Fuel 89 (2010) 265–274

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Fuel

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Review article

Catalytic upgrading of biorefinery oil from micro-algae

N.H. Tran a,*, J.R. Bartlett a, G.S.K. Kannangara a, A.S. Milev a, H. Volk b, M.A. Wilson b

a School of Natural Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC 1797, Australiab Division of Petroleum Resources, Commonwealth Scientific and Industrial Research Organization, 11 Julius Avenue, North Ryde, NSW 1670, Australia

a r t i c l e i n f o

Article history:Received 9 June 2009Received in revised form 6 August 2009Accepted 6 August 2009Available online 26 August 2009

Keywords:Micro-algaeGrowthFuelCatalystNano

0016-2361/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fuel.2009.08.015

* Corresponding author. Fax: +61 2 9685 9915.E-mail address: n.tran@uws.edu.au (N.H. Tran).

a b s t r a c t

Micro-algae are seen as one of the major future fuel sources. Culture and growth of oil rich micro-algaeand catalytic process for the conversion of their crude oils or biomass is reviewed here. While there is asignificant literature on growth and extraction of oil from the resultant biomass the literature on theproblems of refining these oils is diverse and needs collation. It is clear that previous work has beenfocused on the two green algae Botryococcus braunii and Chlorella protothecoides containing terpenoidhydrocarbons and glyceryl lipids as their major crude oils, respectively, both of which will need differentrefinery technology for upgrading. Studies show a number of conventional catalysts in the petroleumrefining industry including transition metals, zeolites, acid and base catalysts can be used with variableeffect. These have been employed for cracking, hydrocracking, liquefaction, pyrolysis and transesterifica-tion processes to produce diesel, jet fuel and petrol (gasoline). However there is strong evidence that newnano-scale materials containing a high number of active sites and high surface areas may offer morepotential.

� 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2652. Botryococcus braunii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

2.1. Cracking/hydrocracking of botryococcenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2672.2. Cracking/hydrocracking using nano-scale catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

3. Chlorella. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

3.1. Pyrolysis of biomass derived from Chlorella protothecoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2703.2. Transesterification of crude oils derived from Chlorella protothecoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2703.3. Transesterification using nano-scale catalysts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

4. Chaetoceros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2715. Dunaliella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2726. Nannochloropsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2727. Spirulina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2728. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

1. Introduction

Critical issues associated with the supply of fossil fuels and theiradverse impacts on the environment have generated significantinterest in renewable fuels. Bio-ethanol or transesterified fats fromanimals, terrestrial or aquatic plants have been used as fuels orblends (e.g. for fuel extend or octane enhancement [1,2]). While

ll rights reserved.

bio-ethanol, especially from non-food crops has its place, its energydensity (the energy obtained per unit mass or volume, but betterdefined as the Gibbs Free Energy of combustion per unit mass orvolume) precludes it from many applications such as use as an avi-ation fuel (jet A1 and equivalents). Because the products from mi-cro-algae are higher molecular weight and hence have higherenergy densities like diesel, they are a target for diesel replace-ment. However, there are a number of refining issues for algal de-rived oil which need to be explored as well as the choice of algaeand growth conditions.

266 N.H. Tran et al. / Fuel 89 (2010) 265–274

The potentials of algae as the alternative biofuel sources havebeen reviewed [3–7]. Micro-algae generally show fast growth ratesand can achieve high biomass per unit area that achievable withterrestrial plants. Different growing conditions can be exploitedranging from highly controlled photo-bioreactors to less well con-trolled open ponds and race ways. The oil yield may vary greatlybetween species, harvest time and is also dependent on the nutri-ent supply. Not all algae are capable of producing enough bio-crude oils but a number of algae species have been designated ascontaining enough material for industrial production of oils. Nor-mally this constitutes a species capable of producing >50% dryweight of extractable oil [3].

Significantly, higher oil contents are associated with nutrientlimitations and need the algae to live in stressed conditions sothere may need to be tight environmental control. These factorsare not the subject of this review but how this oil may be usedwhich is dependent on the composition of the oil product. It is truethat this may vary with how the product is grown so there may bea need to tailor either the organism growth to the refinery or therefinery to the variability of the oil product. While glyceryl lipidsof variable composition may occur and probably can be treatedwith staged upgrading without major separation technologies afew species such as the chlorophyte Botryococcus braunii producequite different products such as unsaturated polyterpenoid hydro-carbons. Upgrading this product will be a different problem. Fortu-nately, the phytochemistry of algae has been the subject ofnumerous studies stretching over hundred years. Glyceryl lipidsmay have carboxylic acid chain lengths from C11 to C26, althoughC14–C22 is more typical. The fatty acids may be saturated or containup to six double bonds. In a few cases, hydroxyl substituents canalso be found. B. braunii products are complex and contain a widerange of compounds which are variants on botryococcene.

One issue of concern is that in producing a suitable transportfuel from glycerol esters by the process of transesterification, theglycerol part of the ester is replaced with ethyl, methyl or propylfrom ethanol, methanol or propanol, respectively. These productswhile having suitable energy densities may hydrolyse to their par-ent carboxylic acids. This can be a problem for long-term storage ofthe fuel. In addition some carboxylic acids may contain functional-ity in the side chain such as hydroxyl or double bonds which maycause polymerisation. These hydrolysis, oxidation and polymerisa-tion products affect the quality of the fuel and potentially could ad-versely affect engine life. Some exploratory producers market ahydrogenated product called Green Diesel [8] however much workneeds to be done to choose the correct and most efficient types ofcatalyst for such processes.

A second issue concerns transport. Whether the economies aresuch that reforming oil at the point of algal harvesting or at a sep-arate refinery is not clear. It may well be that a hybrid route whichcombines biological and chemical process would be ideal for notonly reducing the costs but will also result in optimum quantityand quality of oils [9]. Either options will require the developmentof specific catalysts designed for different economies of scale.

A number of solid or liquid phase catalysts, widely used in thepetroleum refining industries, can be utilised for the upgrading ofthe algae crude oils. The solid heterogeneous catalysts have hadadvantages over the liquid catalysts as they are almost fully recov-ered from reaction products and are normally easy and safe to dis-pose of and can be applied to many liquid or gas phase reactions[10].

While it is difficult to transform old refineries for new bio-feed-stocks, if refineries are to be part of the facility where algal harvest-ing occurs there is the opportunity for introducing new processingmethods. Upgrading of crude oils involves a thermal chemical con-version process, which usually involves fast pyrolysis, solventextraction and/or a catalyst. Transesterification or upgrading ter-

penoids are quite a different process even if conventional catalystsmay work. There are advantages therefore of using specifically de-signed catalysts such as nano-scale materials as new types of het-erogeneous catalysts and/or catalytic supports when newrefineries are being designed.

Some work has been done on exploring conventional catalysts.Amongst those oil rich algae, much more of the conversion isknown on the chlorophytes B. braunii and Chlorella protothecoides,which as already noted, accumulate large fractions of terpenoidhydrocarbons and glyceryl lipids, respectively. Upgrading mainlyinvolved transesterification of the triacylglycerols (triglycerides)derived from the lipids and cracking/hydrocracking of the long car-bon chains. To a lesser extent, results of the catalytic upgrading ofbiomass from the other algae derived feedstocks have been re-ported [11–16].

This review summarises the growth and culture of micro-algae,which have potentials for liquid fuel production due to their highgrowth rate and/or crude oil contents (Table 1). But the review fo-cuses more on the catalytic process and potentials of using nano-materials as a new improved catalyst for upgrading the algae crudeoils and biomass. Table 1 summarises the algae crude oil composi-tion and yields, upgrading route, fuel oil composition and someother relevant properties.

2. Botryococcus braunii

The green colonial micro-alga B. braunii contains an unusuallyhigh level of hydrocarbons (up to 80% of the dry mass), making itpotentially capable as the primary biofuel source [17–19].

Botryococcus can grow in brackish waters, which can be advan-tageous for countries where the availability of fresh water is lim-ited (e.g. Australia). In Australia, blooms of B. braunii reportedlyhad hydrocarbon oil contents of between 27% and 40% of the drymass [20].

Study of the energy balance and CO2 mitigating effect of a liquidfuel production process has shown, if a 100 MW thermal plantusing coal would be replaced by liquid fuel produced from B. brau-nii, the quantity of CO2 mitigation could be 1.5 � 105 tons year�1

and 8.4 � 103 ha of micro-algal cultivation area could be necessary[21]. Similarly, GreenFuel Technologies in Cambridge, Massachu-setts is developing the algal bioreactors that tap into the CO2

streams from coal plants [2]. Nevertheless, a number of critical is-sues such as the slow growth rate of B. braunii must be addressedin order to fully realize its potential as one of the primary sourcesof the third generation biofuels [18].

The growth and strategies for improving the relatively slowgrowth rate of B. braunii and the hydrocarbon contents have beenreviewed [18,19,22]. Particularly, the air enriched conditions with1% CO2 highly enhanced growth, with a mean doubling time of thebiomass in approximately two days and with hydrocarbon produc-tion increased fivefold [23]. Hydrocarbon synthesis was also fa-voured by light intensity irradiance in the range of 40–90 W m�2.Optimisation of light intensity resulted in a twofold increase inbiomass and hydrocarbon production [24]. On the other hand,entrapment of B. braunii colonies in calcium alginate beads re-sulted in an increase in hydrocarbon production but the rate of bio-mass production decreased [25]. A part from these processes, thecloning of the squalene synthase gene from a strain of B. brauniiin Escherichia coli [26] offered promising prospects for the produc-tion of hydrocarbons by fast-growing micro-organisms.

Crude oil from B. braunii has been extracted using a simple sol-vent extraction [27,28], which does not require a catalyst or hightemperature or high pressure conditions but does involve an exces-sive amount of solvents. Nevertheless, oil may also be extractedwithout a solvent by a simple physical process [29] although yields

Table 1Potential micro-algae for liquid fuel production. Summary of their crude oil composition and yields, upgrading route, fuel oil composition and some other relevant properties.

Micro-alga Crude oil (maximised yield, wt%) Upgrading method (catalyst) Oil property (maximised yield, wt%) Refs

Botryococcus braunii Terpenoid hydrocarbons (75%) Liquefaction (Na2CO3) Botryococcene (78%) high-octanegasoline

17-23, 27-32, 34

Cracking (H-zeolite) Aromatics, alkyl benzenes

Hydrocracking (Ni-Mo, Co-Mo) Diesel (15%), jet fuel (15%), gasoline(67%)

Chlorella protothecoides Lipid (55%), carbohydrate (15%) Pyrolysis Fuel oil (58%), viscosity, density,heating value comparable to fossil fuels

44-53, 55, 59

Transesterification(HCl, NaOH in MeOH)

Diesel (80%), complied with the ASTMbiodiesel standards

Chaetoceros muelleri Lipid consisting of free fatty acid(36%), triacylglycerols (14%)

Cracking (zeolite H-ZSM5) High-octane, aromatic gasoline(total organics 36%)

12, 55, 63-64

Transesterification (HCl, NaOH inMeOH)

Diesel (68%)

Dunaliella tertiolecta Glycerol (23%) Liquefaction Fuel oil (42%), compatible to 15, 20

(Na2CO3) Japanese Industrial Standard

Nannochloropsis Lipid (55%) Pyrolysis (H-ZSM5) Alkenes (50%), aromatics (15%) 18, 67

Spirulina Liquefaction in organic solvents andwater (Fe(CO)5-S)

Fuel oil (78%) 14, 16

Fig. 1. Molecular structure of one of the C34 botryococcenes, major crude hydrocar-bons extracted from Botryococcus braunii race B. This particular botryococcene wasisolated from a wild sample. A total of 50 botryococcenes has been identified andamongst these, about 15 structures have been determined. About five C34 isomershave been identified (Metzger et al. [22]. Reproduced by permission of Springer).

CH

CH3H3C

CH2

-H

α-H

β-H

γ-H

CH4 +

+Alkyl

Cyclization, Isomerization

Dehydrogenation

Hydrogenolysis

CH

CH3H3C

CH2

-H

α-H

β-H

γ-H

CH4 +

+Alkyl

Cyclization, Isomerization

Dehydrogenation

Hydrogenolysis

Fig. 2. Schematic diagram of the adsorption of isobutane molecules on thetransition metal surfaces leading to the formation of the surface alkyl intermediates(sec-butyl). Subsequent reforming of the intermediates via a-, b- or c- eliminationsleads to different products. The alkyl surface formation determines the activity butthe selectivity is more dependent on the rate of the hydride elimination steps (Zaera[37]. Reproduced by permission of the American Chemical Society).

N.H. Tran et al. / Fuel 89 (2010) 265–274 267

are lower. Interestingly, researchers at the University of California,Berkeley have recently studied strategies that can produce theB. braunii oil in a form that can be continuously collected [2].

The hydrocarbons in B. braunii have been studied extensivelyand are classified as n-alkadienes, trienes, triterpenoid and tetrat-erpenoid hydrocarbons, depending on the particular race of Botryo-coccus studied. B. braunii race B consists of botryococcenes havingelemental composition of C30–36H48–64 and average molecularweight of 408–496 [22]. Ozonolysis and nuclear magnetic reso-nance have been used for determining the absolute configurationof the most typical botryococcene structure having C34 chainlength (Fig. 1) [30].

In principle this hydrocarbon may function as a transport fuel.However the presence of alkenic compounds may result indiscoloration and polymerisation leading to sludge. This may makeBotryococcus crude oil unacceptable to current users whomnormally require discrete specification parameters for exampleAmerican Society for Testing and Materials (ASTM) D975-08ae1.Thus treatment with conventional cobalt–molybdenum catalystsused in petroleum refineries for cracking mineral oil or coal de-rived liquids have been used since the products will meet knownspecification requirements. Treatments were carried out usingsimple conventional reactors [31].

Nevertheless, there have been some developments of oil extrac-tion via liquefaction, which involves either wet or dry materialsand does not necessarily require a drying process. Experimentshave been carried out using crude B. braunii biomass with highmoisture contents and with or without the Na2CO3 catalyst[17,32,33] (maximum loading 5 wt.%). The recovery of hydrocar-bons was maximized (>95%) at 300 �C.

The liquefied oil of B. braunii was fractionated into three frac-tions by silica gel column chromatography [34]. The yields of thethree fractions based on organics were approx. 5% of lower molec-ular weight (197–281) hydrocarbons, 27% of botryococcenes (MW438–572), and 22% of polar substances (MW 867–2209). The yieldof botryococcenes recovered from the oil using the sodium carbon-ate catalyst was 78%, greatly improved compared to that without acatalyst (48%). This clearly shows the catalysts play a significantrole in the extraction of botryococcenes.

2.1. Cracking/hydrocracking of botryococcenes

Early hydrocracking study of the distillate derived from theDarwin River Reservoir bloom using the conventional cobalt–molybdenum catalysts at the temperatures between 400 and440 �C has resulted in a 67 wt.% petrol fraction, 15% jet fuel and

Fig. 3. A proposed mechanism of self-cracking of a C34-botryococcene andsubsequent ring formation. Step (1): cracking (broken lines showed the potentialcracking sites). (2) Cyclization (Modified from Fig. 12 of reference Kitazato et al.[38], by permission of the Japan Petroleum Institute).

268 N.H. Tran et al. / Fuel 89 (2010) 265–274

15% diesel fuel [31,35,36]. The petrol and jet fuel fractionsconsisted of low molecular weight, straight alkanes and isomerisedalkanes, respectively.

The mechanism of reforming of isobutane on transition metalsurfaces has been described (Fig. 2) [37]. If this is extrapolated tobotryococcene hydrocracking it would suggest the activity isdependent on the initial interactions between the metal surfacesyielding the alkyl surface intermediates.

From this model, cracking of the C34 chains and the petrol for-mation would mainly involve the elimination of the a-hydrogenatoms (from the carbon atoms adsorbed on the surface), which re-sulted in straight alkanes. However, the jet fuel formation wouldmainly involve the elimination of c-hydrogen (from the neigh-bouring carbon atoms), which resulted in cyclization or isomerisa-tion products. The selectivity between petrol and jet fuels isdetermined by the rate of the eliminations of these hydrogenatoms.

Interestingly, a different cracking study of botryococcenes usingH-zeolite as catalysts resulted in an increased aromatic hydrocar-bons, mainly xylenes and trimethyl benzenes exhibiting highoctane numbers [38]. The proposed mechanism involved consecu-tive cracking and subsequent ring formation via cyclo-aromatiza-tion without methyl transfers (Fig. 3).

2.2. Cracking/hydrocracking using nano-scale catalysts

It has long been known that metal particle size is critical incatalysis. Nevertheless, nanotechnology has allowed an under-standing of the process and created new catalysts [39]. Theoreti-cally, the activity and selectivity of the metal nano-particles arestrongly connected with the particle size. This is mainly becausethe surface area of the nano-particles increases and their surfacestructure and electronic properties change greatly with the de-crease in size.

For example, the noble metal gold which has been so called be-cause of its inert characteristics. As a nano-particle gold exhibitscatalytic activity toward oxidation of hydrocarbons or carbon mon-

oxide. Only particles with the average diameter below 3 nm wereactive and those as small as 1.4 nm (55-atom cluster) were evenmore powerful catalysts [40].

Similarly, the activity of the free-standing ruthenium catalystsfor carbon monoxide hydrogenation (Fischer–Tropsch reactions)slightly improved when their sizes reduced from about 4 to2.5 nm, but remarkably improved when the sizes further reducedto 2 nm [41]. It is very interesting to note such a small change ofnano-scale can lead to a dramatic improvement of the catalyticproperties.

In most cases, a catalytic support is essentially required in orderto stabilize such small particles. However, the chemistry of thesupport materials and strong electronic interactions between themand the catalysts will greatly influence the catalytic behaviours.Nevertheless, the industrial process of the bio-oil productions hasincreasingly involved the catalytic supports based on the transitionmetals, noble metals and a wide range of inorganic oxides [42].

Transition metal nano-particles are very attractive as catalystsand/or support materials due to their high surface-to-volume ratioand their high surface energy, which makes their surface atoms veryactive. The use of cobalt (or molybdenum) as a support would im-prove the hydrogenation sites of the bimetallic cobalt–molybde-num catalysts. To further improve the catalyst activity duringhydrogenation of botryococcenes, a possible scenario will involvereducing the average particle size of cobalt (or molybdenum) withinthe catalysts. This process will increase the surface-to-volume ratiosof the catalysts, which therefore improve their activity. Techniquessuch as sol–gel [43] or thermal decomposition [44] could be used tosynthesize cobalt–molybdenum with the average size of molybde-num (or cobalt) particles varying in the range from 1 to hundredsof nm. Mechanisms such as the formation of the molybdate inter-mediate phase has stabilized the very small cobalt particles [45].

Inorganic oxides, such as zeolites and natural clays, which carrya number of Bronsted or Lewis acids on their surfaces, have widelybeen utilised as the support materials. In general, the oxide addi-tion will change the balance between the hydrogenation sitesand the acidic sites of the whole catalysts, which will govern theirbehaviours.

A smectite clay such as saponite can be used as a catalyst sup-port that alters the selectivity of the cobalt–molybdenum catalysts[46]. Saponite consists of the octahedral AlO6 layers sandwichedbetween the tetrahedral SiO4 layers while the clay surfaces consistof a number of hydroxyl groups. Substitution of Si by Al atomswithin the clay structures will create extra hydroxyl groups andtherefore create further acidic sites on the clay surfaces. In the caseof hydrocracking of the n-decane molecules, the selectivity of thesaponite incorporated cobalt–molybdenum catalysts toward theC3 products markedly increased at low Si/Al atomic ratios, e.g.Si/Al = 6 (Fig. 4) [46]. This can be explained as due to the increaseof the acidic sites, which facilitate the secondary cracking ofthe primary cracking products to lower molecular weighthydrocarbons.

It is very interesting that the selectivity of the saponite incorpo-rated cobalt catalysts remained unchanged as the Si/Al ratios var-ied. These catalysts all exhibited the product selectivity similar tothat of the saponite–cobalt–molybdenum samples with the Si/Alratio of 6. This result clearly showed the selectivity is stronglydependent on the support materials.

On the other hands, the uses of high Si/Al ratios (e.g. Si/Al = 39)resulted in the selectivity toward higher hydrocarbons and eventhe C10 products. This can be explained as due to lack of the acidicsites at high Si/Al ratios, the primary cracking products are not fur-ther cracked but preferentially leave the catalyst surfaces. Similareffects were also observed during the hydrocracking experimentsof the n-decane molecules using the H-zeolite incorporated plati-num catalysts with varying the Si/Al atomic ratios [47].

Fig. 4. Selectivity toward C1–C10 hydrocarbons of the saponite incorporated cobalt–molybdenum catalysts in n-decane hydrocracking as the Si/Al atomic ratio varied. Theselectivity toward C3 products dropped from approximately 37–22% when the Si/Al ratio varied from 6 to 39 (Leliveld et al. [46]. Reproduced by permission of Elsevier).

N.H. Tran et al. / Fuel 89 (2010) 265–274 269

Using this model, it can be predicted that during hydrocrackingof botryococcenes, reducing the Si/Al atomic ratios within the sap-onite incorporated cobalt–molybdenum catalysts should result inan increase of petrol/jet fuel fractions; however, increasing theSi/Al ratios would favour the selectivity of the diesel fractions.

3. Chlorella

Species of the green micro-algal genus Chlorella sp. have beenmassively cultured in many countries and also widely distributedin ponds and lakes. Their oil contents are usually ranged from25% to 32% dry weight [3], even though some early work predictedChlorella pyrenoidosa could content up to 85 wt.% crude oils basedon elemental compositions [48]. However, the majority of work oncultivation, processing and biofuel production was systematicallyperformed on C. protothecoides (containing up to 55 wt.% crudeoil [49]) by the research group at the Tsinghua University, China.

The oils produced by C. protothecoides are generally differentfrom those of B. braunii, being primarily glyceryl lipids, proteins

Fig. 5. Biomass concentration of Chlorella as a function of glucose consumption under pglucose concentration; closed circle, biomass concentration). In the primary cultivationglucose concentration was below 10 g l�1. In the improved cultivation, glucose was addecontrolled less than 24 g l�1 (Xiong et al. [53]. Reproduced by permission of Springer).

and carbohydrates. Under the autotrophic growth conditions, thechemical composition of C. protothecoides was mainly protein(51%), lipids (14%), carbohydrate (10%), ash (7%) and moisture(11%).

Through addition of a common organic carbon source (glucose)to the medium together with a decrease in the concentration ofinorganic nitrogen sources, the growth mode of C. protothecoidescan be changed from photoautotrophic to heterotrophic [50]. Het-erotrophic growth of C. protothecoides resulted in not only thereduction of chlorophyll in the cells, but also an accumulation ofhigh lipid. Heterotrophic growth resulted in an increase of the lip-ids (up to 55.2%, about four times higher compared to that from theconventional autotrophic growth) [49,51]. Accordingly, the proteincontent was reduced from 51% to 10%. The fatty acid mainly con-sisted of oleic acid (18:1), linoleic acid (18:2) and palmitic acid(16:0).

A more effective cultivation at pilot scale and even at commer-cial scale bioreactors (from 5 to 11,000 l stirred tanks) has beendeveloped [52,53]. While no inoculation densities have been

rimary (a) and improved (b) cultivation conditions in a 5 l bioreactor (open square,, glucose solution (100 g l�1) was added into culture medium six times when thed according to its consumption rate, and the highest concentration in medium was

270 N.H. Tran et al. / Fuel 89 (2010) 265–274

reported, using the 5 l bioreactor, the biomass concentrationreached approx. 17 g l�1 after 184 h culture and then slightly de-creased to 15 g�1 in the subsequent 2 h culture (Fig. 5) [53]. Themechanisms of the decrease of biomass concentration need to befurther studied. Nevertheless, in the improved cultivation, theresultant biomass concentration reached as high as 51.2 g l�1 in168 h cultivation, almost three times higher than that from the ini-tial cultivation (Fig. 5). This also resulted in the improved lipid con-tents of up to 58% of the cell dry weight [53]. These combinedresults are important for industrial-scale production of liquid fuelsfrom C. protothecoides.

Pyrolysis and transesterification have been used for conversionof the dry biomass and the crude lipids, respectively.

3.1. Pyrolysis of biomass derived from Chlorella protothecoides

Pyrolysis involves thermal treatment of biomass in the absenceof oxygen, and usually results in charcoal, tar, a mixture of liquidfuels and gaseous products. Pyrolysis does not necessarily involvea catalyst. But the non-catalytic process usually results in the oilsconsisting of highly oxygenated complex mixtures, which makethem polar, viscous, corrosive and unstable. These oils have limiteduse for transports and further upgrading utilizing a catalyst (e.g. fordeoxygenation) will be necessary.

Nevertheless, the non-catalytic pyrolysis particularly carriedout in conjunction with a basic thermal gravimetric analyzer [54]could be exploited for fast screening of the alga biomass. Relativelyquick screening experiments have showed that the C. prototheco-ides derived biomass is a potential feedstock for the oil productionvia thermal chemical process [55–58]. It showed the decomposi-tion of the C. protothecoides biomass resulted in the oils with theoxygen contents being lower than that in the oils from wood [59].

Particularly, fast pyrolysis of the biomass derived from the het-erotrophic algae resulted in a maximum of approx. 58 wt.% oilyield. Under the similar temperature conditions, pyrolysis of bio-mass derived from the higher plants such as pine wood, cottonstraw and stalk and sunflower usually gave a maximum oil yieldof 49 wt.% [50]. A more detailed comparison showed the oil viscos-ity (0.02 Pa s), density (0.92 kg l�1) and heating value (41 MJ kg�1)were comparable to those of fossil oil. The integrated approach ofheterotrophic growth followed by pyrolysis has been shown to bevery effective in producing high quality oils, however, the final oilyield needs to be improved.

The low oil yield was attributed to a large amount of the unpy-rolyzed algae powder, found to be adhered onto the reactor wallsprobably via the electrostatic forces, which further resulted in ahigh yield of char. In order to improve the yield, improving thereactor design and engineering will be necessary.

The low oil yield was also due to the formation of the gaseousproducts, which were complex and usually consisted of low hydro-carbons such as methane, ethylene, ethane, butane, isobutane,pentane, together with carbon dioxide and hydrogen. The gaseouscomposition was dependent on the temperature conditions but

Table 2Comparison of the surface areas of a number of the commercial metal oxide catalysts, theirthe American Chemical Society).

Catalyst Surface areas (m2/g) Type Acid/base site strength (H_)

MgO 157.4 Basic 11 < (H_) < 15CaO 61.39 Basic 10.1 < H_) < 11ZnO 6.17 Amphoteric 6.8 < (H_) < 8.2PbO 4.04 Amphoteric 6.8 < (H_) < 8.2PbO2 0.98 Amphoteric 6.8 < (H_) < 8.2Pb3O4 0.55 Basic 6.8 < (H_) < 8.2Tl2O3 0.38 Basic 10.1 < (H_) < 11

this work has not been thoroughly explored. Eventually, the emis-sion of carbon dioxide during pyrolysis also needs to be minimized.

The use of catalysts may also improve the overall oil yield anddecrease the oxygenated compounds. It has recently been demon-strated for the pyrolysis of other biomass feedstocks and energycrops, which involved the MCM-41 based catalysts with uniformmesopores from 1.4 to 10 nm in size [59]. Another advantage ofthe use of the catalysts (e.g. zeolite) during pyrolysis is the forma-tion of the high octane, aromatic gasoline products [18,43].

3.2. Transesterification of crude oils derived from Chlorellaprotothecoides

Crude oils were initially extracted from the dry biomass usingnon polar solvents, e.g. n-hexane. Other solvents (n-butanol, etha-nol, etc.) may slightly change the extracted lipid yields [60].

The acid catalysed transesterification has been performed usingdifferent quantity of sulphuric acid (from 255% to 100% H2SO4

based on actual oil weight) and molar ratios of methanol to oil(from 30 to 56:1 M ratio). Preliminary experiments suggested thatthe basic type catalyst (e.g. alkali) was not suitable for the transe-sterification of micro-algal oil probably because of the high acid va-lue of the oil (8.97 mg KOH g�1). But other transesterificationinvolving strong base such as sodium hydroxide in methanol hasalso been reported [61].

Transesterification showed the lowest value of specific gravity(0.862) was obtained at 90 �C in the presence of 100% catalystquantity (based on oil weight). On the other hand, the yield waslowest at these levels, probably because the relatively high tem-perature and concentration of H2SO4 could oxidise some of theoil. Nevertheless, the optimised condition involved the use of100% catalyst quantity and 56:1 M ratio of methanol to oil at tem-perature of 30 �C, which reduced product specific gravity from aninitial value of 0.912 to a final value of 0.864 in about 4 h of reac-tion time.

Gas chromatography analysis showed the biodiesel, preparedfrom the acidic transesterification of the crude lipids from the het-erotrophic growth, consisted of nine components, in which themost abundant component was oleic acid methyl ester [49]. Thetotal yield obtained was over 80%. Initial comparison showed thebiodiesel has complied with the ASTM biodiesel standardrequirements.

3.3. Transesterification using nano-scale catalysts

There are opportunities of using various inorganic oxides a newtype of less toxic, heterogeneous catalyst for improving the transe-sterification, particularly when the process involves the nanometersized oxide particles. Singh et al. have carried out a systematicstudy of the transesterification of the tryacylglycerols derived fromsoybean oil using a number of commercial oxide particles, and as afunction of their surface area, acid/basic site strength and acid/basicproperties (Table 2) [62]. Lead dioxide particles exhibited highest

site strength and acidity/basicity values (Singh et al. [62]. Reproduced by permission of

Acidity (mmol of NaOH/g of catalyst) Basicity (mmol of HCl/g of catalyst)

46.0516.2412.25 32.3535.747 7.5817.86 7

14.54515.93

Fig. 6. Fatty acid methyl ester (FAME) yield during transesterification of triglyc-eride derived soybean oil using different oxide catalysts and as a function ofoperating temperature (Singh et al. [62]. Reproduced by permission of the AmericanChemical Society).

Fig. 7. (A) Influence of the annealing temperatures on the catalytic activity oftungstated zirconia during the transesterification of triacetin. (B) X-ray diffractionpattern showing changes of crystalline structures of the catalysts with thetemperatures. At the temperatures between 400 and 800 �C, primarily thetetragonal zirconia phase (t-ZrO2) were formed. Increased temperatures resultedin the additional formation of the monoclinic phase (m-ZrO2) and the bulk-liketungstated oxides (WO3). Note the surface areas of the catalysts decreased fromabout 235 to 58 m2 g�1 as the temperatures increased from 400 to 900 �C (Lopezet al. [64]. Reproduced by permission of Elsevier).

N.H. Tran et al. / Fuel 89 (2010) 265–274 271

catalytic activity toward the methyl ester formation (89% conver-sion) at approximately 150 �C (Fig. 6). Although it was noted PbO2

had lowest surface areas amongst the oxides examined (Table 2),but it is more appropriate to compare the surface areas and cata-lytic behaviours of the same catalysts, i.e. PbO2 in this case. Never-theless, the behavioural performances of these solid oxides areinteresting, and their mechanisms need to be studied.

Correlations between the transesterification, nano-structuresand nano-sizes of the oxide catalysts have been examined. Thetransesterification of triglyceride derived from soybean oil withmethanol using calcium oxide nano-particles with the averagecrystallite size of 20 nm and a specific surface area of 90 m2 g�1

has resulted in >99% conversion [63]. Under the same conditions,commercial CaO (average crystallite size 43 nm, specific surfacearea 1 m2 g�1) resulted in only 2% conversion. The 20 nm sized par-ticles with high concentration of the active sites (calcium methox-ides formed on the particle surfaces during exposure to methanol)showed high catalytic performance in multiple cycles (as many as9 cycles), whereas the performance of those larger particles readilyreduced after 3 cycles.

Likewise, the transesterification of triacetin (a synthetic triglyc-eride, C9H14O6) as a model compound using the tungstated zirconianano-catalysts showed a strong relationship between the struc-tural properties of the catalysts, their surface densities and theircatalytic activity [64]. Annealing of the catalysts from 400 to800 �C resulted in an optimised activity but then it decreased shar-ply as the temperatures further increased (Fig. 7A). This was inter-preted as due to the formation of the catalytically active sitestetragonal zirconia at 6800 �C (Fig. 7B). Above this, the monocliniczirconia structures were formed and partially inhibited the reac-tions. Tungstated oxides also served as either active or non-activesites, depending on their crystalline structures which also variedwith the temperatures.

Nano-scale catalysts are also weight efficient as expected, forexample the transesterification of the soybean oil could be carriedout with the concentration of magnesium oxide nano-particles inmethanol being as little as 0.5 wt.% [65]. Although the biodieselproduction yield was optimised as the catalytic concentration in-creased to 5 wt.%.

4. Chaetoceros

The marine diatom Chaetoceros sp. contains up to 57% lipids andfatty acids [66,67]. Particularly, Chaetoceros muelleri exhibited avery rapid growth rate (up to 4.0 doublings/per day [68]). The totallipid contents during cultivation under the nitrogen depleted con-ditions reached as high as 500 mg l�1. Likewise, the lipid contentsalso increased during growth under the silica deficient conditions

[69]. The neutral lipids extracted from the biomass using butanolcontained approx. 36% fatty acids and 14% triglycerides [12].

Transesterification of the lipids derived from C. muelleri showedthe optimised yield of methyl esters (approx. 68%) was achievedusing 0.6 N HCl in MeOH and at 70 �C [60]. Pyrolysis of lipids fromC. muelleri over zeolite H-ZSM5 catalysts was also performed [12].In comparison to transesterification, zeolite upgrading is virtuallyindependent on the glycerol lipids, as it involves cracking whichusually results in aromatics and light hydrocarbons (as observedin B. braunii) and coke formation. Particularly, the use of the med-ium-pore zeolite for pyrolysing the butanol extracted lipids re-sulted in a high octane, aromatic gasoline (yield of alkanes,alkenes and the aromatic fractions were approx. 9, 12 and15 wt.%, respectively).

272 N.H. Tran et al. / Fuel 89 (2010) 265–274

5. Dunaliella

The green micro-algae Dunaliella sp. can be found over an extre-mely wide range of salinities and contains glycerol as the majorcrude oil component [19]. Particularly, Dunaliella tertiolecta hasthe oil (glycerol) contents of up to 23 wt.%. Sawayama et al. havestudied the energy inputs for the fuel production using liquefactionof the D. tertiolecta biomass (20.5 dry wt.%) and with Na2CO3 (max-imum 5 wt.%) as catalysts [21]. The catalytic behaviours of Na2CO3

need to be further examined. It is interesting that deoxygenationincreased with the temperatures although the nitrogen content re-mained approx. 7 wt.%, which eventually releases considerable NOx

during oil burning [15]. Nevertheless, the oil obtained at 340 �Cand holding time 60 min had a viscosity between 150 and330 mPa s and a calorific value of 36 kJ g�1, compatible to thoseof fuel oil in the Japanese Industrial Standard.

The maximum oil yield and the heating energy for liquefactionwas approx. 42 wt.% and 11.94 MJ kg�1 oil. The yield of the liquidfuel produced from D. tertiolecta was lower than that of B. braunii(64 wt.%) and the heating energy was higher (6.69 MJ kg�1 oil).Nevertheless, the large scale cultivation of D. tertiolecta is wellestablished and is easier than that compared to B. braunii.

Ginzburg et al. have carried out pyrolysis of the biomass derivedfrom the alga Dunaliella parva (glycerol content up to 30 wt.%)using a number of transition metal salts as catalysts [70]. Amongstthe catalysts used, Cr2(SO4)3 resulted in lowest hydrocarbons (ap-prox. 18 wt.%) while the use of NiSO4 resulted in a maximum yieldof hydrocarbons (27 wt.%). But the use of these transition metalsalts as catalysts did not result in a significant yield improvementsince the non-catalytic pyrolysis already resulted in 17 wt.% hydro-carbons (toluene, light hydrocarbons [71]). The non-catalytic pyro-lysis has also resulted in ammonium carbonate as the major by-products, completely isolated from these hydrocarbons.

6. Nannochloropsis

The oil produced from the eustigmatophyte alga Nannochlorop-sis sp. are mainly glyceryl lipids, which ranged from 31% to 68% dryweight [3]. Like Chlorella protothecoides, the lipid productivity ofone of the Nannochloropsis (Nanno-Q) was maximized (approx.55 wt.%) by reducing the nitrogen concentration to as low as2 vol.% during the alga cultivation [72].

The Nanno-Q derived lipids, extracted from CHCl3–MeOH–H2O,were pyrolysed over the H-ZSM5 catalysts from 400 to 520 �C un-der low and high pressure [18]. Under the partial pressure condi-tions, pyrolysis resulted in a large amount of alkenes (up to50 wt.%) and aromatics (up to 15 wt.%) but alkanes were notformed. The yield of the total organic product was approx.70 wt.%, significantly greater than that compared to the pyrolysisof Chaetoceros muelleri derived biomass (approx. 39 wt.%) usingthe same catalysts.

7. Spirulina

Suzuki et al. have reported the effect of the Fe(CO)5–S catalysts(S/Fe = 2) during liquefaction of Spirulina derived biomass [16]. Liq-uefaction under nitrogen atmosphere in tetralin without catalystsresulted in approx. 52 wt.% of oil. Experiments carried out underhydrogen with addition of highly dispersed Fe(CO)5–S catalysts re-sulted in an increased oil yield (approx. 67 wt.%). Liquefaction un-der hydrogen without a catalyst resulted in only 0.28 wt.% ofhydrogen adsorbed from the gas phase, whereas hydrogen adsorp-tion increased to 1.2 wt.% when experiments involved the cata-lysts. This suggests hydrogen activated by the dispersed catalystcontributed to an increase in oil yield.

The oil yield also increased considerably from 54 to 64 wt.% asthe concentration of the Fe(CO)5–S catalysts increased to a maxi-mum of 1.0 mmol. It is interesting that the gas fraction (mainlycomposed of methane) remained virtually unchanged as the cata-lysts concentration increased, but it remarkably increased withtemperatures. X-ray diffraction showed the presence of pyrrhotiteresidues (Fe1�xS) after liquefaction, which possibly served as thecatalytic active sites, similar to that in coal liquefaction.

The solvents have also shown to influence the oil yield. Non-catalytic liquefaction in water resulted in a maximum of 78 wt.%of oil, slightly higher than that using toluene (70 wt.%). But theuse of toluene resulted in the oil fractions with higher carbon con-tent and lower oxygen content compared to those using water. As aconsequence, the heating values of the oil using water (26 MJ kg�1)was lower than compared to that using toluene (up to 33 MJ kg�1).Although toluene resulted in a better oil quality, but the use of tol-uene or any other fossil fuel derived solvents will not lead to 100%renewable fuels and therefore water should be a preferred solvent,where appropriate.

Fourier transform infrared spectroscopy of the oil samples ob-tained from the non-catalytic process suggested the process in-volved thermal decomposition of polypeptides in the biomassfeedstocks. Additional gel permeation chromatography of the oilsproduced using toluene, hydrogen and the catalysts showed thelow molecular weight fractions (MW 400–490), suggesting lique-faction involved cracking/hydrocracking, similar to those forupgrading B. braunii crude oils.

Subsequently, the authors have examined the possibility of co-liquefaction of Spirulina (also other micro-algae Chlorella, Littorale)with coals using Fe(CO)5–S catalysts (S/Fe ratio varied) and othercarbonyl precursors [14]. The results of Spirulina (and Liitorale)was similar to those for Chlorella, from which the yield of the oils(hexane soluble fractions) improved from 42 to 55 wt.% as the S/Fe ratios increased from 2 to 4. Probably the excess amount of sul-fur increased the concentration of the active pyrrohotite specieswhich increased the liquefaction rate.

8. Summary

The catalytic process of liquid fuel productions from algae isstill at the very early stage of development compared to that usingother biomass derived feedstocks. Future work should focus on alarge number of micro-algae and macro-algae (marine seaweeds)having high oil contents.

The work summarised here has shown that Botryococcus ex-tracts have potential. Work using the conventional cobalt–molyb-denum catalysts has resulted in a 67 wt.% petrol fraction, 15% jetfuel and 15% diesel fuel but this could be considerably improvedby applying new nano-scale catalysts and support materials. Like-wise strain of algae Chlorella is very promising. High quality diesel(80 wt.%) was achieved from transesterification of the lipids de-rived from Chlorella protothecoides. Other oil rich micro-algae con-taining glycerol and/or glyceryl lipids need to be investigated.

There is clear evidence that nano-materials, particularly oxidenano-particles, may act as better catalysts during transesterifica-tion. Calcium oxide nano-particles with the average crystallite sizeof 20 nm and a specific surface area of 90 m2 g�1 have been suc-cessful in comparison to conventionally sized (43 nm) particles.The crystallographic properties of the tungstated zirconia nano-catalysts have been demonstrated to greatly influence the catalyticbehaviours. To this end, it is important that the nano-catalystbehaviours, fuel properties and coke formation during conversionof crude oils and biomass derived from algae are to be understood.

Work should focus on not only improving the liquid fuel pro-ductions, but also need to improve the productions of hydrogenfuels from the algae biomass or their lipids since hydrogen would

N.H. Tran et al. / Fuel 89 (2010) 265–274 273

be needed for refining at the place of harvesting. Moreover sincehydrogen is currently produced mainly from mineral oil andchange in world oil production will also change hydrogen avail-ability. Electrolysis is possible but it is expensive and may begreenhouse unfriendly if the electricity is produced from coal.

Acknowledgments

Funding for this work was supported by the Australian ResearchCouncil through the ARC Centre of Excellence for Functional Nano-materials, the Commonwealth Scientific Industrial Research Orga-nization through the Flagship Collaborative Projects, and a numberof the start-up grants from the University of Western Sydney, Aus-tralia. The authors wish to thank Dr. John Volkman, CSIRO Marineand Atmospheric Research, for his valuable inputs and suggestions.

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