Bioresource Technology 102 (2011) 26–34

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Bioresource Technology

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Mechanism and challenges in commercialisation of algal biofuels

Anoop Singh a, Poonam Singh Nigam b,*, Jerry D. Murphy a

a Biofuels Research Group, Environmental Research Institute, University College Cork, Cork, Irelandb Faculty of Life and Health Sciences, University of Ulster, Coleraine BT52 1SA, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 March 2010Received in revised form 7 June 2010Accepted 9 June 2010Available online 6 July 2010

Keywords:Algal biomassBiofuelMechanismTechnological challengesFuture prospects

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.06.057

* Corresponding author.E-mail addresses: apsinghenv@gmail.com (A. Sing

Nigam), jerry.murphy@ucc.ie (J.D. Murphy).

Biofuels made from algal biomass are being considered as the most suitable alternative energy in currentglobal and economical scenario. Microalgae are known to produce and accumulate lipids within their cellmass which is similar to those found in many vegetable oils. The efficient lipid producer algae cell masshas been reported to contain more than 30% of their cell weight as lipids. According to US DOE microalgaehave the potential to produce 100 times more oil per acre land than any terrestrial plants. This articlereviews up to date literature on the composition of algae, mechanism of oil droplets, triacylglycerol(TAG) production in algal biomass, research and development made in the cultivation of algal biomass,harvesting strategies, and recovery of lipids from algal mass. The economical challenges in the productionof biofuels from algal biomass have been discussed in view of the future prospects in the commercialisa-tion of algal fuels.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Several biofuel candidates were proposed to displace fossil fuelsin order to eliminate the vulnerability of energy sector (Korreset al., 2010; Prasad et al., 2007a,b; Singh et al., 2010a,b,c; Pantet al., 2010). The biofuels produced from crop seeds have come un-der major controversy as food vs. fuel competition (Nigam andSingh, 2010) as they require land for their production, whereas al-gae can be grown in the submerged area and also in the sea water(Singh et al., 2010c). The algal cultivation not only provides thebiofuel but also provides greenhouse gas (GHG) saving as it utilizedlarge amount of CO2 during the cultivation.

Algae range from small, single-celled organisms to multi-cellu-lar organisms, some with fairly complex and differentiated form.Algae are usually found in damp places or bodies of water and thusare common in terrestrial as well as aquatic environments (Wag-ner, 2007). Algae include seaweeds (macroalgae) and phytoplank-tons (microalgae). Many are eukaryotic organisms but the term isoften used to also include cyanobacteria (blue-green algae), whichare prokaryotic (Packer, 2009). Like plants, algae require primarilythree components to produce biomass, i.e., sunlight, CO2 andwater. The existing large-scale natural sources of algae includebogs, marshes, swamps, etc. (Wagner, 2007). Algae essentially har-ness energy via photosynthesis. They capture CO2 and transform itinto organic biomass which can be converted to energy (Bruton

ll rights reserved.

h), p.singh@ulster.ac.uk (P.S.

et al., 2009). Algae can be either freshwater or marine, some growoptimally at intermediate saline levels and some in hypersalineconditions. Seaweeds are macroscopic multicellular algae thathave defined tissues containing specialised cells. Many are unicel-lular and can be motile or non-motile depending on the presence offlagella. Where multi-cellular conglomerations exist, very littlespecialisation of cell types occurs, distinguishing them from sea-weeds. There are a huge range of different types of microalgaeincluding dinoflagellates, the green algae (chlorophyceae), thegolden algae (chryosophyceae) and diatoms (bacillariophyceae)(Packer, 2009).

Algae contain complex long-chain sugars (polysaccharides) intheir cell walls. These carbohydrate cell walls account for a largeproportion of the carbon contained in these organisms (Packer,2009), though many species contain quite high levels of variouslipids and for some species under certain situations this has beenquoted as up to 80% oil by wet weight (Singh et al., 2010c). Diatomsare a group including approximately 100,000 organisms many ofwhich are marine and dominate the marine phytoplankton. Theyhave silicate cell walls and have been of considerable interest inthe biofuel production as they can accumulate very high levels oflipid. Diatoms, like many other organisms, use the triacylglycerollipid molecules (TAGs) as energy storage molecules that can beeasily transesterified to biodiesel, but a large percentage of the lip-ids contained in diatoms are phospholipids which are structurallydissimilar to TAGs and do not convert well to biodiesel using tradi-tional transesterification procedures. Coccolithophores, that havecalcareous external plates called coccoliths, also include somesingle celled flagellated algae and are also important in natural

A. Singh et al. / Bioresource Technology 102 (2011) 26–34 27

oceanic carbon capture (Packer, 2009). Keeping in view the abovefact, this paper highlights the mechanism of biofuel productionfrom algae and also summarizes the key points involved in thecommercialisation of algal fuels.

Table 1Chemical composition (% dry matter basis) of selected microalgae (Bruton et al.,2009).

Protein Carbohydrate Lipids Nucleic acid

Freshwater algal speciesScenedesmus obliquus 50–56 10–17 12–14 3–6Scenedesmus quadricauda 47 – 1.9 –Scenedesmus dimorphus 8–18 21–52 16–40 –Chlamydomonas rheinhardii 48 17 21 –Chlorella vulgaris 51–58 12–17 14–22 4–5Chlorella pyrenoidosa 57 26 2 –Spirogyra sp. 6–20 33–64 11–21 –Euglena gracilis 39–61 14–18 14–20 –Spirulina platensis 46–63 8–14 4–9 2–5Spirulina maxima 60–71 13–16 6–7 3–4.5Anabaena cylindrica 43–56 25–30 4–7 –

Marine algal speciesDunaliella bioculata 49 4 8 –Dunaliella salina 57 32 6 –Prymnesium parvum 28–45 25–33 22–38 1–2Tetraselmis maculata 52 15 3 –Porphyridium cruentum 28–39 40–57 9–14 –Synechoccus sp. 63 15 11 5

2. Importance of algal fuel

The use of fossil fuels as energy is now widely accepted asunsustainable due to depleting resources and also due to the accu-mulation of GHGs in the environment. Renewable and carbon neu-tral biodiesel are necessary for environmental and economicsustainability. Biodiesel demand is constantly increasing as thereservoir of fossil fuel are depleting. Unfortunately biodiesel pro-duced from oil crop, waste cooking oil and animal fats are not ableto replace fossil fuel. The viability of the first generation biofuelsproduction is however questionable because of the conflict withfood supply. Production of biodiesel using microalgae biomass ap-pears to be a viable alternative (Khan et al., 2009). The idea of mic-roalgae utilization as a fuel source is being taken seriously becauseof the rising price of petroleum and more significantly, the emerg-ing concern about global warming that is associated with burningof fossil fuels (Gavrilescu and Chisti, 2005). Recent research initia-tives have proven that microalgae biomass appear to be the one ofthe promising source of renewable biodiesel which is capable ofmeeting the global energy demand and it will also not compromiseproduction of food, fodder and other products derived from crops.

Microalgae appear to be the only source of biodiesel that hasthe potential to completely displace fossil diesel. Unlike other oilcrops, microalgae grow extremely rapidly and many are exceed-ingly rich in oil. Microalgae commonly double their biomass within24 h. Biomass doubling times during exponential growth are com-monly as short as 3.5 h (Chisti, 2007). Oil content in microalgae canexceed 80% by weight of dry biomass (Metting, 1996; Spolaoreet al., 2006).

Similar to other biomass resources algal biofuel is also a carbonneutral energy source.

There may be opportunities for applying biorefinery-type pro-cesses to extract and separate several commercial products frommicroalgal biomass. Besides lipids, microalgal biomass offers oppor-tunities for obtaining additional commercial materials. These in-clude fermentation to obtain ethanol and biogas. It is also possibleto produce protein-rich feed for both animal and human consump-tion. Poly-unsaturated fatty acids (PUFAs) are a potential co-productof biodiesel production from microalgae. PUFAs are alternative tofish oils and other oils rich in omega-3 fatty acids (Bruton et al.,2009). Bulk markets for the co-products are potentially available.

The microalgal oil contain high proportions of long chain fattyacids (i.e., C-20, C-22) with a high degree of un-saturation (20:5).These very long chain-poly-unsaturated fatty acids are importantin aquaculture applications as they improve the nutritional qualityof feed (Packer, 2009). There is much speculation that integratedbiorefinery solutions would allow sufficient scale to enable eco-nomic production of fuel from macroalgae. The only industrialproduct of significance from macroalgae is hydrocolloids. Extrac-tion of energy from wastestreams is a valid commercial biorefineryconcept. If the cost of seaweed permits, a dual production of etha-nol and biogas is also possible. There are many other opportunitiesfor extraction of high-value niche products from seaweeds. Eachwould have to be assessed on commercial terms and demonstratethe feasibility for co-production of energy alongside the higher-va-lue product, with particular attention to whether the scale of oper-ation is appropriate (Bruton et al., 2009).

There has been a great deal of analysis done on the land re-quired to produce microalgae for biofuels production (Chisti,2007). Although most of these studies are in the context of using

North American saline aquifers, it is sufficient to say that theseanalyses suggest that there is certainly more than enough non-ara-ble land suitable for mass algal cultivation for biofuel production tomeet the needs of that country (Packer, 2009). Drawing from thesestudies it is also probable that several countries like New Zealand,Canada, etc. have enough land that does not compete with foodproduction that is also close to industrial CO2 sources to meetthe liquid fuel requirements.

The ocean has already absorbed nearly half of the anthropo-genic CO2 generated since the industrial revolution and the absorp-tion of CO2 has an induced effect on the water acidity, which isnegatively affecting marine life including microalgae (Riebesellet al., 2007). However, it has also been suggested that increasedlevels of CO2 on the atmosphere might actually stimulate the bio-logical pump involving growth of some algal species for the trans-port of carbon to long-term deep ocean storage (Arrigo, 2007;Riebesell et al., 2007). The LOHAFEX (LOHA is Hindi for iron, Fstands for Fertilization EXperiment) an Indo-German iron fertiliza-tion experiment in the Southwest Atlantic Sector of the SouthernOcean conducted for rapid growth of the minute, unicellular algaethat not only provide the food, sustaining all oceanic life, but alsoplay a key role in regulating concentrations of the CO2 in the atmo-sphere. The development of such algal bloom on its environmentand the fate of the carbon sinking out of it to the deep ocean mightplay a crucial role in popularization of algal biofuels.

3. Composition and structure of algae

Microalgae biomass has a chemical composition which varies al-gal use (Table 1). Microalgae are being widely researched as a fueldue to their high photosynthetic efficiency and their ability to pro-duce lipids (a biodiesel feedstock). Macroalgae do not generallycontain lipids and are being considered for the natural sugars andother carbohydrates that can be fermented to produce either biogasor ethanol (Bruton et al., 2009). It can be rich in proteins or rich inlipids or have a balanced composition of lipids, sugars and proteins.On the basis of minimal nutritional requirements the approximatemolecular formula of the microalgal biomass is estimated asCO0.48H1.83N0.11P0.01 (Chisti, 2007). Species selection should bemade according to the desired biofuel route. A characteristic of mic-roalgae is to have significant lipid content and even very high lipidcontent under certain stress conditions (Bruton et al., 2009).

Several researchers have reported that algae produced more oilin stressed condition in comparison to optimal growth condition

28 A. Singh et al. / Bioresource Technology 102 (2011) 26–34

(Grant, 2009; Hu et al., 2008). Under optimal growth conditions, al-gae synthesize fatty acids principally for esterification into glycerolbased membrane lipids, which constitute about 5–20% of dry cellweight. Fatty acids include medium-chain (C10–C14), long-chain(C16–C18) and very-long-chain (PC20) species and fatty acidderivatives. But under unfavourable environmental or stress condi-tions, many algae alter their lipid biosynthetic pathways towardsthe formation and accumulation of neutral lipids (20–50% dry cellweight), mainly in the form of TAG. Lipid accumulation in algae be-gins when it exhausts a nutrient from the medium (usually nitro-gen), but an excess of carbon is still assimilated by the cells and isconverted into TAG, while lipid is synthesized during the balancephase of growth at nearly the same rate. As the limitation supplyof nitrogen, it means that cell proliferation is prevented and theformed lipid has to be stored within the existing cells which canno longer divide (Meng et al., 2009). Unlike the glycerolipids foundin membranes, TAGs do not perform a structural role but insteadserve primarily as a storage form of carbon and energy. After beingsynthesized, TAGs are deposited in densely packed lipid bodies lo-cated in the cytoplasm of the algal cell, although formation andaccumulation of lipid bodies also occur in the inter-thylakoid spaceof the chloroplast in certain green algae (Hu et al., 2008). The fattyacid composition of typical oil from microalgae is given in Table 2.It is mainly composed of mixture of unsaturated fatty acids, such aspalmitoleic (16:1), oleic (18:1), linoleic (18:2) and linolenic acid(18:3). Saturated fatty acids, palmitic (16:0) and stearic (18:0)are also present to a small extent (Meng et al., 2009).

Algae are primitive plants (thallophytes), i.e., lacking roots,stems and leaves, have no sterile covering of cells around thereproductive cells and have chlorophyll a as their primary photo-synthetic pigment (Lee, 1980). Algae structures are primarily forenergy conversion without any development beyond cells, andtheir simple development allows them to adapt to prevailing envi-ronmental conditions and prosper in the long term (Falkowski andRaven, 1997). Prokaryotic cells (cyanobacteria) lack membrane-bound organelles (plastids, mitochondria, nuclei, golgi bodies,and flagella) and are more akin to bacteria rather than algae.Eukaryotic cells, which comprise of many different types of com-mon algae, do have these organelles that control the functions ofthe cell, allowing it to survive and reproduce (Brennan andOwende, 2010). Eukaryotes are categorised into a variety of classesmainly defined by their pigmentation, life cycle and basic cellularstructure (Khan et al., 2009). The most important classes includegreen algae (Chlorophyta), red algae (Rhodophyta) and diatoms(Bacillariophyta). Algae can either be autotrophic or heterotrophic.Some photosynthetic algae are mixotrophic, i.e., they have the abil-ity to both perform photosynthesis and acquire exogenous organicnutrients (Lee, 1980). For autotrophic algae, photosynthesis is akey component of their survival.

4. Mechanism of triacylglycerol (TAG) production in algalbiomass

Microalgae are eukaryotic cells that mean they contain a nu-cleus and other membrane-bound organelles and use sophisticated

Table 2Fatty acid composition of microalgal oil (Meng et al., 2009).

Fatty acid Chain length: no.of double bonds

Oil composition(w/total lipid)

Palmitic acid 16:0 12–21Palmitoleic acid 16:1 55–57Stearic acid 18:0 1–2Oleic acid 18:1 58–60Linoleic acid 18:2 4–20Linolenic acid 18:3 14–30

control mechanisms and post-translational biosynthetic processes.The flexible metabolic repertoire affords a greater choice and speedof response in metabolic approaches to different situations (Packer,2009). Microalgae are able to survive heterotrophically, exogenouscarbon sources offer prefabricated chemical energy, which the cellsoften store as lipid droplets (Ratledge, 2004). Another naturalmechanism through which microalgae can alter lipid metabolismis the stress response owing to a lack of bioavailable nitrogen (Tor-nabene et al., 1983). Although nitrogen deficiency appears to inhi-bit the cell cycle and the production of almost all cellularcomponents, the rate of lipid synthesis remains higher, which leadsto the accumulation of oil in starved cells (Sheehan et al., 1998).

Many microalgal cells possess the enzyme pyruvate formatelyase (key enzyme in bacterial fermentation pathways), which iswidespread in bacteria, but seldom found in eukaryotes (Hem-schemeier and Happe, 2005). The presence of this enzyme in mic-roalgae allows fermentative behaviour when oxygen is low(important in the metabolism of hydrogen production by microal-gae). There is evidence that metabolic pathways involving the en-zyme glucose-6-phosphatase and ATP transesterification behavedifferently in algal cells partly contributing to their greater produc-tivity over terrestrial species (Woodward et al., 2000). Anotherpossible contributing factor to algal efficiency is the way they actu-ally fix carbon. There are three main carbon fixation mechanismsemployed by plants; the C3, C4 and crassulacean acid metabolism(CAM). Algae, because of their ancient origin and single-celled nat-ure, have always been thought to rely on C3 carbon fixation, butthere is evidence in marine diatoms that the C4 pathway is func-tional and important (Reinfelder et al., 2004).

Over-production of the diatom enzyme acetyl CoA carboxylase(ACCase), which catalyses a key metabolic step in the biosynthesisof lipids, did not lead to increased oil production (Sheehan et al.,1998). More recently the squalene synthase gene was cloned fromthe green algae Botryococcus braunii and over-expressed in the bac-terial cell Escherichia coli, a common laboratory organism that isused widely for molecular biology over-expression work. The genewas expressed but failed to have activity in the foreign environ-ment of the bacterial cell (Banerjee et al., 2002). Hsieh and Wu(2009) concluded in a study with Chlorella sp. on biomass and lipidproduction that mass production of lipids from microalgae for bio-fuel production can be successfully accomplished by using a semi-continuous process with replacement of limited amounts of ureaduring the cultivation.

Observations of intracellular oil droplet-formation under stressand especially nutrient deprivation, lead to much effort trying tofind a ‘lipid switch’ or ‘lipid trigger’ where a simple manipulationmight be able to greatly increase oil biosynthesis. Nutrient stress,especially nitrogen and also silicate for diatoms, was shown togreatly increase oil production in microalgae but at the expenseof total biomass production. This can be explained in that the stres-ses lead to decreased cell division, nitrogen being important forprotein biosynthesis and silicate involved in the cell wall struc-tures of diatoms, and in the absence of increasing numbers of cellsthe microalgae stored up extra lipid reserves (Packer, 2009).

Recently, Xiong et al. (2010) studied a photosynthesis–fermen-tation model to merge the positive aspects of autotrophs and het-erotrophs. In this model microalga Chlorella protothecoides wasgrown autotrophically for CO2 fixation and then metabolized het-erotrophically for oil accumulation. They recorded 69% higher lipidyield on glucose at the fermentation stage and released 61.5% lessCO2, compared to typical heterotrophic metabolism. An elemen-tary flux mode study suggested that the enzyme Rubisco-catalyzedCO2 re-fixation, enhancing carbon efficiency from sugar to oil.Immunoblotting and activity assay further showed that Rubiscofunctioned in sugar-bleaching cells at the fermentation stage. Bymeans of double CO2 fixation in both photosynthesis and fermen-

A. Singh et al. / Bioresource Technology 102 (2011) 26–34 29

tation stages, it simultaneously achieved the enhancement of car-bon efficiency for biofuel synthesis and the reduction of green-house gas emission. This strategy lowers the consumption ofsugar substrates largely, thereby opening a door for cost-effectivebiodiesel production from microalgae.

5. Utilising or storing the biomass

5.1. Cultivation of algal biomass

There are two main cultivation systems, i.e., open pond andclosed photobioreactor (PBR). Open pond refers to a simple opentank or natural ponds. Algae are grown in suspension with addi-tional fertilizers. Gas exchange is via natural contact with the sur-rounding atmosphere and solar light. The highest productivity inopen pond systems is obtained in raceway systems. A shallowdepth pond with an elliptical shape (like a raceway) is mechani-cally mixed with a paddle wheel. This moves the water along theraceway, ensures vertical mixing of water to avoid algae settle-ment and to maximize gas exchange (Bruton et al., 2009). Largeindustrial production facilities currently use raceway systems forcultivation of algal biomass. The raceway entails comparativelylow capital investment. Operational costs are also low as weeklymonitoring is enough to survey the biomass and nutrients. Energyis mainly consumed in the mixing operation. Some raceways werealso designed with artificial light, but this design is not practicaland economicaly feasible for commercial production. The low pro-ductivity is the main drawback of the raceway. High light intensitycauses cell mortality and contamination by fast growing microorganisms often happens. High biomass density cannot beachieved with these systems. In PBR system also, algae are culti-vated in suspension, but the system is closed and water is circu-lated by pumps. In existing commercial applications, artificiallight and sometimes heat is used. Only solar light and waste heatare being considered for the biofuel production purposes. Nutrientand gas levels need to monitored and adjusted them continuously.The PBRs have the advantages of high productivity, low contamina-tion, efficient CO2 capture, continuous operation, and controlledgrowth conditions (Bruton et al., 2009). The major drawbacks arethe high capital and operating costs. There are many design andoperational challenges which need to be resolved before commer-cial production of microalgae using PBR can be considered. Foulingand cleaning of PBR of both external and internal walls is a bigtrouble. Over time accumulation of dirt (external) or algae (inter-nal) will prevent light. Mixing to ensure optimum photosyntheticefficiency is also a major challenge. In order to maintain turbulentflow, energy needs to be supplied, generally for pumping, or forsparging with gases (Bruton et al., 2009). Any parasitic energy loadneed to be minimised in order to keep a positive energy balance onthe overall process. Intermediate systems have also been designed,such as open ponds under greenhouses allow a more controlledenvironment. In the same way designers of photobioreactors havereduced costs by using simple materials, such as transparent pipes,using natural solar light and gravity feeding of the growth medium.Mixing by CO2 bubbling is another way of maximizing CO2 captureand reducing mixing costs.

The large scale demand for microalgae may results in fertilizershortages. At concentrations below 0.2 lmol P/l availability ofphosphates in the growth medium will be a growth-limiting factor.Equally nitrates availability will be a problem for growth whenconcentrations are below 2 lmol N/l (Bruton et al., 2009). For dia-toms, in addition to N and P, silicate is essential. Silicon washed outfrom land to sea by freshwater run-off, will under normal condi-tions be available in sufficient amounts. Silicon will be a limitingfactor for growth of diatoms in concentrations lower than 2 lmol

Si/l. Carbon is a key requirement, as the composition of microalgaeis about 45% carbon. This is generally supplied as CO2. For eachkilogram of microalgae, at least 1.65 kg of CO2 are required basedon a mass balance (Berg-Nilsen, 2006). A two-step cultivation pro-cess has been developed that involves a combination of racewayand photobioreactor designs. The first step is the fast cultivationof biomass in the PBR and the second step is stress cultivation inopen ponds. A photoreactor first step allows good protection ofthe growing biomass during early stages by maximizing the CO2

capture. After that the microalgae suspension is transferred toopen ponds with low nitrogen nutrients and maintaining highCO2 levels. The open raceway in the second step has some prob-lems because higher density of algal biomass is more resistant toexternal contamination and this nutrient deficit phase avoidedthe growth of contaminating species (Bruton et al., 2009). Thecombination of photoreactor and open pond cultivation has provedefficient for astaxanthin production (Huntley and Redalje, 2007). Itis currently being tested by companies developing biofuel applica-tions. The University of Florence has undertaken considerable re-search into this topic (Rodolfi et al., 2008).

In the natural carbon cycle some oceanic species of microalgaesuch as the diatom Chaetoceros spp. and coccolithophores likeIsochrysis spp. and Pavola spp. naturally sink to eventually becomefossilised. Dead microalgae coalesce to form semi-solid structurescalled transparent exopolymer particles (TEPs) which are stickyand facilitate the aggregation and increased sinking of other organ-ic particles transporting carbon to deep water (Arrigo, 2007). Theseobservations have led to the possibility that these natural cyclesmight be enhanced. There are two mechanisms proposed. One isthe large-scale use of oceanic pumps to cause upwelling of nutri-ent-rich water to surface waters for stimulating microalgal produc-tion (Lovelock and Rapley, 2007). The second approach stems fromthe understanding that natural algal growth is maximal where ter-restrial sources bring nutrients to the sea. Of note were the pio-neering experiments in the Southern Ocean stimulatingmicroalgal growth with iron fertilization (Boyd et al., 2000).

The main advantages and limitation of open pons and PBR sys-tems are summarized in Table 3 (Brennan and Owende, 2010).Compared to closed photobioreactors, open pond is the cheapermethod of large-scale algal biomass production. Open pond pro-duction does not necessarily compete for land with existing agri-cultural crops, since they can be implemented in areas withmarginal crop production potential (Chisti, 2008). They also havelower energy input requirement (Rodolfi et al., 2008), and regularmaintenance and cleaning are easier (Ugwu et al., 2008) and there-fore may have the potential to return large net energy production(Brennan and Owende, 2010; Rodolfi et al., 2008). Open pond sys-tems, require highly selective environments due to inherent threatof contamination and pollution from other algae species and proto-zoa (Pulz and Scheibenbogen, 1998). Monoculture cultivation ispossible by maintenance of extreme culture environment,although only a small number of algae strains are suitable (Bren-nan and Owende, 2010). In respect to biomass productivity, openpond systems are less efficient when compared with closed photo-bioreactors (Chisti, 2007). This can be attributed to several deter-mining factors, including, evaporation losses, temperaturefluctuation in the growth media, CO2 deficiencies, inefficient mix-ing and light limitation (Brennan and Owende, 2010). Althoughevaporation losses make a net contribution to cooling, it may alsoresult in significant changes to ionic composition of the growthmedium with detrimental effects on algae growth (Pulz and Schei-benbogen, 1998). Temperature fluctuations due to diurnal cyclesand seasonal variations are difficult to control in open ponds (Spo-laore et al., 2006). Potential CO2 deficiencies due to diffusion intothe atmosphere may result in reduced biomass productivity dueto less efficient utilization of CO2. Poor mixing by inefficient stir-

Table 3Advantages and limitations of open ponds and photobioreactors (Brennan and Owende, 2010; Ugwu et al., 2008).

Production system Advantages Limitations

Raceway pond Relatively cheapEasy to cleanUtilises non-agricultural landLow energy inputsEasy maintenanceGood for mass cultivation

Poor biomass productivityLarge area of land requiredLimited to a few strains of algaePoor mixing, light and CO2 utilisationCultures are easily contaminatedDifficulty in growing algal cultures for long periods

Tubular photobioreactor Large illumination surface areaSuitable for outdoor culturesRelatively cheapGood biomass productivities

Some degree of wall growthFoulingRequires large land spaceGradients of pH, dissolved oxygen and CO2 along the tubes

Flat plate photobioreactor High biomass productivitiesEasy to steriliseLow oxygen build-upReadily temperedGood light pathRelatively cheapEasy to clean upGood for immobilization of algaeLarge illumination surface areaSuitable for outdoor cultures

Scale-up require many compartments and support materialsDifficult temperature controlSmall degree of hydrodynamic stressSome degree of wall growth

Column photobioreactor CompactHigh mass transferLow energy consumptionGood mixing with low shear stressEasy to sterilizeHigh potentials for scalabilityReadily temperedGood for immobilization of algaeReduced photoinhibition and photo oxidation

Small illumination areaExpensive compared to open pondsShear stressSophisticated constructionDecrease of illumination surface area upon scale-up

30 A. Singh et al. / Bioresource Technology 102 (2011) 26–34

ring mechanisms may also result in poor mass CO2 transfer ratescausing low biomass productivity (Ugwu et al., 2008). Light limita-tion due to top layer thickness may also incur reduced biomassproductivity. However, enhancing light supply is possible byreducing layer thickness; using thin layer inclined types of culturesystems, and improved mixing can minimise impacts to enhancebiomass productivity (Brennan and Owende, 2010; Chisti, 2007;Pulz, 2001; Ugwu et al., 2008). Jorquera et al. (2010) studied theenergy life-cycle for biomass production using the oil-rich microal-gae Nannochloropsis sp. in raceway ponds, tubular and flat-platephotobioreactors, and found that the net energy ratio (NER) of hor-izontal tubular photobioreactors (PBRs) is not economically feasi-ble. The NER for ponds and flat-plate PBRs could be raised tosignificantly higher values if the lipid content of the biomass wereincreased to 60% dw/cwd.

5.2. Harvesting of algal biomass

Harvesting methods depends primarily on the type of algae. Thehigh water content of algae must be removed to enable harvesting.Macroalgae harvesting employs manpower whereas, microalgaecan be harvested by sedimentation, filtration, flotation and centri-fugation. Macroalgae grow either on a solid substrate or free-float-ing in water. The harvesting of free floating algae can be madesimply by rising installed net in the pond, with a large energy sav-ing with respect to microalgae, which need filtration for their sep-aration. The algae harvesting is the concentration of diluted algaesuspension into a thick algae paste. Normally harvesting of micro-algae can be a single or two step process which involves harvestingand dewatering. Harvesting microalgae is difficult because of thesmall size of the algae. The selection of harvesting process for aparticular strain depends on size and properties of algal strain (Oil-gae, 2010). Pre-treatment of the biomass may also be necessary(e.g. flocculation) to improve harvesting yield. The aim of harvest-ing is to obtain slurry with at least 2–7% algal suspension on dry

matter basis. When operated on raceway cultures, the algal con-centration in ponds is typically 0.02–0.06% total solid matter (Bru-ton et al., 2009). The sedimentation and flotation harvestingtechniques mainly apply to open pond cultivation systems whilefilteration and centrifugation apply to PBRs. The simple sedimenta-tion system is suitable for microalgae which have naturally highsedimentation rates. This is performed in thickeners or clarifiers,standard processes in water treatment plants. The capital andoperation costs are low. If the strain has poor sedimentation prop-erties, a flocculation agent can be used. The flocculation processesare aided by cell flocculation, either through the addition of chem-ical flocculants or through culture autoflocculation. Flocculationcauses the cells to become aggregated into larger clumps whichare more easily filtered and/or settle more rapidly. There arenumerous inorganic and organic flocculants available in the mar-ket having negative or positive charges and working at differentpH levels. Some algal strains naturally float at the surface of thewater. Oxygen production in the photosynthesis by algae generatesgas bubbles that assist the flotation. Some chemicals can be addedto modify the surface tension of particles in order to increase bub-ble attachment and the fine air bubbling at the bottom of the pondcan also increase flotation behaviour. The other interesting charac-teristic is that as the microalgal oil content increases, the algaetend to float. Compared to sedimentation the flotation process isvery fast, it only requires a few minutes instead of hours for sedi-mentation. Capital and operating costs are also low, but the effi-ciency may be poor in shallow-depth ponds (Bruton et al., 2009).

Biomass cultivated in PBR is generally concentrated by filtrationor centrifugation. Centrifugation is an accelerated sedimentationprocess. It can operate with rotating walls or with fixed walls insystems called hydrocyclones. Capital and operation costs are high,but efficiency compared to natural sedimentation is much higher.Filtration process can range from simple screening or micro-strain-ers to dewatering up to complex vacuum or pressure filtration sys-tems. The more complex system costs more. The main limitation of

A. Singh et al. / Bioresource Technology 102 (2011) 26–34 31

filtration is plugging that can be solved by vibrating screens or tan-gential filtrations. Deep bed filtration is also commonly used toavoid plugging, but it requires mixing the solution with sand. Somecombined systems use pressing and screening belts, having theadvantage of continuous operation (Bruton et al., 2009).

Cells are more dilute in pond cultures in comparison to BPR. Themethod chosen greatly depends on the final product and the pro-cesses used for biofuel production. Some processes require the al-gae to be completely dewatered, whilst others may requiremoderate dewatering. The presence of other chemicals such asflocculants must also be taken into consideration. Filtration is mostcommonly used method to harvest algal biomass. Micro-strainers,rotating screen filters with a backwash, are widely used for collect-ing algae such as Spirulina but it is unlikely that this would be eco-nomic for collecting algae for non-food products (Packer, 2009).Flocculation, where multivalent cations are added to overcomethe negative charge carried on the surface of most microalgae thatnormally prevents them sticking together in suspension, is a rela-tively low-cost method (Eisenberg et al., 1981). It is often com-bined with dissolved air floatation (DAF). Both are maturetechnologies used in sewage ponds and wastewater treatment.DAF uses tiny bubbles that are injected under high pressure intothe water column and as they rise to the surface they drag organicmolecules and cells with them. Efficiency is increased using flocc-ulants but their addition can cause problems, depending on thedownstream process of the biomass utilization (Packer, 2009).

Collection of algae by centrifugation is only really feasible forrelatively high value products (Molina Grima et al., 2003), thoughcontinuous centrifugation has been explored which might be moreeconomic on a large scale (Briggs, 2004). A variation of centrifuga-tion that has not been explored a great deal for use with algal bio-mass collection, but is widespread in the petroleum and miningindustries, is the use of hydrocyclones. In these devices water con-taining the particles, in this case algal cells, is channelled in a spiralfashion creating centripetal forces causing the denser particles tobe spun out of the traversing liquid. Although the technique worksfor removing dense particles from liquid streams and for separat-ing oil from water, their application to soft algal cells is experimen-tal (Packer, 2009). However, their simplicity and fewer movingparts avail their potential for large-scale economic application.

5.3. Processing biomass to biofuels

Among macroalgae, the Laminaria spp. and Ulva spp. are themost important prospects from an energy perspective (Brutonet al., 2009). Biogas production is a long-established technologyand previous trials have indicated that anaerobic digestion (AD)of seaweed is technically viable. The lack of easily fermented sugarpolymers such as starch, glucose or sucrose makes fermentationprocess difficult as there is little point in pursuing standard sugarfermentation processes. The polysaccharides that are present willrequire a new commercial process to break down into their con-stituent monomers prior to fermentation, or a direct fermentationprocess will have to be developed. The traditional markets for sea-weed products sustain a much higher price for raw material thanthat for biofuel production.

Microalgae have the ability to produce lipids that can be usedfor the production of biodiesel. Existing chemical esterificationprocesses require a lipid-rich material without water. So dryingof microalgae biomass is considered in some processes. From 15%to 25% algal concentration, at least a 90% concentration shouldbe obtained. Drying requires a lot of energy and is the economicalbottleneck of the entire process that can account for 70% of the to-tal cost. Whatever the technology, evaporating 1 kg of water willalways require at least 800 kcal of energy. Several technologiesare available for drying like spray drying, rotating drum dryer

and flash drying are normally considered. A very important issuein biomass treatment is the preservation of chemical quality of al-gal biomass. After harvesting, chemicals in the biomass may besubject to degradation induced by the process itself and also byinternal enzyme activity in the microalgae (Bruton et al., 2009).For instance, lipase enzymes are well known to hydrolyse cellularlipids to free fatty acids after cell death. This reaction is fast enoughto significantly reduce the part of the lipid content suitable for bio-diesel production.

The basic chemical reaction required to produce biodiesel is theesterification of lipids, either triglycerides or oil, with alcohol andreaction performed at high pH. The result is a fatty acid alkylesterand glycerol as by-product. This chemical reaction is sensitive tothe presence of water, as saponification reactions occur in the pres-ence of water, which affects yield and quality of biodiesel. Freefatty acids also cause similar problems during the reaction. Themain limitation of microalgae oil is the unsaturated fatty acid con-tent. The levels of unsaturated fatty acids in microalgae are some-times very high (up to 30%) and excess unsaturated fatty acidlevels are a major problem for biodiesel production, because theymay induce cross linking of fatty acid chains, causing tar formation(Bruton et al., 2009).

There are several approaches to process the harvested microal-gal biomass from the ocean or from bioreactors or from ponds, i.e.,chemical, biological, thermolytic and non-lethal extraction or‘milking’. For the production of biodiesel interest has been focusedon higher lipid-producing algae, so that lipids can be extracted andprocessed to biodiesel. The most straight forward chemical conver-sion involves the transesterification of TAGs to biodiesel. Somemicroalgae have capacity to produce high levels of TAGs but mostoften with lesser growth rate (Packer, 2009). Many marine speciesproduce higher levels of phospholipids than TAGs. Phospholipidsdo not act optimally in the transesterification process. The interestin these marine species is increased due to their ability to grow inthe saline aquifers and keeping away from any conflict with freshwater use. Biological conversion includes fermentation, yieldingproducts such as ethanol and butanol. A promising approach to de-crease processing steps would be to use high-productivity marinealgal species, where osmotic shock with fresh water would liberateall the cellular constituents making them available and adjust thesalt concentration at the same time suitable for fermentation(Packer, 2009). New approaches would be required for continu-ously removal of butanol from a continuous fermentation. Thermo-lytic techniques offer the conversion of total algal biomass tobiofuels, which includes traditional pyrolysis where biomass isconverted to biofuel in the absence of oxygen, but this is highly en-ergy intensive and might produce small amounts of potentiallytoxic by-products (Packer, 2009). While worldwide several projectsare developing less extreme thermolytic processes that combine achemical transformation with less severe heat that overcome someof these problems (Huber et al., 2005; Rostrup-Nielsen, 2005).

Non-lethal extraction, or ‘milking’ the algae, for specific mole-cules has the potential advantage of greater efficiency in conver-sion to product because only the molecule of interest is beingremoved from the cell, which can then go onto make more product(Packer, 2009). The appropriate engineering solutions are neededto develop for targeting more specific useful molecules; this willimprove the environmental impact to utilize the microalgae.

5.4. Extracting lipid from algal biomass

There are several approaches to extract lipids from harvestedalgal biomass, including solvent extraction, osmotic shock, ultra-sonic extraction and critical point CO2 extraction. Hexane is themain solvent used for lipid extraction, either alone or in combina-tion with an oil expeller or press. After the oil extraction using an

32 A. Singh et al. / Bioresource Technology 102 (2011) 26–34

expeller, the leftover pulp can be mixed with cyclohexane to ex-tract the remaining oil. The oil dissolves in the cyclohexane andthe pulp is filtered out from the solution. The oil and cyclohexaneare separated by means of distillation process. These two stages(cold press and hexane solvent) together are able to derive morethan 95% of the total oil present in the algae (Packer, 2009). Osmo-tic shock is a sudden reduction in osmotic pressure causing cells torupture and release cellular components including oil. Some mar-ine species of algae such as Dunaliella sp. that lack a cell wall areparticularly suitable for this process. They grow in hypersaline con-ditions and then can be easily ruptured by diluting in non-saltywater (Williams et al., 1978). Ultrasonic extraction can greatlyaccelerate extraction processes. In this process, ultrasonic wavescreate cavitation bubbles in a solvent and by collapsing of thesebubbles near to the cell walls the shock waves developed cause cellwalls to break and release the oil into the solvent (Packer, 2009).Critical point gas/fluid extraction is probably the most efficientmethod for complete extraction of oils. The use of CO2 for this pur-pose is the most developed technology. The CO2 utilized in thisprocess is recycled (Packer, 2009) but higher energy demandmakes it unfit for oil extraction.

Recently, Samorì et al. (2010) proposed a new procedure to ex-tract hydrocarbons from dried and water-suspended samples ofthe microalga B. braunii by using switchable-polarity solvents(SPS) based on 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) andan alcohol. The high affinity of the non-ionic form of DBU/alcoholSPS towards non-polar compounds was exploited to extract hydro-carbons from algae, while the ionic character of the DBU-alkyl car-bonate form, obtained by the addition of CO2, was used to recoverhydrocarbons from the SPS. DBU/octanol exhibited the higheryields of extracted hydrocarbons from both freeze-dried and liquidalgal samples compared to n-hexane and chloroform/methanol.

6. Challenges in commercialisation of algal fuel

Microalgae are the untapped resource with more than 25,000species of which only few are in use (Raja et al., 2008). The maingenera cultivated include: Laminaria, Porphyra, Undaria, Gracilaria,Euchema, Ulva and Chondrus. From the vast number of known mar-ine and freshwater species, only a handful are currently of com-mercial significance. These include Chlorella, Spirulina, Dunaliellaand Haematococcus. Of these only Dunaliella is predominantly amarine species. These are generally cultivated for extraction ofhigh-value components such as pigments or proteins (Brutonet al., 2009). In recent years, microalgae have garnered interestfor producing valuable molecules ranging from therapeutic pro-teins to biofuels, due to there uniqueness as they combine therenewable energy capturing ability of photosynthesis with the

Table 4Comparison of the properties of various large-scale algal culture systems (Borowitzka, 19

Reactor type Light utilizationefficiency

Temperaturecontrol

Gastransfer

Mixing

Unstirred shallow ponds Poor None Poor Very pCircular stirred ponds Fair–good None Poor FairPaddle-wheel

Raceway PondsFair–good None Poor Fair–go

Tanks Very poor None Poor PoorStirred tank reactor Fair–good Excellent Low–high LargelyAir-lift reactor Good Excellent High GeneraBag culture Fair–good Good Low–high VariablFlat plate reactor Excellent Excellent High UniformTubular reactor

(serpentine type)Excellent Excellent Low–high Uniform

Tubular reactor(biocoil type)

Excellent Excellent Low–high Uniform

high yields of controlled microbial cultivation, making them poten-tially valuable organisms for economical, industrial-scale produc-tion processes in the 21st century (Rosenberg et al., 2008). Thevarious large-scale culture systems also need to be compared ontheir basic properties such as their light utilisation efficiency, abil-ity to control temperature, the hydrodynamic stress placed on thealgae, the ability to maintain the culture unialgal and/or axenic andhow easy they are to scale up from laboratory scale to large-scale(Table 4). The final choice of system is almost always a compro-mise between all of these considerations to achieve an economi-cally acceptable outcome (Borowitzka, 1999).

The overarching goal of microalgal biotechnology is to improvethe productivity of these organisms in order to meet the demandsof a rapidly growing market (Spolaore et al., 2006). Large-scaleopen ponds had lower productivity than required for economicdeployment, probably due to low night temperatures in the areaswhere these open ponds were tested. The coupling of waste heatfrom power plants and other industrial sources might also helpto overcome this problem (Packer, 2009).

The complex harvesting and processing procedures combinedwith insufficient production of algal dry mass are limiting factorsfor algal biofuel production (Ahrens and Sander, 2010).

Several landmark projects using ponds and photo-bioreactorsfor the production of microalgae for a maximum biomass produc-tion are existing throughout Germany, USA, NewZealand and sev-eral other countries. The pilot project in Hamburg Reitbrook(Germany) concerns itself with the aspects of photosynthetic CO2

fixation in microalgae as a contribution to reduce greenhouse gases(Ahrens and Sander, 2010). The commercial bioreactor supplierAlgaeLink claim year round productivity of several different spe-cies of algae in the order of 365 t ha�1 yr�1 for one of their systems.Greenfuel Technologies Corporation, based in Massachusetts USA,have several large-scale pilot plants operating and focus on CO2

capture from industrial emitters, demonstrate dry weight produc-tivities between 250 and 292 t ha�1 yr�1 in their sunlight-poweredalgal bioreactors (Packer, 2009). The Aquatic Species Programme(ASP) closeout report states open ponds were able to achieve apeak performance of ‘almost’ 300 t ha�1 yr�1 dry weight biomassproduction, whereas at the beginning of the programme they wereproducing around 50 t ha�1 yr�1 dry weight biomass (Sheehanet al., 1998). In a recent report describing algal biomass for poten-tial production in New Zealand, Heubeck and Craggs (Heubeck andCraggs, 2007) reported high rate algal pond production with CO2

stimulation is between 40 and 75 t ha�1 yr�1.Optimising stress conditions to obtain the highest possible

yields of lipids in the cells is important. There is scope for addi-tional research leading to further increases in yields. Stimulatedevolution is another option commonly used for bacteria. Stressconditions can induce spontaneous mutation in cultivated strains.

99).

Hydrodynamicsstress on algae

Speciescontrol

Sterility Scaleup

oor Very low Difficult None Very difficultLow Difficult None Very difficult

od Low Difficult None Very difficult

Very low Difficult None Very difficultuniform High Easy Easily achievable Difficult

lly uniform Low Easy Easily achievable Difficulte Low Easy Easily achievable Difficult

Low–high Easy Achievable DifficultLow–High Easy Achievable Reasonable

Low–high Easy Achievable Easy

A. Singh et al. / Bioresource Technology 102 (2011) 26–34 33

Selection of these natural mutants can improve production yields.Another option is to select wild local species that are alreadyadapted to local growth conditions. Genetic modification (GM) isanother option to improve production efficiency. One currentexample is the Algenol Company which is developing a strain ofGM cyanobacteria capable of producing ethanol. The microalgawas designed in Canada and the production site is operating inMexico. Improved harvesting technologies are needed. The solu-tion may lie in adapting and refining separation technologies al-ready being used in the food, biopharmaceutical and waste watertreatment sectors.

Lipid extraction prior to esterification is an area for further re-search. It would be an important advance if methods without dry-ing or solvent extraction of the algae slurry could be developed asit would significantly reduce the cost of biomass pre-treatment.This could be overcome if water tolerant downstream processesare developed (Bruton et al., 2009).

The utilization of existing biodiesel production processes re-quires a lipid material free of both water and free fatty acids. Thisleads to high processing costs to dry the microalgae material. Alter-native esterification processes are being investigated using theacidic reaction route or enzymatic reactions. However, they arestill at the research stage. Enzymatic esterification with lipasesmay be worth pursuing as it has the added advantage of runningat low temperatures (60 �C). A key problem with this process isthat esterification generates a glycerol by-product which inhibitslipases (Bruton et al., 2009). Development of lipase for direct ester-ification or other extraction techniques could remove the dryingstep. Studies are being carried out with methylacetate as a sub-strate which avoids glycerol formation and lipase inhibition.Unsaturated fatty acid content is high in algal oils and their pres-ence lowers esterification yields.

There are current discussions of the economics of biodiesel pro-duction in the recent review by Chisti who suggests about 1.5–3times higher productivity is required (Chisti, 2007). The costs inbiofuel production from algal biomass amounts approximately 50€/L that is very away to attract the commercial production of algalbiofuels (Ahrens and Sander, 2010).

7. Future prospects

The costs in biofuel production from algal biomass amountsapproximately 50 €/L that is very away to attract the commercialproduction of algal biofuels. An investigation of cost extensive ap-proaches for the algal biofuel production is needed. One promisingalternative seems to be the production of algal biomass in waste-water, providing a readily available medium for the productionof algal biomass at almost no cost (Ahrens and Sander, 2010),and also the cultivation of algal biomass removed nutrients fromthe wastewater and reduces the environmental pollution. Somecommercial interests into large-scale algal-cultivation systemsare looking to tie into existing infrastructures, such as coal-firedpower plants or sewage treatment facilities. This approach not onlyprovides the raw materials for the system, such as CO2 and nutri-ents but also converts wastes into resources (Wagner, 2007). Themost obvious opportunity for integrated manufacturing is by pro-duction of algae at a power-plant, in order to take advantage ofwaste CO2 and possibly also to utilize the waste heat from thepower-plant.

8. Conclusions

The integration of microalgae cultivation with fish-farms, foodprocessing facilities and waste water treatment plants etc., will of-fer the possibility for waste remediation through recycling of or-

ganic matter and at the same time low-cost nutrient supplyrequired for the algal biomass cultivation. These options could allbe explored as part of an integrated biorefinery concept. For re-gions at higher latitude, it may be possible to identify local strainsof algae requiring low light intensities and lower water tempera-tures with satisfactory growth and yields. Further work is requiredfor an economical process since the dry lipids are necessary foresterification.

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