly benefit thetechnological development involved in biodiesel production. For autotrophic microalgae,by using genetic engineering in capture, storage, and conversion of solar energy throughphotosynthesis, either biomass yield of oleaginous algae or oil yield of fast-growing algaemight be enhanced. The underlying mechanisms of stress-induced accumulation ofneutral lipid need to be elucidated, from which new approaches might be figured out toattenuate adverse effects of stress treatments on growth. For heterotrophic microalgae,more deep insights into the conversion from glucose to lipid might be generated that willhelp in seeking substitutes of costly glucose—either cheaper nutritional or regulatorysubstances. In addition, mixotrophic pathway may provide a unique strategy for algae toutilize both sunlight and chemical energy. The details of mixotrophic features should becharacterized in the near future.

(b) A systems approach is required to study the whole chain of the process from algal straindevelopment to utilization of all biomass components for the reduction of the costs. Basedon the fast progresses that have been made in heterotrophic microalgae that could be

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411REFERENCES

fitted into the infrastructure of mature fermentation industry with high efficiency,heterotrophic microalgae might be placed on the priority list of the algal biodieselproduction.

(c) Integration and optimization of the whole processing of algal biomass to acquiremaximal benefits and increasing competiveness over other feedstock-based processing.Coproduct extraction might be coupled in parallel or in tandem with oil extraction fromheterotrophic microalgae. After extraction, algal remnants might be turned to biogasthrough the anaerobic fermentation, or directly used as feeds and fertilizer. As a long-termgoal, the remnantsmight be subject to the physical treating like pyrolysis or liquefaction toproduce bio-oils which in turn are refined to various biofuels when the biorefining isapplied to commercial production.

Acknowledgments

This study was supported by the projects 30970224 and 41030210 from the NSF of China, the MOST 863 projects2009AA064401 and 2010AA101601, the MOST supporting project 2011BAD14B05. We apologize for those progressesmade in the field that could not be cited owing to the limited space assigned to the article.

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