Comparative Analysis of Fatty Acid Composition in Seven Plant Seed Oils

Page 1

Comparative Analysis of the Fatty Acid Compositionof Microalgae Obtained by Different Oil Extraction Methodsand Direct Biomass Transesterification

Aline Terra Soares & Dayane Cristine da Costa & Bruna Ferreira Silva &

Rafael Garcia Lopes & Roberto Bianchini Derner &

Nelson Roberto Antoniosi Filho

Published online: 23 March 2014# Springer Science+Business Media New York 2014

Abstract One of the main challenges for the successful pro-duction and use of microalgae for biodiesel production is toobtain a satisfactory level of fatty acid methyl esters (FAME).The aims of this study are to identify the best method of lipidextraction and provide high FAME levels and to evaluate theirfatty acid profiles. Six lipid extraction methodologies in threemicroalgae species were tested in comparison with the directtransesterification (DT) of microalgal biomass method. Thechoice of extraction method affected both the oily extract yieldand the FAME composition of the microalgae and consequentlymay affect the properties of biodiesel. The efficiency of differentlipid extraction methods is affected by the solvent polarity,which extracts different target compounds from lipid matrix.Dichloromethane/methanol extraction and Folch extraction pro-duced the largest oil extract yields, but extraction with hexane/ethanol resulted in the best ester profile and levels. PerformingDT reduces the volume of extractor solvent, the time and cost ofFA composition analysis, as well as, presents less steps for fattyacid quantification. DT provided biomass FAME levels of 50.2,636.4, and 258.2 mg.g−1 in Nannochlorophisis oculata,Chaetoceros muelleri, and Chlorella sp., respectively. On thebasis of an analysis of the fatty acids profiles of different species,C. muelleri is a promising microalga for biodiesel production.Depending on the extraction method, Chlorella sp. and

N. oculata can be considered as an alternative in obtainingarachidonic (Aa) and eicosapentaenoic (EPA) acids.

Keywords Microalgae . Extraction . Directtransesterification . Fatty acids . Biodiesel

Abbreviations

Aa Arachidonic acid (C20:4 ω6)ANP Brazilian National Agency for

Petroleum Natural Gas and BiofuelsCFPP Cold filter plugging pointCN Cetane numberDCM DichloromethaneDT Direct transesterificationCO2 Carbon dioxideEPA Eicosapentaenoic acid (C20:5 ω3)GC-HRMS Gas Chromatography -High resolution

mass spectrometryFA Fatty acidsFAME Fatty acid methyl estersFID Flame ionization detectorPAR Photosynthetically active radiationPUFA Polyunsaturated fatty acidsSFA Saturated fatty acidsSV Saponification valueTAG TriglyceridesUSFA Unsaturated fatty acids

Introduction

Currently, commercial biodiesel is derived mainly from ole-aginous plants, and soybeans are the most widely used fatty

A. T. Soares :D. C. da Costa :B. F. Silva :N. R. Antoniosi Filho (*)Laboratory of Methods of Extraction and Separation (LAMES),Chemistry Institute, Federal University of Goiás, Campus II,Samambaia, Cx, 131, 74001-970 Goiânia, Goiás, Brazile-mail: [email protected]

R. G. Lopes : R. B. DernerLaboratory of Cultivation of Algae (LCA), Department ofAquaculture, Agricultural Sciences Center, Federal University ofSanta Catarina, Servidão dos Coroas, n. 503, 88061-600 Barra daLagoa, Florianópolis, Santa Catarina, Brazil

Bioenerg. Res. (2014) 7:1035–1044DOI 10.1007/s12155-014-9446-4

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raw material in Brazil and other countries [1]. Soybeans, likemost other oilseeds, food grains, and forage used for biodieselproduction, present certain drawbacks: (a) an extensive culti-vation area, (b) competition with food grain production, and(c) inability to meet the global demand for biodiesel [2].

Although biodiesel production frommicroalgae is not yet acommercial reality, there are reasons that may indicate thatthis will occur in the future. Like oilseeds, microalgae arecapable of converting sunlight into a metabolite-rich biomassand are thus a promising raw material for biofuel productionsince they are a source of oil rich in triglycerides (TAGs). Thenumerous advantages of microalgae over oilseeds result inhigher per-hectare oil production than that of oilseeds com-monly used to produce biodiesel [2].

A number of challenges need to be addressed for thesuccessful production and use of microalgae-derived biodie-sel. One of the main challenges is obtaining a satisfactory levelof fatty acid methyl esters (FAME) for biodiesel production,which is different from the quantity of soluble extract innonpolar or medium-polarity solvents, which can be obtainedfrom this fatty raw material, and therefore, the lipid extractionand transesterification stages are of great importance.Furthermore, it is important to select high-quality microalgaewith a suitable fatty acid profile because these factors willdetermine production feasibility and biodiesel quality [3].

The fatty acid (FA) content determined in microalgaevaries depending on the species, growing conditions andextraction methods used [4]. The Folch and Bligh and Dyerextraction methods and adaptations that modify solvent con-tent are the most widely studied and used methods forextracting the lipid portion of microalgal biomass [5,6].However, due to the high efficiency and low selectivity ofthese methods, they can result in the extraction of pigments,proteins, and carbohydrates [4], which are not suitable forbiodiesel production and can compromise analytical resultsin quantifying lipid content.

In order to eliminate the oil extraction and purification stages,some authors suggest the direct transesterification (DT) ofmicroalgal biomass [7-10]. Various methods, original or adap-tations, such as those of Hartman and Lago [11] and Lepage andRoy [12], have been used for DT or in situ FAME synthesis, inwhich catalysis involves the use of an organic solvent, alcohol,and sample heating. The term in situ is used when catalyticreactions occur without the prior step of lipid portion extraction[13], that is, in DT lipid portion extraction and alcoholysis andesterification reactions occur simultaneously. In this process, asingle type of catalyst, basic or acidic, is often used.

The efficiency of the catalysis will depend on the ability of thesolvent to solubilize the lipids and form a single phase thatisolates the lipid-derived components from the other phase con-taining the remaining reagents. The combination of two catalysessuch as basic catalysis followed by acid catalysis, for example,tends to increase the efficiency of transesterification. Many

studies have demonstrated the efficiency of DT in quantifyingthe fatty acid profile [14,10]; although, this has not been dem-onstrated using two types, basic and acidic, catalysis.

The objective of this study was, thus, to compare theefficiency of direct transesterification of microalgal biomassusing basic catalysis followed by acid catalysis with that of oilextract transesterification using six extraction methods onthree microalgae species in regard to oil extract content, fattyacid methyl ester content, and fatty acid profile, assessing eachprocedure’s potential for biodiesel production.

Materials and Methods

Organisms

In this study, dry and lyophilized biomass from the followingmarine microalgae were used: Nannochloropsis oculata,Chaetoceros muelleri, and Chlorella sp. (courtesy of Prof.Dr. Roberto Bianchini Denner, Federal University of SantaCatarina-UFSC). The culture media for cultivating microalgaewere f/2 [14] for N. oculata and C. muelleri and BG-11 [15]for Chlorella sp.

The cultures were grown in glass fiber cylinders containing100 L of the respective culture media. Fixtures with fluorescentlamps were used, and photosynthetically active radiation (PAR)was measured. Lighting was 150 μmol m−2 s−1 for a completephotoperiod. Pressurized atmospheric air continuously injectedat a flow rate of 10 Lmin−1 into each cylinder was enrichedwith1 % CO2. The temperature of the cultivation room was kept at25 °C (±2 °C) using air conditioners. The biomass was separatedby adding polycationic flocculant at a final concentration of5 ppm (500 mg flocculant/100 L of microalgae culture). Afterflocculation and sedimentation, the biomass was filtered througha screen and dried in an oven at 50 °C for 24 h.

Oil Extraction

The means of extraction were:

i. Hexane (TEDIA®)ii. Hexane/ethanol (4:1 v/v—TEDIA®)iii. Chloroform/methanol (2:1 v/v—TEDIA®)—Adaptation

of the method of Folch et al. [16]iv. Methanol/chloroform/water (2:1:0.5 v/v—TEDIA®)—

Adaptation of the method of Bligh and Dyer [17]v. Dichloromethane/methanol (2:1 v/v—TEDIA®)vi. Adaptation of the Rose Gottlieb method [18]

To compare the six oil extraction methods with directtransesterification of biomass, the lyophilized biomass of themicroalgae N. oculata, C. muelleri, and Chlorella sp. wasextracted and about 1.0 g of biomass was weighed into a test

1036 Bioenerg. Res. (2014) 7:1035–1044

Page 3

tube, to which 12.0 mL of extraction solvent was added. Thebiomass and solvent mixture in the test tube was vortexed for30 s, which was followed by stirring in a shaker for 2 h atroom temperature. At the end of the extraction, the tubes werecentrifuged at 3000 rpm for 5 min, and the organic phase wastransferred to pre-weighed vials for subsequent solvent evap-oration. The residual biomass was extracted again by addinganother aliquot of 12.0 mL of organic solvent, vortexing againfor 30 s and extracting for 1 h. The test tubes were centrifuged,and the liquid phase was added to the vial in which the firstaliquot of extract was stored. The vials were left open in alaminar flow for continued solvent evaporation and determi-nation of the extract obtained. All oil extractions or directtransesterifications were carried out in five replicates fromthe same microalgal biomass.

Transesterification of Microalgae Oil Extracts and DirectTransesterification (DT) of Microalgal Biomass

Yield and fatty acid methyl ester composition were deter-mined using the Hartman and Lago method adapted to micro-scale [19]. Initially, an esterifying mixture, which was usedduring the transesterification process, was prepared. For thismixture, 2.0 g of ammonium chloride (Merck®) was added to60.0 mL of methanol (TEDIA®) followed by the addition of3.0 mL of concentrated sulfuric acid (Merck®). In a roundbottom flask adapted to a condenser, the mixture was refluxedunder manual agitation for 15 min. The reagent obtained wasthen stored in a 100 mL volumetric flask with a glass stopper.

Transesterification of the Oily Extract

Thirty five milligrams of microalgae oil extract was weighedinto an autoclavable 20 mL test tube. Then, 0.5 mL of0.5 mol L−1 sodium hydroxide solution (Merck®) was placedin dry methanol (TEDIA®), and the test tube was heated for10 min in a water bath at 90ºC. The test tube was cooled in anice bath and 1.5 mL of the esterifying mixture previouslyprepared according to the procedure described above wasadded. The test tube was heated again for 10 min in a waterbath at 90 °C. It was then cooled in an ice bath, and 5.0 mL ofn-heptane (TEDIA®) and 10.0 mL of distilled water wereadded. The test tube was shaken a few times, and the systemwas allowed to stand until phase separation. The upper phaseconsisted of fatty acid methyl esters dissolved in n-heptane.After that phase, this was collected using a Pasteur pipette andtransferred to a screw-cap vial, and high resolution gas chro-matography was used to determine its FAME composition.

Direct Transesterification (DT) of Biomass

Approximately 200 mg of microalgal biomass was weighed.Next, 3.0 mL of 0.5 mol L−1 sodium hydroxide solution

(MERCK®) in dry methanol (TEDIA®) was added, and thetest tube was heated for 10 min in a 90ºC water bath. The testtube was cooled in an ice bath, and 9.0 mL of the esterifyingmixture previously prepared according to the procedure de-scribed above was added. The test tube was heated again for10 min in a 90 °C water bath. The test tube was cooled in anice bath, and 5.0 mL of n-heptane (TEDIA®) and 2.0 mL ofdistilled water were added. The organic phases were collectedwith a Pasteur pipette and analyzed by high resolution gaschromatography.

Chromatographic Analysis

An Agilent 7890 gas chromatograph equipped with a FlameIonization Detector (FID) and a split/splitless injector wasused to analyze FAME composition. The capillary columnwas the DB-WAX (30 m×0.25 mm×0.25 μm). Initial tem-perature of the oven was 70ºC, and it was heated at a rate of10ºC min−1 to 240 °C and maintained at this temperature for13 min. It was heated again at a rate of 5 °C min−1 to 250ºC.The injector temperature was kept at 310ºC in split mode witha split ratio of 10:1 for an injection volume of 2 μL. The oventemperature was maintained at 310 °C. Hydrogen (5.0) wasused as the carrier gas at a linear velocity of 42 cm s−1, andnitrogen was used as the auxiliary gas at a rate of20 mL min−1.

The FAMEs were identified by comparison with the reten-tion times of samples of known composition such as soybeanoil, peanut, and crambe (Crambe abyssinica) by analyzingFAME reference standards (Nu-Check-Prep®) and by gaschromatography coupled with high resolution mass spectrom-etry (GC-HRMS) using a Shimadzu model 17A chromato-graph coupled with a Shimadzu QP-5050 mass spectrometer.The carrier gas was helium with a linear velocity of 42 cm s−1.The GC-FID operating conditions (oven, injector, interface,and capillary column) were maintained for GC-HRMS.

To calculate the relative yield in esters, the ester peak areasof each of the chromatograms of each microalga were addedup. The totals that generated the highest values for eachmicroalga were designated as the maximum esters obtainedand given a relative value of 100 %. The percentages obtainedin the other chromatograms for each microalga were calculat-ed in reference to these values.

Results and Discussion

Analysis of Oily Extract Content by Extraction Methods

The yields of oily extracts after using different extractionmethods for microalgae N. oculata, C. muelleri, andChlorella sp. are shown Fig. 1a. The extraction according toFolch et al. (1957) and the dichloromethane/methanol method

Bioenerg. Res. (2014) 7:1035–1044 1037

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showed similar effectiveness in oil extracted inN. oculata andC. muelleri, but Folch’s method provided the highest extractyields for these microalgae. Dichloromethane/methanol wasthe best extractor solvent in Chlorella sp.

The lowest yields were obtained with hexane, which pro-vided the best separation of the extraction solvent from themicroalgal biomass and with which the clearest extract color-ation was obtained. The fact that hexane generated low ex-traction yields indicates that neutral lipids, consisting primar-ily of acylglycerides, especially triglycerides, were obtained.They are a minority among the nonpolar and medium-polaritycomponents extracted with different solvents. Thus, the

microalga N. oculata displayed a small quantity of neutrallipids given its low extraction yield in hexane.

The addition of ethanol to hexane increased the amount ofextract obtained which indicates that increasing the polarity ofthe mixture should lead to a greater quantity of extract. This isevidenced by the results obtained using the Folch’smethod and the dichloromethane/methanol mixture. As thepolarity of the solvent mixture increases, so does the greenishcolor of the extract due to the extraction of pigments, espe-cially chlorophyll pigments, and some lipid classes, such ashigh-polarity phospholipids and glycolipids, may be extracted[5].

This indicates that fatty acid structures in microalgae mustcorrespond to molecules that are more polar thantriacylglycerides and may consist mainly of free fatty acids,monoacylglycerides, diacylglycerides, or steroid esters withfatty acid and a minority of higher polarity molecules such asphospholipids, or acylated glycoside steroids, which are also asource of fatty acids for biodiesel production [20].

The adapted Rose Gottlieb method provided extractionyields that fell between those of hexane/ethanol and theadapted Bligh and Dyer method. The Rose Gottlieb methodis widely used in lipid extraction from food. It is based on thehydrolysis of protein–lipid binding by alcohol and ammoniumhydroxide, with extraction of lipids using ethyl and petroleumether [18]. Although favoring a cleavage in the lipid–proteinbond, the petroleum ether and ethyl ether solvents were notvery effective in extracting the lipid portion from microalgalbiomass and gave low extraction yields. Also, this may indi-cate that most of the lipids present in the microalgae are notassociated with proteins.

The Bligh and Dyer method, traditionally used by manyresearchers to publicize high oil extraction yields frommicroalgae [21-27], generated extract mass values intermedi-ate to those obtained with other solvents. The reason for this isthat the solvent used in the extraction method contains meth-anol, a polar solvent that assists in the removal of lipids whichare in contact with aqueous phases, and which, therefore, aremore polar than neutral lipids.

Thus, given the highly complex biochemistry ofmicroalgae, different extraction systems may generatecompletely different results, making it difficult to assess thequality of a particular microalga for use as a fatty raw materialfor biodiesel production via conventional lipid extractionmethods. For biodiesel production, the fatty acid contentprovides more suitable data than lipid content. To obtain thatinformation, a DT method was employed which allowedobtaining fatty acid content, with reproducibility, in fewersteps and in an inexpensive way. It is very important toobserve that all the liquid/solid extraction systems will pro-vide extracts with different compositions, which will dependon the polarity of the solvents employed. Thus, DT appears tobe an accurate method to obtain this information.

0

50

100

150

200

250

300

Yie

ld e

xtra

ct(m

g E

xtra

ct/g

bio

mas

s)A Nannochloropsis oculata

Chaetoceros muelleri

Chlorella sp.

0

20

40

60

80

100

120

Rel

ativ

e fa

tty a

cid

met

hyl

este

rs c

onte

nt (

%)

B

Fig. 1 Yield of oil extracts and relative esters content based on differentextraction methods for microalgae Nannochloropsis oculata,Chaetoceros muelleri, and Chlorella sp. Shown are average values±SEof five technical replicate extractions from the same biomass

1038 Bioenerg. Res. (2014) 7:1035–1044

Page 5

Comparison Between FAME Content and the Applicabilityof Direct Transesterification

Evaluation of relative fatty acid methyl ester yield was obtain-ed through the transesterification of oils extracted by the sixmethods and by direct transesterification. Despite these alter-native methods, Folch’s and the dichloromethane/methanolmethod provided the best yields for oily extract, while theconversion of lipids to FAME was lower than when usinghexane/ethanol and Bligh and Dyer (Fig. 1b). Extraction withhexane and ethanol proved to be an excellent alternative forachieving high levels of esters and consequently of biodiesel,since higher yields of N. oculata and C. muelleri esters wereobtained using this solvent mixture.

Bligh and Dyer extraction was the most efficient in theconversion of oily extract into esters in Chlorella sp. Thisindicates that in this microalga, the molecules of the chemicalspecies that provide fatty acids must contain mainly polarportions such as phospholipids or acylated steroid glycosides.

Also, the data in Fig. 1b show that fatty acids resulted byneutral lipids (hexane extract) correspond to only 24.1 % ofthe fatty esters obtained by N. oculata, 30.2 % of those fromC. muelleri, and 5.7 % of fatty esters from Chlorella sp.

The relative FAME content obtained by Rose Gottlieb’smethod in N. oculata and Chlorella sp. was similar to thatobtained by DT in microalgae biomass. Furthermore, it gavethe lowest ester yield inC.muelleriwhen comparedwith othermethodologies.

Direct transesterification eliminates the oil extraction andpurification stages and thus reduces the volume of solventused in extraction as well as biodiesel production time andcost. However, DT FAME yield with microalgae species ismuch lower (12 %, 37.3 %, and 34.6 %) than the values of theextraction methods with the greatest extract yields (hexane/ethanol and Bligh and Dyer).

Nevertheless, the chromatograms obtained from the directtransesterification of microalgal biomass and the data fromTable 1 show that the percentage composition of saturated andunsaturated fatty acids obtained for the transesterification ofthe extracts using hexane/ethanol and Bligh and Dyer solventsis similar to that obtained by direct transesterification.

Evaluation of the Effectiveness of the Extraction Methodsand DT for the Microalgae

Among the microalgae species studied, in relation to the oilyextract content, C. muelleri had the highest content of extractfor all extraction methods tested (Fig. 1a), with Folch’s ex-traction method being the most efficient in removing the lipidportion from the biomass.

Absolute values about which microalga provided thehighest ester content can be determined using data obtainedvia extraction and direct transesterification. For this purpose,

the sums of the FAME peak areas in each of the chromato-grams were compared with each other. The hexane/ethanolextraction system and the adapted Bligh and Dyer methodprovided the largest FAME peak areas for the microalgaeC. muelleri and N. oculata and the microalga Chlorella sp.,respectively.

Table 2 compares these values relatively (as a percentage)and absolutely (sum of FAME peak area units for highercapacity extraction systems). The microalga C. muelleri pro-vided the most fatty acid esters, both via transesterificationafter solvent extraction and by direct transesterification. Thetwo methods produced similar yields for Chlorela sp. (61.4 %and 56.9 %). However, for the microalga N.oculata, percent-ages of the highest ester yields for the two methods showed nosimilarity (49.4 % and 15.8 %). This demonstrates that directtransesterification is not an appropriate methodology for de-termining if one microalga is more suitable than another forbiodiesel production from the point of view of biodiesel yield.

It can be concluded that direct transesterification is fasterthan other methods in providing a sample’s fatty acid compo-sition, and this is important for assessing the suitability of afatty raw material for biodiesel production. DT does not allowan absolute determination of how much lipid is possible toextract from microalgal biomass, but it is appropriate forcomparing the fatty acid composition of one microalga withthat of another to determine suitability for biodieselproduction.

An alternative worth testing for increasing directtransesterification yield is the use of sodium ethoxide insteadof sodium methoxide to improve lipid extraction frommicroalgae, which would lead to the formation of fatty acidmethyl esters.

Fatty Acid Composition

The profile of fatty acids that make up oils can affect biodieselphysical properties such as kinematic viscosity, oxidativestability, cold filter plugging point (CFPP), and cetane number(CN), among others [3,28]. Oils consisting of saturated acidshave higher viscosity and high CFPP. An increase in thedegree of unsaturation decreases CN, causing a delay inignition in addition to favoring biodiesel oxidationprocesses.These properties are also affected by carbon chainlength. The longer the chain, the greater the viscosity andCFPP, but this parameter tends to favor the CN.

The fatty acid composition of the three microalgae studiedus ing d i f f e r en t ex t r ac t ion methods and d i r ec ttransesterification of biomass are shown in Table 1, andFig. 2 displays the chromatogram of the microalgaeC. muelleri obtained by extraction with hexane/ethanol (a)and direct transesterification (b), N. oculata obtained by ex-traction with hexane/ethanol (c), andChlorella sp. obtained byextraction with Bligh and Dyer (d).

Bioenerg. Res. (2014) 7:1035–1044 1039

Page 6

Tab

le1

FAMEcompositio

nfrom

Nannochloropsisoculata,Chaetoceros

muelleriandChlorellasp.w

ithhexane,hexane/ethanol,adaptatio

nof

themethodof

RoseGottlieb

extractio

n,adaptatio

nof

the

methodof

Folchetal.,dichloromethane/m

ethanol,adaptatio

nof

themethodof

Blig

handDyerextractio

n,anddirecttransesterification

Fatty

acids

FAMEcompositio

n(%

)

Hexane

Hexano/Ethanol

RoseGootilieb

Folch

etal.

Dichlorom

ethane/m

ethanol

Blig

handDyer

Directtransesterificatio

n

a No

bCm

c Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

C10:0

––

––

––

––

––

––

––

––

––

–1.0

–

C12:0

0.5

0.2

–0.5

0.2

––

––

––

–0.8

0.2

–0.4

–0.1

–0.5

–

C14:0

5.1

24.6

–5.9

22.7

2.0

1.6

5.1

0.7

3.3

23.7

2.6

6.8

20.1

23.1

4.0

23.9

1.7

5.4

23.1

0.7

C14:1

cis9

1.9

0.6

–3.1

0.9

4.2

–2.2

2.8

2.0

–4.5

2.7

0.9

1.0

1.9

0.5

3.3

1.9

1.7

1.7

C15:0

1.5

1.8

–1.5

1.3

0.3

––

1.4

2.6

1.4

–1.7

1.1

1.1

1.0

1.5

0.2

1.4

1.2

–

C15:1

cis9

–0.9

–0.6

0.3

0.7

––

1.8

––

0.9

0.6

0.3

0.7

0.4

0.4

0.6

2.1

–0.5

C16:0

30.6

36.9

10.7

28.2

26.5

13.0

20.7

18.6

46.9

25.4

29.7

16.7

39.1

23.4

23.9

30.6

35.5

14.0

33.2

25.2

20.1

C16:1

cis7

0.7

–1.6

2.4

1.4

17.0

3.0

2.1

0.8

2.1

0.7

7.4

0.5

1.3

3.0

1.7

0.3

11.0

2.4

2.3

6.2

C16:1cis9

7.2

2.7

1.7

10.2

30.0

0.9

2.9

1.5

1.0

7.7

28.0

1.3

4.5

27.6

26.9

8.7

23.1

1.1

9.6

28.3

1.3

C16:1

cis11

0.5

––

1.3

3.1

0.3

––

2.1

0.6

3.2

0.6

0.8

2.8

3.1

0.5

2.6

0.6

1.2

2.8

0.9

C16:2

cis7,10

0.6

––

1.5

1.1

5.0

–6.4

1.5

2.4

1.0

5.7

0.9

1.1

1.5

0.9

0.9

5.5

0.9

1.1

5.0

C16:3

cis4,7,10

–4.6

––

–10.5

–8.2

––

–7.3

––

––

–8.2

––

5.3

C17:0

2.8

2.3

–1.7

1.6

2.9

–1.7

2.0

0.7

1.6

1.7

1.1

1.9

1.6

2.2

1.5

1.8

1.0

1.4

1.4

C17:1

cis9

1.4

0.4

–2.4

1.8

2.8

–3.2

0.7

1.8

1.8

2.9

0.8

2.2

2.0

1.8

1.7

3.0

2.3

2.0

2.9

C17:2

cis9,12

0.7

4.3

––

––

––

1.2

2.1

––

0.5

––

––

––

––

C18:0

7.7

3.4

5.8

4.8

1.3

3.8

6.1

1.2

4.8

4.6

1.4

1.9

7.3

1.8

1.0

4.4

1.8

1.4

4.8

1.4

0.8

C18:1

cis7

0.6

0.6

–1.1

1.3

––

–0.7

0.7

2.0

–2.5

2.8

0.9

0.9

1.6

–1.1

1.2

–

C18:1

cis9

16.8

15.4

11.5

12.7

1.5

7.3

30.3

10.6

4.6

17.2

1.4

9.2

5.4

2.3

2.7

20.4

1.0

8.8

15.4

2.2

9.9

C18:1

cis11

3.6

––

5.0

1.7

1.1

2.8

1.7

1.1

3.4

1.7

1.7

2.6

1.5

1.3

4.5

1.5

1.6

4.4

1.7

1.8

C18:2

cis9,12

12.9

1.3

8.2

6.9

1.0

13.0

29.0

19.5

6.5

16.9

0.7

18.4

1.7

1.1

2.6

8.7

0.6

18.0

7.2

1.9

19.5

C18:3

cis6,9,12

––

–0.6

0.2

0.5

––

–0.9

–0.6

–0.2

––

–0.6

––

0.5

C18:3

cis9,12,15

2.8

–32.0

3.5

–10.4

3.6

15.0

3.5

4.1

–13.8

2.0

0.3

1.0

3.2

–13.5

3.5

–12.8

C18:4

cis6,9,12,15

––

3.0

––

2.8

–3.0

––

–2.8

––

––

–3.1

––

1.9

C19:0

1.3

––

1.3

––

––

––

––

1.2

––

0.9

––

––

–

C19:1

––

–1.0

––

––

4.5

––

––

––

0.6

––

––

–

C20:0

––

––

––

––

2.0

––

––

––

––

––

––

C20:1

cis9

––

––

0.1

––

––

––

–3.4

––

––

––

––

C20:2

cis11,14

––

––

0.2

––

––

––

––

––

––

––

–0.8

C20:4

cis5,8,11,14

––

15.2

1.0

0.3

––

––

–0.4

––

0.3

––

––

––

–

C20:5cis5,8,11,14,17

0.8

–10.3

2.8

0.3

0.8

––

4.8

1.5

1.0

–10.0

2.4

1.1

2.3

1.6

0.7

2.2

1.0

1.1

1040 Bioenerg. Res. (2014) 7:1035–1044

Page 7

In the microalga N. oculata, palmitic acid (C16:0) is pre-dominant in almost all the processes except for RoseGottlieb’s method, in most cases accounting for 25.4 % ofFA composition. The high concentration of this saturated acidhas also been observed in the literature [4].

The extraction with hexane in N. oculata is composedmainly of myristic (5.1 %), palmitic (30.6 %), palmitoleic(7.2 %), stearic (7.7 %), oleic (16.8 %), and linoleic (12.9 %) acids. In this extraction, the levels of saturated fatty acids(SFA; 49.5 %) and unsaturated fatty acids (USFA; 50.5 %)were very close.

When the polarity of oil extraction in N. oculata wasincreased with the addition of ethanol to hexane, there was areduction in saturated fatty acid content which favored anincrease in unsaturated acids. The presence of arachidonicacid (C20:4 ω6) and an increase in the level ofeicosapentaenoic acid (C20:5 ω3) were noted. Aa and EPA,considered essential in the human diet, are fatty acids belong-ing to the Omega 6 and 3 family [29].

In the microalga N. oculata, the method adapted fromFolch et al. produced a predominance of unsaturated fattyacids (63.4 %), mainly oleic acid (C18:2 cis9, 12) and linoleicacid (C18:2 cis9, 12). The level of linoleic acid was higherthan reported by Fuentes et al. [29], who cultured thismicroalga under different culture conditions and for extractionused chloroform and methanol in a ratio of 2:1 v/v. Thepresence of double bonds favors the CFPP, avoiding thelow-temperature formation of crystals which clog engine fil-ters and nozzles; however, this biodiesel is more susceptible tooxidation.

Extraction with dichloromethane/methanol in N. oculatashowed the greatest amount of SFA (61.1 %), making thisbiodiesel less susceptible to oxidation, but with high viscosityand a high CFPP. The presence of long-chain fatty acids and asubstantial level of EPA (10.0 %) compared to other methodswere also noted.

Characteristics of the biodiesel produced from the oil ex-tracted from N. oculata using the method adapted from Blighand Dyer were similar to those of the biodiesel extracted withhexane and ethanol. However, since it is greenish due to thepresence of chlorophyll, the biodiesel produced from the oil

Table 2 Comparison of absolute and relative esters content for extractionfollowed by derivatization and direct transesterification

Microalgae Systems higher extractioncapacity

Directtransesterification

Absolutea Relative Absolute a Relative

N. oculata 1790 (49.4 %) 214 (15.8 %)

C. muelleri 3626 (100.0 %) 1352 (100.0 %)

Chlorella sp. 2226 (61.4 %) 769 (56.9 %)

aValues are FAME peak area units

Tab

le1

(contin

ued)

Fatty

acids

FAMEcompositio

n(%

)

Hexane

Hexano/Ethanol

RoseGootilieb

Folch

etal.

Dichlorom

ethane/m

ethanol

Blig

handDyer

Directtransesterificatio

n

a No

bCm

c Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

No

Cm

Cs

C22:0

––

––

1.1

––

––

––

–0.5

–0.9

––

0.2

––

0.5

C22:1

cis13

––

––

0.1

0.7

––

1.8

–0.3

––

3.1

0.6

––

0.5

––

2.0

C24:0

––

––

––

––

1.6

––

–2.6

––

––

––

––

C24:1

cis15

––

––

––

––

1.2

––

––

1.3

––

–0.5

––

2.4

∑Saturated

49.5

69.2

16.5

43.9

54.7

22.0

28.4

26.6

59.4

36.6

57.8

22.9

61.1

48.5

51.6

43.5

64.2

19.4

45.8

53.8

23.5

∑Insaturated

50.5

30.8

83.5

56.1

45.3

78.0

71.6

73.4

40.6

63.4

42.2

77.1

38.9

51.5

48.4

56.5

35.8

80.6

54.2

46.2

76.5

∑Triisaturated

2.8

4.6

32.0

4.1

0.2

21.4

3.6

23.2

3.5

5–

21.7

2.0

0.5

13.2

–22.3

3.5

–18.6

∑Po

liinsaturated

0.8

–28.5

3.8

0.6

3.6

–3.0

4.8

1.5

1.4

2.8

10.0

2.7

1.1

2.3

1.6

3.8

2.2

1.0

3.0

Legend:

aNo:

Nannochlorophisisoculata;

bCm:C

haetoceros

muelleri;c

Cs:Chlorella

sp.

Bioenerg. Res. (2014) 7:1035–1044 1041

Page 8

extracted by Bligh and Dyer may be more susceptible tooxidation. This is because chlorophyll promotes the degrada-tion of fatty acids during heating [30].

The adaptation of the Rose Gottlieb method used onN. oculata biomass resulted in a profile with high percentagesof oleic acid (30.3 %) and linoleic acid (29.0 %). This extrac-tion method thus generated the highest percentage of unsatu-rated fatty acids (71.6 %) of all the methods, so this biodieselwould have low oxidative stability. The FA composition of oilfrom the direct biomass transesterization of N. oculata, likethat of oil extracted using hexane/ethanol, is characterized bythe presence of myristic, palmitic, palmitoleic, oleic, andlinoleic acids. The oil from DT also shows a balance in SFAand USFA levels.

As reported in the literature, C. muelleri FA composition ischaracterized by a significant presence of myristic, palmitic,and palmitoleic acids [31]. This fatty acid profile is similar tothose obtained in this study, with the exception of FAs obtain-ed by hexane extraction, which showed high levels of oleicrather than palmitoleic acid, and the adapted Rose Gottliebmethod, whose profile is characterized by palmitic acid andunsaturated long-chain acids such as oleic, linoleic, and

linolenic acids and low levels of myristic and palmitoleicacids.

Fatty acids affect the properties of biofuels. The FA profilesof the biomass of the microalga C. muelleri obtained by directtransesterification, by oil extraction with hexane/ethanol usingan adaptation of the method of Folch et al. anddichloromethane/ methanol are characterized by an SFA con-tent ranging from 48.5 % to 57.8 % and by a balance in thedegree of saturation among the fatty acids.

The microalga C. muelleri contained low levels of long-chain unsaturated fatty acids, especially when compared to thelevels of palmitoleic acid. Some methods of extracting oilf rom C. muel leri showed the presence of EPA.Dichloromethane/methanol was the extraction method withthe highest yield for this polyunsaturated acid (2.4 %), and theonly method that detected the presence of arachidonic acid.Studies have shown that the content of these acids inC. muelleri is affected by culture conditions [32] and, asshown in this study by extraction methods.

The main fatty acids found in Chlorella sp. in relation toextraction methods were C16:0, C16:1, C16:2, C16:3, C18:1,and C18:3 in a profile similar to that demonstrated by [31] for

Fig. 2 Chromatogram representing themajor fatty acids frommicroalgaea Chaetoceros muelleri obtained by extraction with hexane/ethanol and bby direct transesterification, c Nannochloropsis oculata obtained by

extraction with hexane/ethanol, and d Chlorella sp. from extraction withBligh and Dyer’s method

1042 Bioenerg. Res. (2014) 7:1035–1044

Page 9

species of the Chlorophyceae class. As demonstrated by Kisset al. [33], octadecatrienoic acid C18:4 cis6, 9, 12, 15 was alsopresent.

Of the three microalgae species analyzed, Chlorella sp. ischaracterized by a high level of EPA (10.3%) and Aa (15.2%)when oil is extracted with hexane. Thus, the extraction of oilfrom Chlorella sp. using hexane in an ultrasonic systememerges as an alternative process for production of the essen-tial acids Aa and EPA.

The FAME profile for the oil extracted fromChlorella sp. with hexane, hexane/ethanol, and usingFolch et al., Bligh and Dyer, and the DT of biomassis characterized mainly by unsaturated acids, whichwould make the biodiesel produced by these methodsmore susceptible to oxidation processes.

Unlike the other methods, oil extraction from Chlorella sp.using dichloromethane/methanol and Rose Gottlieb’s methodpresented a profile with similar levels of saturated and unsat-urated acids. However, biodiesel from the oil extracted bydichloromethane/methanol would have better physical prop-erties given the high level of C16:1 cis9.

EN 14214 [34] sets the upper limit for linolenic acidcontent at 12 % and that for fatty acids with more than threedouble bonds (PUFAs) at 1 %. In Brazil, however, ANPResolution No. 14/2012 does not specify limits for theseparameters [35].

The oils extracted from the microalga N. oculata by sixmethods and DT, thus, do not exceed maximum permittedlevels of C18:3. However, only extraction with hexane pro-duced PUFA levels below the maximum allowed.

For microalga C. muelleri, C18:3 content from the DTprocess and extracted oils was below the established 12 %maximum except for the oil from the adapted Rose Gottliebmethod, which had 15 % linolenic acid content. Importantly,most methodologies used with this microalga provided verylow C18:3 yields with zero levels of this unsaturated carbonchain reported in some cases. The methodologies with under-the-limit PUFAs in C. muelleri are hexane, hexane/ethanol,and direct biomass transesterification.

The microalga Chlorella sp. is mainly composed of unsat-urated fatty acids. The adapted Rose Gottlieb methodologyprovided oil with a 3.5 % linolenic acid content but 4.8 %PUFA content. Extraction with dichloromethane and metha-nol produced 1% of C18:3 and 1.1% of PUFAs, near the levelestablished by norm EN 14214. Other methodologies pro-duced C18:3 and PUFA yields above the establishedEuropean norm.

In this research assessing biodiesel potential, themicroalga C. muelleri was the most promising of thethree species of microalgae studied due to its higherester content from biomass and because its fatty acidcomposition is in accordance with specifications laiddown by norm EN 14214.

Conclusion

In this study, the choice of extraction method affected both theextract yield and the FAME composition of the microalgae inN. oculata, C. muelleri, and Chlorella sp. Fatty acid should beextracted from microalgae with a non-polar solvent such ashexane mixed with another solvent that can solubilize more-polar molecules such as ethanol. Thus, both neutral lipids andpolar lipids may be solubilized and converted into the fattyacid esters that constitute biodiesel.

Most of the microalgae displayed triunsaturate andpolyunsaturate levels above those established in internationallegislation. For this reason, the search for microalgae to serveas the fatty raw material for biodiesel production should becarried out in environments, in the case of plants, hot climates,where microalga metabolism generates less unsaturated fattyacids. Particularly for microalgae, tropical freshwater bodies,which are environments with little temperature variation andwarm temperatures, could become the best alternative in thesearch for species that can be used for biodiesel production.

According to their FA profiles and aiming at biodieselproduction, the microalga C. muelleri is the most promisingamong the three species of microalgae analyzed, by highercontent of esters from biomass, and due to the composition offatty acids that are in concordance with the specifications laiddown by the standard EN 14214.

Considering the benefits that the essential fatty acids (Aaand EPA) offer to human health, the lipids extraction withhexane in Chlorella sp. provides high quantities of these acidsand, therefore, can be considered as an alternative in obtainingthese acids. The methodology with dichloromethane andmethanol gave high content of EPA in N. oculata.

Acknowledgments The authors would like to express their apprecia-tion to the Ministry of Science Technology and Innovation (MCTI) forfinancial support provided through FINEP (Agreement No.01.10.0457.00) and CNPq (Case No. 574796/2008-8), to CAPES for ascholarship awarded to Aline Terra Soares, to CNPq for a researchproductivity scholarship awarded to Nelson Roberto Antoniosi Filho,(Case No. 309832/2010-1) and to FUNAPE for management of financialresources.

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