Fuel 128 (2014) 46–52

Contents lists available at ScienceDirect

Fuel

journal homepage: www.elsevier .com/locate / fuel

Efficacy of drying and cell disruption techniques on lipid recovery frommicroalgae for biodiesel production

http://dx.doi.org/10.1016/j.fuel.2014.02.0590016-2361/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +27 313732346; fax: +27 313732777.E-mail address: faizalb@dut.ac.za (F. Bux).

Abhishek Guldhe, Bhaskar Singh, Ismail Rawat, Krishan Ramluckan, Faizal Bux ⇑Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa

h i g h l i g h t s

�Microwave and sonication techniques are compared for efficient lipid extraction.� Qualitative lipid variation caused by cell disruption and drying methods is studied.� Energy consumption calculated for drying and lipid extraction processes.� Lipid quality has been assessed for its suitability for biodiesel production.� Sun drying with efficient microwave extraction could be possible processing step.

a r t i c l e i n f o

Article history:Received 18 October 2013Received in revised form 8 January 2014Accepted 25 February 2014Available online 12 March 2014

Keywords:BiodieselDryingMicrowaveLipidMicroalgae

a b s t r a c t

Downstream processing of microalgal biomass presents a significant challenge to large scale biodieselproduction. Scenedesmus sp. which is known to be a potential feedstock for biodiesel production was cul-tivated in an open circular pond. The biomass productivity peaked at day 21 with a yield of 1.16 g L�1.Biomass was harvested by gravitational settling followed by centrifugation. Harvested biomass was driedusing the freeze drying, oven drying and sun drying processes followed by lipid extraction which yielded29.65%, 28.63% and 28.33% lipid g�1 DCW (dry cell weight) respectively. Lipids were extracted from mic-roalgal biomass dried by selected drying techniques using microwave and sonication for cell disruption inthe presence of mixture of chloroform and ethanol (1:1). Microwave assisted extraction of sun dried bio-mass yielded 28.33% lipid g�1 DCW, as compared to 18.9% lipid g�1 DCW achieved by sonication assistedextraction. The saponification and acid values of the lipid obtained from Scenedesmus sp. dried by selecteddrying techniques showed high saponification and acid value indicating presence of high free fatty acidcontent. Effect of different drying and cell disruption technique on fatty acid profile of lipids extractedfrom Scenedesmus sp. biomass was also studied. These values indicate promising potential of the oilproduced for conversion into biodiesel.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae have emerged as promising feedstocks for biodieselproduction. Microalgal biodiesel has several advantages over cropbased biodiesel such as high potential for biomass production withminimal land and freshwater requirement; no arable land require-ment, no food security concerns; a higher carbon-dioxide seques-tration potential; wastewater utilization during cultivation; andthe production of value-added products from de-oiled microalgalbiomass. The production of a sustainable and economically viablebiodiesel from microalgae is however still a challenge [1–6].

Biodiesel production from microalgae is a multi-step processincluding cultivation, harvesting and dewatering of microalgal bio-mass, extraction of lipids from biomass, and conversion of lipids tobiodiesel. Photoautotrophic cultivation of microalgae is widely ac-cepted as a more economically viable method for large scale mic-roalgal biomass production. Microalgae essentially require light,carbon dioxide, inorganic nutrients, and water for their growth[7]. Microalgae are either cultivated in open raceway ponds or inphoto-bioreactors for a high biomass and lipid production. Thechoice of technique for cultivation and production of microalgalbiomass with high quantity of lipids depends upon microalgalstrain selection, culture conditions, land area availability, naturallight and production scale [2].

Harvesting and dewatering of microalgal biomass are crucialsteps in commercial production of microalgae. The harvesting

A. Guldhe et al. / Fuel 128 (2014) 46–52 47

method is dictated largely by the microalgal strain and the cultiva-tion procedure. The common harvesting methods currently em-ployed include flocculation, filtration, flotation and centrifugationor any combination of these methods [8]. Harvesting is followedby drying of the wet biomass. Drying of harvested biomass is nec-essary to increase the viability of biomass for lipid extraction. Dry-ing methods may include natural sun drying or using advancedtechniques like freeze drying, drum drying, oven-drying, spray-drying and fluidized bed-drying. Despite sun drying being amongstthe slower methods, it is cost and energy effective as compared toother techniques. Freeze drying is widely used for dewatering ofmicroalgal biomass. Freeze drying is a gentle process in which allthe cell constituents are preserved without rupturing the cell wall[8–10]. Extraction of lipids from microalgae is carried out by usingeither physical (mechanical expeller) or chemical methods (solventextraction) or by using a combination of these methods. Cell dis-ruption followed by solvent extraction is a widely-used methodfor oil extraction. Disruption techniques like autoclave, bead-beat-ing, sonication, microwave and osmotic shock are usually coupledwith solvent extraction for improved lipid yield. Conventionalsoxhlet extraction is a time consuming process taking severalhours for complete extraction of lipids. The extracted lipids arethen converted to biodiesel via transesterification [8,11–13]. Dueto the vast diversity of microalgae, investigating suitable harvest-ing, drying, cell disruption, and extraction techniques for a partic-ular microalga becomes necessary. Development of effective andeconomical drying, cell disruption and extraction techniques arerequired to address the challenge of scaling up of the biodieselproduction process from pilot to industrial scale.

The present research compares drying and cell disruptiontechniques for effective extraction of lipids from fresh waterScenedesmus sp. grown in an open circular pond. The effect ofdrying and cell disruption techniques on lipid quality has also beenassessed for its suitability for biodiesel production. The conversionof microalgal lipids to fatty acid methyl esters (FAME) was carriedout using acid catalyzed transesterification reaction.

2. Materials and methods

2.1. Chemicals and reagents

A mixed FAME standard comprising of 37 componentswas obtained from Sigma–Aldrich, USA. All organic solvents(chloroform, ethanol, methanol, iso-propanol, dichloromethane,hexane and toluene) and other chemicals purchased from Sigma–Aldrich were of analytical grade.

2.2. Cultivation of microalgae for production of biomass

A strain of Scenedesmus sp. isolated from the Durban region,Kwa-Zulu Natal, South Africa was used for the presentstudy. Scenedesmus sp. was grown under natural light (400–1200 lmol m�2 s�1) and water temperatures ranging from 18 to27 �C in an open circular pond of 8000 L capacity for biomass pro-duction. BG11 medium [14,15] was used as a nutrient medium toculture the microalgae. Mixing and aeration of the suspended algalbiomass was accomplished by submersible pumps having flow rateof 110 L min�1. Lipid accumulation in Scenedesmus sp. cells wasmonitored using Nile Red staining [2,16]. The biomass yield ofScenedesmus sp. was monitored by gravimetric analysis [17].Dewatering is required to obtain thick biomass slurry. Harvestingof biomass was initially done on day 21 by gravitational settlingto remove the bulk amount of water followed by centrifugationto obtain thick slurry.

2.3. Drying techniques

The harvested wet microalgal biomass was dried using threedifferent drying techniques (sun drying, oven drying, and freezedrying). Thick slurry of wet biomass obtained from 20 L microalgalculture after gravitational settling followed by centrifugation wasused for different drying techniques. Microalgal biomass was sundried on a drying bed lined with white plastic of 1500 lm thick-ness at ambient temperature (25–30 �C) for 72 h [18]. For freezedrying, wet biomass was frozen overnight at �84 �C and lyophi-lized using freeze dryer (Mini lyotrap, LTE scientific Ltd., UnitedKingdom) [15]. Oven drying was carried out using a hot air ovenfor 12 h at 60 �C [19]. The dried microalgal biomass was crushedin a mortar and pestle and the dry microalgal powder was storedin desiccator to avoid moisture absorption. The harvesting anddrying techniques were repeated to accumulate substantial driedbiomass for further experiments. Energy consumption of processis calculated using following equation:

E ðkWÞ ¼ P � t=1000 ð1Þ

where P is the power (W) consumption of all the instruments used, tthe operating time (h) of instruments.

2.4. Cell-disruption and extraction of lipids

Cell disruption and extraction of Scenedesmus sp. biomass wascarried out by slight modification in methods used by Lee et al.[15] for microwave extraction and Kumari et al. [20] for sonicationextraction. The dried biomass (2 g) was added to a 40 ml mixtureof chloroform and ethanol (1:1, v/v) and subjected to cell disrup-tion by microwave (Milestone S.R.L., Italy, output power 1200 W)at 100 �C for 10 min at 1000 W. Mixture was centrifuged to sepa-rate solvent mixture and cell debris. The mixture was vacuum fil-tered followed by distillation of solvent in rotary evaporator at70 �C. The amount of crude lipid was quantified gravimetricallyand the lipid yield (%) was calculated. For sonication assistedextraction, microalgal biomass (2 g) was mixed in solvent mixture(20 ml) and the oil was extracted using sonicator (MisonixXL-2000-010, output power 100 W, output frequency 22.5 kHz)in 50 ml tubes at 15 kHz for 2 min. The solvent mixture was centri-fuged to separate solvent mixture with lipids and residual biomassin the form of a pellet. Residual biomass was again mixed with sol-vent mixture (20 ml) and subjected to sonication followed by cen-trifugation. Solvent mixture was pooled together and filteredthrough a vacuum filter. The solvent was removed in a rotary evap-orator at 70 �C to obtain the microalgal oil. The crude microalgal li-pid was measured gravimetrically and the lipid yield (%) wasquantified. The lipid yields obtained from biomass dried by differ-ent drying processes using the two techniques viz. microwave andsonication were compared. The energy consumption of themicrowave and sonication extraction process was calculated byusing Eq. (1).

2.5. Lipid qualitative analysis and conversion to biodiesel

The conversion of microalgal lipids to fatty acid methyl esters(FAME) was carried out by acid catalyzed transesterification reac-tion [21]. The lipid quality was assessed by determination ofsaponification value and acid value using ASTM methods D5558-95, Reapproved 2011; and D664-07 respectively. The potentiomet-ric titration for determination of acid and saponification value wascarried out by using an automatic titrator (TIM 855 Titration man-ager, Radiometer analytical, Titralab, France). The extracted lipidswere then subjected to simultaneous esterification and transesteri-fication using sulfuric acid as catalyst and methanol as acyl accep-tor for its conversion to biodiesel. The reaction conditions were

48 A. Guldhe et al. / Fuel 128 (2014) 46–52

30:1, methanol to oil molar ratio; temperature: 60 �C; catalyst con-centration: 10% w/w of oil; time: 4 h; hexane: 1 ml [21]. The rate ofstirring was kept constant at 200 rpm in orbital shaker incubator(Model TU-454, mrc Ltd., Israel). The purification of crude biodieselwas conducted by adding hexane and distilled water to the reactionmixture to remove the residual methanol and acid catalyst. A twophase system was formed comprising of an organic and an aqueousphase. Biodiesel was dissolved in hexane and the unreacted meth-anol and H2SO4 was solubilized in water. The organic phase con-taining biodiesel was collected for further analysis. The fatty acidmethyl esters (FAME) were then analyzed with gas chromatograph(Shimadzu GC-2014, Japan) equipped with a flame ionizationdetector and a capillary column (SP2380, Supelco Analytical,USA). The oven temperature was programmed to start at 60 �Cand kept at hold for 2 min, then initially increased to 160 �C at aramp rate of 10 �C min�1 and then to 240 �C at a ramp rate of7 �C min�1 and again kept at hold for 1 min. The injector and detec-tor temperature was 250 �C and nitrogen was used as carrier gas.The molecular weight of lipid was calculated from the lipid profileobtained from the GC using the equation given by Komers et al.[22]. Flow diagram of experimental process of microalgae cultiva-tion to biodiesel production in present study is depicted in Fig. 1.

2.6. Statistical analysis

The data was expressed as mean value ± SE (Standard Error).The results were tested for significance at the 0.05 level by com-paring mean values obtained from experiments for different dryingand cell disruption techniques employing one-way analysis ofvariance.

3. Results and discussion

3.1. Biomass production

The cultivation of microalgae in an open ponds have a greatpotential for large scale economical production of microalgal

Lipid characterization (fatty acid profile, saponification and acid value)

Conversion to biodiesel (by acid catalyst)

Cell Disruption and lipid exttraction

Sonication Microwave

Biomass Drying

Sun drying Oven drying Freeze drying

Biomass harvesting

Gravitational settling followed by centrifugation

Cultivation of Microalgae

Scenedesmus sp. in open circular pond (8000 L) with BG11 media

Fig. 1. Flow diagram of experimental process of microalgae cultivation till biodieselproduction in present study.

biomass. The microscopic observation showed dominance ofScenedesmus sp. (>95%) in the culture grown in open pond. Photo-autotrophic growth of Scenedesmus sp. under natural sunlight sup-plemented with BG11 as nutrient medium produced substantialamount of biomass. Fig. 2 depicts lipid accumulation in Scenedes-mus sp. by Nile Red staining, where neutral lipid bodies in cytosolare stained yellow in color. The biomass production yield of Scene-desmus sp. reached 1.16 g L�1 on 21st day of cultivation.

3.2. Effect of drying technique

Water has to be removed from microalgal biomass slurry to in-crease its viability for effective lipid extraction. Fig. 3 shows the li-pid productivity of biomass obtained from the selected dryingtechniques. In the microwave assisted solvent extraction, the lipidyields of Scenedesmus sp. biomass dried by freeze drying, oven dry-ing and sun drying were found to be 29.65 ± 1.05%, 28.63 ± 0.42%,and 28.33 ± 1.37% lipid g�1 DCW respectively. Whereas, in the caseof sonication assisted solvent extraction, lipid yields by freeze dry-ing, oven drying and sun drying were found to be 19.85 ± 0.35%,18.8 ± 0.1% and 18.9 ± 0.5% respectively. Thus, no significantdifference in the lipid yield was observed among the three dryingtechniques (p > 0.05). Similar findings were reported by Balasubra-manian et al. [23] where biomass of Nannochloropsis sp. was driedusing oven drying, freeze drying and sun drying and there was nosignificant effect of drying technique on the total lipid yield afterextraction. Among the three techniques, freeze drying and ovendrying are energy intensive methods. The time taken for removalof water for the three techniques varied. Sun drying took substan-tially longer (72 h) to remove water from the microalgal biomassas compared to oven drying (12 h) and freeze drying (24 h). How-ever, the process of drying microalgal biomass via oven and freezedrying were energy intensive. The energy consumption by ovenand freeze drying was 6 kW and 21.96 kW respectively (Table 1).Based on energy consumption and sophisticated instrumentation;the overall cost was in the following increasing order: sun dry-ing < oven drying < freeze drying. The choice of drying techniquealso has an influence on the scale of microalgae production. Theeconomical feasibility offered by sun drying makes it a preferredchoice for a large scale production of dry biomass from the wetmicroalgal biomass. However, for sun-drying, requirement of largeland area may be a constraint. Nevertheless, the unused land areanear the site of harvested microalgal biomass could be utilized forsun-drying of microalgae.

3.3. Effect of cell disruption techniques and lipid extraction

The commonly utilized extraction methods viz. Bligh and Dyerand Folch method employ a mixture of chloroform and methanolas solvent [20]. However, methanol is known to be highly toxicand hence was substituted with ethanol. A mixture of chloroformand ethanol was used as solvent for the extraction of lipids [24].The extraction of lipid from microalgae by solvent assisted withcell disruption technique (microwave or sonication) results in abetter yield as these techniques facilitates the contact betweencellular lipids, polar lipids, and solvents. Both microwave and son-ication techniques of cell disruption avoids use of toxic chemicalsand also greatly reduce the time of lipid extraction when comparedto conventional chemical cell disruption techniques and soxhletextraction. In sonication technique, sound waves forms cavitationbubbles in liquid medium which after collapsing generates chem-ical and mechanical energy that disrupts the cell wall of microalgae[25]. Sonication enhances lipid extraction from microalgal cells byfacilitating solvent access through cell disruption and proper masstransfer. In microwave, the microwave energy facilitates rotationof molecular dipole and thus disrupting weak hydrogen bonds.

Fig. 2. Microscopy images of Scenedesmus sp. (A) Scenedesmus sp. without Nile Red staining (B) Nile Red stained Scenedesmus sp. viewed under fluorescence microscope.Neutral lipid globules in the cytosol are stained yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)

Freeze drying Oven drying Sun drying0

10

20

30

Microwave Sonication

Drying technique

Lipi

d yi

eld

(%)

Fig. 3. Effect of drying and cell disruption techniques on lipid yield (%) frommicroalgal biomass.

Table 1Energy consumption and time required for different drying and cell disruptiontechniques.

Drying technique Energy consumption (kW) Time required (h)

Freeze drying 21.96 24Oven drying 6 12Sun drying Nil 72Microwave extraction 1.18 –Sonication extraction 1.43 –

A. Guldhe et al. / Fuel 128 (2014) 46–52 49

This effect causes movement of dissolved ions which increase thesolvent penetration into microalgal biomass and improves lipidextraction [13]. In the present study, the effect of microwave andsonication on the lipid yield has been studied where a compara-tively higher yield of lipid was observed with microwave (Fig. 3).The microwave mode of lipid extraction gave substantial high yieldof 29.65 ± 1.05% lipid g�1 DCW as compared to sonication wherethe yield observed was 19.85 ± 0.35% lipid g�1 DCW in case offreeze dried microalgal biomass. Microwave technique showedsimilar trend of higher lipid yields in oven dried (28.63 ± 0.425%)and sun dried (28.33 ± 1.37%) biomass compared to oven dried(18.8 ± 0.1%) and sun dried (18.9 ± 0.5%) biomass extracted bysonication technique. Microwave assisted lipid extraction gave49.37–52.29% better yield as compared to sonication assisted lipidextraction of biomass dried by selected drying techniques. Higheryields by microwave technique can be attributed to several factors.

In microwave, electromagnetic waves penetrate the cell matrixand interact at molecular level, which makes heating more effi-cient [26]. In conventional solvent extraction, direction of masstransfer is from inside to outside while heat transfer direction isfrom outside to inside, however in case of microwave both massand heat transfer occurs from inside cell matrix to outside solvents.This causes the efficient cell disruption as well as lipid extraction[27]. Results from the present study are in compliance with a pre-vious study by Lee et al. [15] where the microwave method hasbeen shown to extract higher amounts of lipids from Scenedesmussp. as compared to sonication. In a previous study by Iqbal andTheegala [27], microwave assisted lipid extraction and soxhletextraction was studied for lipid extraction of Nannochloropsis sp.Using chloroform and ethanol as solvent, soxhlet extractionyielded 49% lipid g�1 DCW while microwave assisted extractiongave slightly higher lipid yield of 53% lipid g�1 DCW. The energyconsumption of overall extraction process by microwave and son-ication was 1.18 kW and 1.43 kW respectively (Table 1). Themicrowave technique has an easy mode of operation and thuscan be scaled up for industrial scale of lipid extraction.

3.4. Characterization and fatty acid composition of lipid

Lipid characteristics viz. saponification value, acid value andfatty acid profile has an influence on the choice of conversion tech-nique to be adopted for the production of biodiesel. It also subse-quently influences the fuel properties of biodiesel. Table 2 depictsthe saponification and acid value of extracted lipids from sun dried,freeze dried and oven dried Scenedesmus sp. biomass by microwaveassisted extraction. A high saponification value of lipids extractedfrom Scenedesmus sp. (172.52 ± 1.16–177.2 ± 0.82 mg KOH g�1)showed that it can be used as efficient feedstock for biodiesel pro-duction. A high acid value (21.01 ± 0.52–29.75 ± 0.54 mg KOH g�1)indicated the presence of high amount of free fatty acids. Resultsshown in Table 2 demonstrated that drying method has significanteffect on amount of free fatty acid content in total lipids (p < 0.05).Highest acid value was determined in lipids extracted frombiomass dried using sun drying (29.75 ± 0.54 mg KOH g�1) fol-lowed by oven drying (26.17 ± 0.84 mg KOH g�1) and freeze drying(21.01 ± 0.52 mg KOH g�1). This high acid value of lipids extractedfrom sundried microalgal biomass could be because of lipid oxida-tion caused by long exposure to light and UV irradiation andenzyme degradation [23]. Results in present study are in compli-ance with study by Balasubramanian et al. [23], where they foundsimilar trend in FFA content of lipids extracted from Nannochlorop-sis sp. dried using sun drying, oven drying and freeze drying.

Table 2Saponification and acid value analysis of Scenedesmus sp.

Property (unit) Freeze dried Oven dried Sun dried

Saponification value (mg KOH g�1) 177.2 ± 0.82 172.52 ± 1.16 175.45 ± 1.62Acid value (mg KOH g�1) 21.01 ± 0.52 26.17 ± 0.84 29.75 ± 0.54

50 A. Guldhe et al. / Fuel 128 (2014) 46–52

The fatty acid profile of the lipids extracted from Scenedesmussp. biomass by microwave assisted extraction is depicted inFig. 4 and that by sonication is depicted in Fig. 5. Palmitic acid(C16:0) was found to be the most dominant fatty acid in the lipidsextracted from Scenedesmus sp. despite using different drying andcell disruption techniques contributing from 31.67 ± 0.32% to55.18 ± 2.07% to the total lipid constituents. The other pro-minent fatty acids identified in the lipid extracts wereC16:1 (4.40 ± 0.09% to 8.86 ± 0.32%), C18:1 (6.87 ± 0.48% to16.66 ± 0.85%), C18:2 (6.88 ± 0.17% to 12.72 ± 0.08%), C18:3(8.84 ± 0.12% to 15.30 ± 0.13%) and C20:1 (1.41 ± 0.04% to8.85 ± 2.48%). In this study fatty acid profile result has shown thatthere is variation in percentage composition of saturated fattyacids, monounsaturated fatty acid and polyunsaturated fatty acidsdepending upon the drying as well as cell disruption technique(Figs. 4 and 5). Oven dried and freeze dried biomass has shownhigher percentage of saturated fatty acid (46.43–58.44%) thansun dried biomass (44.33–44.98%). Microalgal lipid componentsspecifically polyunsaturated fatty acids show swift response tovarious factors like temperature, pH, light, nutrient depletionetc. [28,29]. Lipids extracted by microwave technique showed39.61–54.1% of unsaturated fatty acids while lipids extracted bysonication technique showed 47.65–52.85% of unsaturated fattyacids. High temperature used in microwave technique as well asfree radical formation during sonication technique could causethe oxidative cleavage of unsaturated bonds [30,31]. More detailedresearch is needed to understand the effect of drying and cell dis-ruption techniques on the quantitative lipid yield as well as varia-tion in fatty acid components in lipids of different microalgalspecies which can be employed as potential biodiesel feedstock.

Fatty acid composition has a profound effect on the fuel prop-erty of biodiesel. There exists an inverse relation between the oxi-dation stability and cold flow property of biodiesel [6,32]. A highcontent of saturated fatty acid is desirable for a better oxidationstability of biodiesel. This will be beneficial for the industry as bio-diesel could be stored for a longer period. On the other hand, a highcontent of unsaturated fatty acid is beneficial for cold flowproperties (cloud point, cold filter plugging point, and pour point)

C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C0

30

60

Fatty

aci

d co

mpo

sitio

n (%

)

Fatt

FM O SM

Fig. 4. Lipid profile obtained by microwave technique (FM- freeze drie

of biodiesel. This would lead to the possible usage of the fuel evenin cold countries and in cold months in the tropical countries.Though, the saturated fatty acids could be removed from the oil/li-pid by winterization and antioxidants could be added to retard theoxidation of the fuel, these processes (of winterization and addi-tion of antioxidants) will enhance to the overall cost of the fuel.Thus, it is desirable that there is a mixture of both saturated andunsaturated fatty acids in the lipid/oil so that both the oxidationstability and cold flow property strike a balance. The concentrationof linolenic acid in lipid should not exceed 12% [6]. In the presentstudy the concentration of linolenic acid (C18:3) varied from8.84 ± 0.12% to 15.30 ± 0.13%. A high concentration of linolenic acidmay lower the oxidation stability of the fuel and lead to its rancid-ity, if the fuel is not supplemented with antioxidants. In the pres-ent work, the concentration of linolenic acid is greater (rangingfrom 8.84% to 15.3%) than the specified limit (12%) in lipid ex-tracted through sonication. Whereas, in the microwave assisted li-pid extraction, the content of linolenic acid (ranging from 9.32% to11.96%) is within the specified limit of 12%. Hence, it may be con-cluded that the lipid extracted through microwave is superior tothat from sonication for its suitability for biodiesel feedstock.

3.5. Conversion of microalgal oil to biodiesel

The use of an alkali catalyst for transesterification of feedstockwith high value of FFA (i.e. >4 mg KOH g�1) leads to saponification[25,32]. Hence, the simultaneous esterification of free fattyacids (FFA) and transesterification of fatty acids was doneby H2SO4 (10% by weight) due to the high acid value(29.75 ± 0.5355 mg KOH g�1) of the lipids extracted from Scenedes-mus sp. The biodiesel yield obtained was 91.75 ± 0.25% from lipidextract of Scenedesmus sp. The acid catalyst H2SO4 converts FFA aswell as triglycerides from the lipid extract. D’Oca et al. [21] reported98.4% biodiesel yield using H2SO4 as a catalyst to convert lipid ex-tracted from the microalgae, Chlorella pyrenoidosa. The presenceof solvent (hexane) during conversion reaction facilitated the prop-er mass transfer and a high conversion of the reactants to products.

18:3 C22:0 C24:0 SFA MUFA PUFAOTHERS --y acids

M

d biomass, OM- oven dried biomass and SM- sun dried biomass).

C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C18:3 C22:0 C24:0 SFA MUFA PUFAOTHERS0

20

40

60

Fatty

aci

d co

mpo

sitio

n (%

)

Fatty acids

FS OS SS

Fig. 5. Lipid profile obtained by sonication technique (FS- freeze dried biomass, OS- oven dried biomass and SS- sun dried biomass).

A. Guldhe et al. / Fuel 128 (2014) 46–52 51

4. Conclusion

Cultivation of microalgae in an open pond is easy to set-up andresulted in a high biomass generation. Drying methods and celldisruption techniques which are crucial for effective downstreamprocessing in microalgal biodiesel synthesis were studied. The se-lected drying methods did not show a significant difference in theextraction of lipid. Microwave cell disruption resulted in a higherlipid yield after extraction when compared to sonication. Lipidsextracted from Scenedesmus sp. showed desirable saponificationvalue, acid value, and lipid profile as a feedstock for biodiesel syn-thesis. Lipids extracted from Scenedesmus sp. were subjected totransesterification and produced 91.75 ± 0.25% yield of biodiesel.Thus, it may be concluded that an easy and economical sun dryingmethod with an efficient microwave cell disruption techniquecould be possible processing steps in further scaling up of biodieselsynthesis from Scenedesmus sp.

Acknowledgements

The authors hereby acknowledge the National Research Foun-dation (South Africa), Durban University of Technology and Ethek-wini Municipality for financial contributions.

Reference

[1] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294–306.[2] Mutanda T, Ramesh D, Karthikeyan S, Kumari S, Anandraj A, Bux F.

Bioprospecting for hyper-lipid producing microalgal strains for sustainablebiofuel production. Bioresour Technol 2011;102:57–70.

[3] Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, et al. Biodieselfrom algae: challenges and prospects. Curr Opin Biotechnol 2010;21:277–86.

[4] Huang G, Chen F, Wei D, Zhang X, Chen G. Biodiesel production by microalgalbiotechnology. Appl Energy 2010;87:38–46.

[5] Rawat I, Ranjith Kumar R, Mutanda T, Bux F. Biodiesel from microalgae: acritical evaluation from laboratory to large scale production. Appl Energy2013;103:444–67.

[6] Singh B, Guldhe A, Rawat I, Bux F. Towards a sustainable approach fordevelopment of biodiesel from plant and microalgae. Renew Sustain EnergyRev 2014;29:216–45.

[7] Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. Cultivation, photobioreactor designand harvesting of microalgae for biodiesel production: a critical review.Bioresour Technol 2011;102:71–81.

[8] Brennan L, Owende P. Biofuels from microalgae—a review of technologies forproduction, processing, and extractions of biofuels and co-products. RenewSustain Energy Rev 2010;14:557–77.

[9] Chen P, Min M, Chen Y, Wang L, Li Y, Chen Q, et al. Review of the biological andengineering aspects of algae to fuels approach. Int J Agric Biol Eng2009;2:1–30.

[10] Halim R, Danquah MK, Webley PA. Extraction of oil from microalgae forbiodiesel production: a review. Biotechnol Adv 2012;30:709–32.

[11] Halim R, Gladman B, Danquah MK, Webley PA. Oil extraction from microalgaefor biodiesel production. Bioresour Technol 2011;102:178–85.

[12] Sharma YC, Singh B, Korstad J. A critical review on recent methods used foreconomically viable and eco-friendly development of microalgae as a potentialfeedstock for synthesis of biodiesel. Green Chem 2011;13:2993–3006.

[13] Cravotto G, Boffa L, Mantegna S, Perego P, Avogadro M, Cintas P. Improvedextraction of vegetable oils under high-intensity ultrasound and/ormicrowaves. Ultrason Sonochem 2008;15:898–902.

[14] Ehimen EA, Sun ZF, Carrington CG. Variables affecting the in situtransesterification of microalgae lipids. Fuel 2010;89:677–84.

[15] Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM. Comparison of several methods foreffective lipid extraction from microalgae. Bioresour Technol 2010;101(Suppl.1):575–7.

[16] Moazami N, Ranjbar R, Ashori A, Tangestani M, Nejad AS. Biomass and lipidproductivities of marine microalgae isolated from the Persian Gulf and theQeshm Island. Biomass Bioenergy 2011;35:1935–9.

[17] Mutanda T, Karthikeyan S, Bux F. The utilization of post-chlorinated municipaldomestic wastewater for biomass and lipid production by Chlorella spp. underbatch conditions. Appl Biochem Biotechnol 2011;164:1126–38.

[18] Rwehumbiza VM, Harrison R, Thomsen L. Alum-induced flocculation ofpreconcentrated Nannochloropsis salina: residual aluminium in the biomass,FAMEs and its effects on microalgae growth upon media recycling. Chem Eng J2012;200–202:168–75.

[19] Cheirsilp B, Torpee S. Enhanced growth and lipid production of microalgaeunder mixotrophic culture condition: effect of light intensity, glucoseconcentration and fed-batch cultivation. Bioresour Technol 2012;110:510–6.

[20] Kumari P, Reddy CR, Jha B. Comparative evaluation and selection of a method forlipid and fatty acid extraction from macroalgae. Anal Biochem 2011;415:134–44.

[21] D’Oca MGM, Viêgas CV, Lemões JS, Miyasaki EK, Morón-Villarreyes JA, PrimelEG, et al. Production of FAMEs from several microalgal lipidic extracts anddirect transesterification of the Chlorella pyrenoidosa. Biomass Bioenergy2011;35:1533–8.

[22] Komers K, Stloukal R, Machek J, Frantšiek S. Biodiesel from rapeseed oil,methanol and KOH3. Analysis of composition of actual reaction mixture. Eur JLipid Sci Technol 2001;103:363–73.

[23] Balasubramanian RK, Yen Doan TT, Obbard JP. Factors affecting cellular lipidextraction from marine microalgae. Chem Eng J 2013;215–216:929–36.

[24] Ramluckan K, Moodley KG, Bux F. An evaluation of the efficacy of usingselected solvents for the extraction of lipids from algal biomass by the soxhletextraction method. Fuel 2014;116:103–8.

[25] Vyas AP, Verma JL, Subrahmanyam N. A review on FAME production processes.Fuel 2010;89:1–9.

[26] Manco I, Giordani L, Vaccari V, Oddone M. Microwave technology for thebiodiesel production: Analytical assessments. Fuel 2012;95:108–12.

[27] Iqbal J, Theegala C. Microwave assisted lipid extraction from microalgae usingbiodiesel as co-solvent. Algal Res 2013;2:34–42.

[28] Borges L, Morón-Villarreyes JA, D’Oca MGM, Abreu PC. Effects of flocculantson lipid extraction and fatty acid composition of the microalgaeNannochloropsis oculata and Thalassiosira weissflogii. Biomass Bioenergy2011;35:4449–54.

52 A. Guldhe et al. / Fuel 128 (2014) 46–52

[29] Gao Y, Yang M, Wang C. Nutrient deprivation enhances lipid content in marinemicroalgae. Bioresour Technol 2013;147:484–91.

[30] Viswanathan T, Mani S, Das KC, Chinnasamy S, Bhatnagar A, Singh RK, et al.Effect of cell rupturing methods on the drying characteristics and lipidcompositions of microalgae. Bioresour Technol 2012;126:131–6.

[31] Gerde JA, Montalbo-Lomboy M, Yao L, Grewell D, Wang T. Evaluation ofmicroalgae cell disruption by ultrasonic treatment. Bioresour Technol2012;125:175–81.

[32] Sharma YC, Singh B, Upadhyay SN. Advancements in development andcharacterization of biodiesel: a review. Fuel 2008;87:2355–73.