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Research Article

Response surface modeling and optimization of biodieselproduction from Cynara cardunculus oil

Inês Sengo1, Jorge Gominho2, Lourenco d’Orey3, Miguel Martins4, Elizabeth d’Almeida-Duarte4,Helena Pereira2 and Suzana Ferreira-Dias1

1 Instituto Superior de Agronomia, Centro de Estudos de Engenharia Rural, Technical University of Lisbon,Lisbon, Portugal

2 Instituto Superior de Agronomia, Centro de Estudos Florestais, Technical University of Lisbon, Lisbon, Por-tugal

3 Fundacão Maria Manuela e Vasco de Albuquerque d’Orey, Lisbon, Portugal4 Instituto Superior de Agronomia, Unidade de Investigacão de Química Ambiental, Technical University of

Lisbon, Lisbon, Portugal

Cardoon (Cynara cardunculus L.) is a perennial spontaneous thistle grown in Mediterranean countriesand well adapted to marginal lands, recently considered as a non-food energy crop. Their seeds contain24% of oil (dry basis). In this study, modeling and optimization of the production of fatty acid methylesters (FAME) from cardoon oil for biodiesel uses was performed at laboratory scale, via response surfacemethodology, following a central composite rotatable design. FAME were obtained by transesterificationof crude cardoon oil with methanol in the presence of a catalyst (sodium methoxide) for 120 min. Thetemperature ranged from 26 to 94 7C, the amount of sodium methoxide varied between 0.12 and 2.5 wt-%and the molar ratio methanol/oil from 0.95 : 1 to 11 : 1. The estimated yield of FAME (97%) was obtainedafter 30 min, at 52 7C, for a molar ratio of 6.4 : 1 and 1.4 wt-% of catalyst. In laboratory-scale modelvalidation experiments, 94% of FAME yield was obtained after 30 min of reaction. Transesterification wasperformed in a 30-L reactor, under previously optimized conditions: A yield of 88% FAME was obtainedafter 90 min of reaction time, due to mass transfer limitations. After purification, the biodiesel showed highquality according to DIN EN 14214 standard specifications.

Keywords: Biodiesel / Biofuels / Energy / Modeling / Vegetable oils

Received: June 11, 2009; accepted: September 14, 2009

DOI 10.1002/ejlt.200900135

310 Eur. J. Lipid Sci. Technol. 2010, 112, 310–320

1 Introduction

In the last years, the use of renewable energies has increaseddue to (i) environmental problems related with the use of fos-sil carbon fuels, together with (ii) increasing shortage in fossilenergy fuels and (iii) the political instability in producingcountries. In order to comply with the national and EUrenewable energy targets and international agreements (Kyotoand EU White Paper), Portugal is implementing the produc-tion and use of renewable energies. The energy market is an

emerging market in Portugal. In fact, the installation of newcrops for bioenergy production will promote the creation ofnew market opportunities. This will also lead to rural devel-opment, resulting from population fixation, with an increasein social well-being and local prosperity. Also, the sustain-ability of small- to medium-scale industries is expected.

Concerning biodiesel, Portugal meets with the EU target,incorporating 5% of biodiesel in diesel fuel. However, in Por-tugal, as in several other countries, the agro-based oil-bearingcrops are not self-sufficient for edible and non-edible pur-poses. Therefore, energy crops must be non-edible cropsadapted to marginal lands, in order not to compete with theedible crops. In tropical regions, non-edible oils such as castor(Ricinus communis L.), jatropha (Jatropha curcas L.) andBrassica carinata A. Braun oils have been successfully used forbiodiesel production [1–4].

Correspondence: Suzana Ferreira-Dias, Instituto Superior de Agrono-mia, Centro de Estudos de Engenharia Rural, Technical University of Lis-bon, Tapada da Ajuda, 1349-017 Lisbon, Portugal.E-mail: [email protected]: 1351 21 3653195

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Eur. J. Lipid Sci. Technol. 2010, 112, 310–320 Biodiesel production from Cynara cardunculus oil 311

In the Mediterranean countries, Cynara cardunculus L. oilmay be a good alternative to conventional edible oils for bio-diesel production. Cynara cardunculus L., a thistle currentlyknown as cardoon, belongs to the Asteraceae family (Composi-tae). It is a perennial and spontaneous plant that presents ahigh regenerative capacity, since its aerial part completelydries in summer, but in autumn, after the first rains, the plantsprouts with the appearance of the first leaves on the stump.Traditionally, their flowers have been used as coagulant agentfor cheese making. However, recent studies have shown thehigh potential of this plant as energy crop: The aerial biomassmay be used as a solid biofuel and the oil from the seeds maybe used for the production of biodiesel [5–10]. The costs of acardoon crop are lower than those required for other energycrops, since cardoon is a perennial crop (15–20 years of pro-ductivity) adapted to harsh soil and climate conditions [8, 9,11]. In 2005, Cynara was recognized by the European Com-mission as an energy crop for biomass and oil production (ECRegulation n71701/2005, October 18).

The direct use of vegetable oils as a fuel may cause ignitionand combustion problems as well as clogging of fuel lines andfilters. Therefore, the oils must be converted into their fattyacid methyl esters (FAME) by transesterification with metha-nol (also named alcoholysis) in order to improve the combus-tion characteristics and to be used as biodiesel. The yield andpurity of the obtained FAME (biodiesel) have been shown todepend on several reaction variables, namely the degree ofrefinement of the oil, the temperature, the molar ratio of alco-hol to vegetable oil, and the type and amount of catalyst used(acid or alkali). When alkali catalysts (e.g. sodium hydroxideor methoxide) are used, the reaction occurs at lower temper-ature and at higher rates and conversion degree than with acidcatalysts [12].

The alkali-catalyzed methylation of oils begins with theattack of triacylglycerol (TAG) molecules by the methoxideion, with the formation of one molecule of diacylglycerol(DAG) and one molecule of FAME. The same sequenceproceeds with DAG converted to monoacylglycerol (MAG)and MAG to FAME and free glycerol. One molecule ofFAME is produced in each step [13]. The reaction occurs in abiphasic system because the oil is almost immiscible withmethanol. Therefore, the reaction is mass transfer limited. Asthe reaction proceeds, the chemically bound glycerol content(in MAG, DAG and TAG) of the biodiesel decreases, but theacidity increases due to the reaction with hydroxide ionscoming from the water molecules in the reaction medium [13].Thus, the water content must be lower than 0.3% [14]. Inaddition, when alkali catalysts are used, the oils must have afree fatty acid (FFA) content lower than 1–2% [14]. SinceFFA will react with the catalyst to form soaps, high levels ofFFA cause a reduction in the amount of available catalyst andin FAME yield, as well as an increase in viscosity or the for-mation of gels which interfere with glycerol separation [14,15]. In fact, the direct use of crude oils as raw material inalcoholysis reactions has been limited due to the presence of

FFA, moisture and unsaponifiable matter which inactivatesodium catalysts [14, 15].

The aims of this study were (i) to model and optimize, viaresponse surface methodology (RSM), the production ofFAME from crude cardoon oil for biodiesel fuels, at labora-tory scale, (ii) to scale up the process to pilot scale, underoptimized conditions, and (iii) to purify and characterize theobtained FAME.

2 Materials and methods

2.1 Materials

The cardoon seeds used were produced in an installed field(about 90 ha) in the South of Portugal (Beja). Anhydrousmethanol was purchased from Merck; sodium methoxide(25% wt/vol) solution in methanol was from Aldrich, and thestandards of palmitic acid methyl ester, triolein, diolein(mixed isomers), monoolein and oleic acid from Sigma. Silicagel 60 (0.25 mm width, 20620 cm) thin-layer chromatogra-phy (TLC) plates were obtained from Macherey-Nagel. Allother chemicals were of analytical grade and obtained fromvarious sources.

2.2 Methods

2.2.1 Oil extraction

For the assessment of oil content, the seeds were ground in ahousehold knife mill (Moulinex) and the oil was extracted bysolvent (n-hexane) in a Soxhlet apparatus, for 8 h. Themoisture content of the seeds was determined by drying at100–105 7C, until constant mass, in an oven (WTB Binder7200).

For biodiesel production, the oil was mechanically ex-tracted in a screw press, Täby Press type 20 (Skeppsta MaskinAB, Sweden), equipped with an outlet nozzle of 9 mm inter-nal diameter. The oil was centrifuged to remove suspendedsolids and directly used in transesterification experiments.

2.2.2 Oil characterization

The acidity, i.e. the FFA content (%), was assayed by titrationwith a 0.1 N sodium hydroxide aqueous solution, using phe-nolphthalein as indicator. Iodine value (IV) and saponificationnumber (SN) were determined according to the Portuguesestandards, NP-941 (1972) and NP-940 (1972), respectively.These values were used to estimate the cetane number (CN),as follows [16]:

CN = 46.3 1 548/SN – 0.225 IV (1)

The CN is an indicator of the auto-ignition capacity of afuel.


312 I. Sengo et al. Eur. J. Lipid Sci. Technol. 2010, 112, 310–320

The fatty acid composition was determined by gas chro-matography, as FAME, following the methodology describedin Commission Regulation (EEC) n7 1989/03 of Novem-ber 6, 2003. A Finnigan TRACE GC Ultra gas chromato-graph (Thermo Electron Corporation), equipped with aThermo TR-FAME capillary column (30 m60.25 mmID60.25 mm film), an autosampler AS 3000 from ThermoElectron Corporation and a flame ionization detector, wasused for FAME analysis. The injector (in splitless mode) anddetector temperatures were set at 250 and 260 7C, respec-tively. Helium was used as carrier gas at a flow rate of1.5 mL/min. Air and hydrogen were supplied to the detectorat flow rates of 350 and 35 mL/min, respectively. The oventemperature program was as follows: 60 7C for 1 min, atemperature increase to 150 7C at 15 7C/min, a plateau at150 7C for 1 min, followed by a temperature increase to220 7C at a rate of 5 7C/min, and a final plateau at 220 7C for10 min.

2.2.3 Production of FAME in laboratory reactors

The transesterification reaction of Cynara oil with anhy-drous methanol was carried out in a 25-mL thermostatedcylindrical glass reactor, closed with a stopper, under mag-netic stirring and atmospheric pressure, using sodiummethoxide as catalyst. In each batch, 11 mL oil was used.The best reaction conditions were established via RSM [17,18]. RSM consists of a set of mathematical and statisticalmethods developed for modeling phenomena and findingcombinations of a number of experimental factors (vari-ables) that will lead to optimum responses. With RSM,several variables are tested simultaneously with a minimumnumber of trials, using special experimental designs thatenable to find interactions between the variables which can-not be identified with classical approaches. The transester-ification experiments were carried out following a centralcomposite rotatable design (CCRD), as a function of threefactors: temperature, amount of catalyst (sodium methox-ide), and molar ratio alcohol/oil. A total of 18 experimentswere carried out: 8 factorial points (coded levels as (11)and (–1)); 6 star points (coded as (1a ) and (–a)); 4 centerpoints (coded as 0) (Table 1). The reaction temperaturevaried from 26 to 94 7C, the amount of catalyst from 0.12 to2.5 wt-% (based on oil amount), and the molar ratio of

methanol/oil varied from 1 : 1 (deficit of methanol) to 11 : 1(large excess of methanol), according to the experimentaldesign followed (Table 1). The repetitions of the centerpoints are used to determine the experimental error, whichis assumed to be constant along the experimental domain.The experiments were performed randomly in order toavoid systematic errors.

For calculation purposes, the molecular weight of trilino-lein (879.38) was considered, since linoleic acid is the majorfatty acid (56–60%) of this oil [6, 8]. After modeling the reac-tion, an additional experiment was carried out under theoptimized conditions in order to validate the model.

2.2.4 Production of FAME in a pilot reactor

A handcraft pilot reactor, from the Fundacão MariaManuela e Vasco de Albuquerque d’Orey, Lisbon, Portugal,was used. The reactor consists of a 30-L stainless-steelcylindrical tank with conical bottom with a valve to collectsamples. Heating is achieved by a thermostatical heaterband. The reaction medium is pumped throughout a PVCexternal recirculating tubing to perform stirring. The trans-esterification was performed under the following, previouslyoptimized conditions (Section 2.2.3): 52 7C, molar ratio ofmethanol/oil of 6.5 : 1, and 1.4 wt-% sodium methoxide.Two different sets of experiments were performed in dupli-cate, as follows:

Experiments 1: The experiments were carried out asdescribed for laboratory-scale experiments. Samples weretaken during a 90-min reaction. After this time, the heatingand stirring were switched off; the reaction medium wasallowed to settle for 24 h at room temperature and the glyceroland FAME phases were removed separately.

Experiments 2: The reaction conditions were similar tothose described for experiments 1, except for the methanoladdition. At the beginning of the reaction, only 70% (vol/vol)of the total methanol was added. After 30 min of reactiontime, the stirring was switched off for 10 min to remove theproduced glycerol, and the remaining methanol was added.After 60 min, the same procedure was followed for glycerolremoval, and the reaction was allowed to pursue for 90 min.The remaining glycerol and the upper phase containingFAME were recovered after 24 h of settling at room tempera-ture.

Table 1. Coded and decoded levels of the experimental factors used in the CCRD.

Coded levels Temperature [7C] Methanol/oil molar ratio Catalyst [wt-%], based on oil mass

(21) 40 3 : 1 0.6(11) 80 9 : 1 2.0(2a) 26 1 : 1 0.12(1a) 94 11 : 1 2.50 60 6 : 1 1.3



2.2.5 FAME separation and characterization

After the conversion of cardoon oil into FAME, in the pilotreactor, the reaction mixture was allowed to stand for a pre-liminary phase separation. The upper phase contains theFAME and non-reacted acylglycerols while the lower phase isrich in glycerol. The excess of methanol and the catalyst weredistributed between the two phases. The residual methanol inthe organic phase was removed in a rotavapor, under reducedpressure at 40 7C. The recovery of methyl esters was achievedby several washings of the organic phase with water at 60 7C toremove both the catalyst and the hydrophilic compounds(glycerol and methanol), followed by phase separation bycentrifugation, until obtaining a clean product. The recoveredproduct was analyzed at the laboratory ASG Analytik-ServiceGesellschaft mbH, Germany, in order to investigate if it meetsthe requirements of the DIN EN 14214 standard.

2.2.6 Analytical methods

Reaction monitoring: During the time course of the reaction(30, 60 and 120 min), 0.5-mL samples were withdrawn forvials and added to equal volumes of distilled water to inacti-vate the catalyst and to stop the reaction [14, 15]. The mixturewas vigorously stirred and centrifuged in a Sigma 3K30 cen-trifuge (B. Braun Biotech International), at 12,000 rpm for10 min. The upper layer was diluted with n-hexane (1 : 10 vol/vol) and spotted on silica gel TLC plates. Elution was carriedout in petroleum ether/diethyl ether/acetic acid (85 : 15 : 1vol/vol/vol) as the mobile phase. Plates were sprayed with 0.2%(wt/vol) 2’,7’-dichlorofluorescein in 95% ethanol andobserved under UV light at 366 nm. The various groups ofcompounds (FAME, TAG, FFA, DAG and MAG) wereidentified by comparison with standards. The relativeamounts of every group of compounds were estimated bymeasuring the areas of the bands on the TLC plates with aLeica Microsystems image analyzer, and expressed as areapercentage.

2.2.7 Experimental design analysis

The results of the CCRD, concerning FAME production,DAG and TAG consumption were analyzed using the soft-ware Statistica™, version 5, from Statsoft (Tulsa, USA). Bothlinear and quadratic effects of each of the factors under study,as well as their interactions, on the transesterification kineticswere calculated. Their significance was evaluated by analysisof variance. A surface, described by a second-order poly-nomial equation, was fitted to each set of experimental datapoints. First- and second-order coefficients of the polynomialequations were generated by regression analysis.

The fit of the models was evaluated by the determinationcoefficients (R2) and adjusted R2 (Radj

2) [17, 18]. The R2

value provides a measure of how much of the variability in theobserved response values can be explained by the experi-

mental factors and their interactions. However, the R2 shouldbe used with caution since it will always increase with theinclusion of a new variable to the model. The use of Radj

2 ispreferred and is related with R2 by the following equation [19]:

R2adj ¼ 1� n� 1

n� pð1� R2Þ (2)

where n is the number of experiments and p is the number ofvariables (factors) in the model. The Radj

2 takes into accountthe fact that the number of residual degrees of freedom in thepolynomial regression changes as the order of the polynomialchanges. Radj

2 is an unbiased estimate of the coefficient ofdetermination and is always smaller than R2. In practice, R2

should be at least 0.75 or greater; values above 0.90 are con-sidered to be very good [18].

3 Results and discussion

3.1. Oil extraction and characterization

The cardoon seeds contained 24.2 wt-% of oil (dry basis,average of three determinations), as determined in the Soxhletapparatus, and 7.76% water (dry basis). The oil content iswithin the variation range reported previously [8]. For bio-diesel production, the oil was extracted in a screw press inorder to lower the extraction costs. A yield of 74% wasobtained in the mechanical extraction of oil.

The crude oil had an FFA content of 0.6%, an IV of 122and an SN of 200. The crude cardoon oil used presented thefollowing fatty acid composition: 0.11% 14:0 (myristic acid),0.02% 15:0, 11.21% 16:0 (palmitic acid), 0.16% 16:1 (palmi-toleic acid), 0.06% 17:0 (margaric acid), 0.02% 17:1 (mar-garoleic acid), 3.43% 18:0 (stearic acid), 22.40% 18:1 (oleicacid), 61.81% 18:2 (linoleic acid), 0.08% 18:3 (linolenic acid),0.33% 20:0 (arachidic acid), 0.12% 20:1 (gadoleic acid),0.10% 22:0 (behenic acid), and 0.15% 24:0 (lignoceric acid).Thus, it contains 84.6% of unsaturated fatty acids, mainlylinoleic and oleic acids.

Due to the low acidity value, the crude oil was directly usedfor biodiesel production with no further treatment.

3.2 Modeling and optimization of FAME production inlaboratory reactors

The alcoholysis of crude cardoon oil with methanol was car-ried out following a CCRD, as a function of the amount ofalkali catalyst (C), reaction temperature (T) and molar ratio ofmethanol/oil (MR). Samples were taken after 30, 60 and120 min of reaction. The amounts (in area percentage onTLC plates) of produced FAME, residual TAG and DAG,after 30 and 60 min of reaction time, are presented in Table 2.After 30 min of transesterification of crude cardoon oil withmethanol, the conversion to FAME varied from 21.5 to93.7%. Also, a quasi-equilibrium state was observed at this



time, since only small changes in relative amounts of com-pounds were detected thereafter. With respect to DAG, theyvaried from 0% to about 21 and 22%, after 30 or 60 min ofreaction time, respectively. The amounts of MAG observedon the TLC plates also varied from 0 to about 20% (data notshown). No FFA from the crude oil (0.6%) were observedsince they might have reacted with the catalyst (sodiummethoxide) with the formation of sodium soaps.

The effects of each factor and the interactions betweenfactors on the various compounds present in the reactionmedia during alcoholysis were calculated. Table 3 shows thelinear and quadratic effects of each variable and of theirinteractions on the amounts of FAME, TAG and DAG in thereaction medium, during the transesterification reaction. Nosignificant effects (p ..0.05) of interactions between thevariables on FAME, TAG and DAG levels were detected, aftereither 30 or 60 min of reaction time. MAG were shown not todepend on the reaction conditions (data not shown).

For the results obtained after either 30 or 60 min of reac-tion time, a negative significant effect of Ton FAME produc-tion indicates that higher T values, within the tested range,correspond to lower conversions into FAME. On the contrary,the increase of C and MR will promote FAME production(significant positive linear effects). The significant negativequadratic effects of T, C and MR on FAME production indi-cate that the experimental results of FAME production can befitted to a four-dimensional convex surface (Fig. 1). Thisfour-dimensional surface presents a stationary point (opti-mum) inside the technological space, which is illustrated bytwo three-dimensional surfaces, where only two of the three

initial factors vary while keeping the remaining factor constantand equal to the value assumed in the central point.

Concerning TAG and DAG levels, significant linear posi-tive effects of Tand negative effects of C and MR were found,in contrast to the observations for FAME production. Also,the significant quadratic positive effects of T, C and MR indi-cate that TAG and DAG can both be described by four-dimensional concave surfaces (Figs. 2, 3). In fact, the synthe-sis of FAME is performed by the consumption of TAG, DAGand MAG. The lowest levels of DAG corresponded to thehighest levels of FAME produced. This has also beenobserved previously by others [14, 20–22].

As reported by Mahajan et al. [13], the oil that diffuses intothe methanol phase is rapidly converted into FAME, via theDAG and MAG pathway. These partial acylglycerols will actas emulsifiers and, for a short time, an emulsion may beformed, facilitating the contact between the reagents. How-ever, the formation of a separated glycerol-rich phase willrapidly dominate. The catalyst will be partly or completelyremoved by this phase, leading to a decrease of the reactionrate, or even to the reaction stop.

A decrease in FAME production with temperature hasalso been reported [13, 20–22]. This may be explained by afaster rate of soap formation than of FAME formation, athigher temperatures.

The response surfaces (Figs. 1–3) fitted to FAME, TAGand DAG amounts can be described by second-order poly-nomial models as a function of the reaction temperature, themolar ratio methanol/oil, and the amount of catalyst (Table 4).In these response surface models, the significant effects

Table 2. CCRD followed in the experiments as a function of reaction temperature, catalyst concentration and molar ratio of methanol/oilused, and respective values of FAME (area %) produced after 30 and 60 min, and residual TAG (area %) and DAG (area %).

ExperimentN7

Temperature[7C]

Catalyst [wt-%] Methanol/oil FAME, 30 min [%] FAME, 60 min [%] TAG, 30 min [%] TAG, 60 min [%] DAG, 30 min [%] DAG, 60 min [%]

1 40 0.60 3 : 1 37.00 36.00 51.56 50.34 8.15 9.162 40 0.60 9 : 1 40.00 39.00 49.00 52.00 7.00 8.003 80 0.60 3 : 1 21.46 21.06 52.37 50.49 21.06 21.964 80 0.60 9 : 1 27.95 27.26 48.94 54.21 16.81 15.045 40 2.00 3 : 1 50.10 60.38 35.83 32.20 14.07 7.426 40 2.00 9 : 1 67.55 54.67 25.70 38.80 6.74 6.527 80 2.00 3 : 1 33.20 34.66 43.04 39.57 17.20 20.068 80 2.00 9 : 1 34.55 33.20 46.06 45.21 14.39 16.359 60 0.12 6 : 1 44.38 43.21 38.44 44.28 13.79 12.5110 60 2.50 6 : 1 49.57 48.46 44.89 44.02 5.54 7.5211 26 1.30 6 : 1 87.01 84.00 0.00 0.00 8.81 8.1712 94 1.30 6 : 1 42.28 37.73 40.63 44.60 17.09 17.6713 60 1.30 1 : 1 31.09 33.30 40.69 37.31 19.75 19.1414 60 1.30 11 : 1 52.48 89.23 41.85 10.77 5.67 0.0015 60 1.30 6 : 1 93.41 92.45 0.00 0.00 0.00 0.0016 60 1.30 6 : 1 91.68 92.41 0.00 0.00 0.00 0.0017 60 1.30 6 : 1 93.72 93.47 0.00 0.00 0.00 0.0018 60 1.30 6 : 1 92.54 93.11 0.00 0.00 0.00 0.00



Table 3. Linear (L) and quadratic (Q) effects and respective significance levels (p) of the tested variables [factors: temperature (T), con-centration of catalyst (C) and molar ratio methanol/oil (MR)] and interactions on FAME yield, residual TAG and DAG after 30 and 60 min oftransesterification of crude cardoon oil with methanol.

Factor FAME (30 min) TAG (30 min) DAG (30 min) FAME (60 min) TAG (60 min) DAG (60 min)

Effect p Value Effect p Value Effect p Value Effect p Value Effect p Value Effect p Value

T (L) –22.36 0.0010a 14.15 0.0231a 6.94 0.0011a –22.21 0.0270a 13.35 0.1063b 8.53 0.0015a

T (Q) –24.14 0.0008a 17.67 0.0098a 9.46 0.0002a –29.36 0.0089a 21.59 0.0221a 9.82 0.0008a

C (L) 9.90 0.0557b – 5.90 0.2759 –2.12 0.1692b 10.00 0.2581b – 7.56 0.3326 – 1.79 0.3492b

C (Q) –36.45 0.0000a 32.61 0.0002a 7.11 0.0012a –39.78 0.0016a 36.85 0.0013a 7.73 0.0032a

MR (L) 9.40 0.0665b – 1.63 0.7548 –5.74 0.0035a 14.08 0.1252b – 3.96 0.6040 – 6.57 0.0064a

MR (Q) –40.23 0.0000a 32.43 0.0003a 9.28 0.0002a –29.02 0.0094a 22.77 0.0173a 7.44 0.0040a

T6C – 5.57 0.3638 6.71 0.3393 –2.99 0.1428b – 5.13 0.6459 2.85 0.7735 0.66 0.7874T6MR – 3.15 0.6007 3.07 0.6543 0.36 0.8508 1.86 0.8666 0.27 0.9779 – 2.14 0.3883C6MR 2.33 0.6979 – 0.27 0.9679 –1.19 0.5359 – 4.09 0.7131 1.72 0.8621 0.87 0.7209

a Significant effects with p �0.05.b Effects with p .0.05 considered in the model.

Figure 1. Response surface fitted to the percentage of FAME produced after 30 min of transesterification of Cynara oil, as a function of(i) catalyst load and reaction temperature and (ii) of the molar ratio of methanol/oil and temperature.

(p ,0.05) and those having a confidence range smaller thanthe value of the effect, or smaller than the standard deviation(data not shown), were included in the model equations ofthese surfaces. It is better to accept factors with p values higherthan 0.05 rather than to take the chance of missing an impor-tant factor [18]. The high values of both R2 and Radj

2 of thesemodels (Table 4) show a close agreement between the experi-mental results and the theoretical values predicted by themodels. Partial differentiation of the polynomial equations forFAME production after 30 and 60 min of reaction time was

used to find the maximum points. The best reaction condi-tions found for 30 or 60 min of transesterification were similarand as follows: reaction temperatures of 51 7C, 1.4 wt-% ofcatalyst, and a large excess of methanol (MR = 6.4 : 1). Underthese conditions, it is expected to obtain 97% FAME after30 min of reaction time. The same conversion value was esti-mated for 60 min of reaction time, which confirms the quasi-equilibrium situation attained after 30 min.

An experiment was carried out under the predicted opti-mum conditions and samples were taken after 15, 30 and



Figure 2. Response surface fitted to the percentage of residual TAG in the reaction medium, after 30 min of transesterification of Cynaraoil, as a function of (i) catalyst load and reaction temperature and (ii) of the molar ratio of methanol/oil and temperature.

Figure 3. Response surface fitted to the percentage of DAG in the reaction medium, after 30 min of transesterification of Cynara oil, as afunction of (i) catalyst load and reaction temperature and (ii) of the molar ratio of methanol/oil and temperature.

60 min of reaction time (Fig. 4). The obtained results validatethe model and show that the reaction equilibrium is attained atleast after 15 min of reaction time.

A quasi-equilibrium situation after 10 min of transester-ification of Cynara oil with methanol has also been reported

[6]. Also, over 80% conversion of refined sunflower oil intoFAME was observed after 5 min of reaction time, catalyzed bysodium hydroxide [23].

RSM was previously used by us for optimization of thereaction conditions for FAME production, catalyzed by



Table 4. Model equations for the response surfaces fitted to the values (%) of FAME, TAG and DAG, after 30 and 60 min of reaction time,as a function of reaction temperature (T), concentration of catalyst (C), and molar ratio of methanol/oil (MR), and respective R2 and Radj

2.

Polynomial model equation R2 Radj2

FAME30 min = –143.76 1 3.063T – 0.03T2 1 103.803C – 37.203C2 1 28.39MR – 2.235MR2 0.943 0.912TAG30 min = 33.85 – 0.632T 1 0.0082T2 1 0.237C2 1 0.059MR2 0.872 0.833DAG30 min = 62.35 – 1.108T 1 0.012T2 – 13.97C 1 7.25C2 – 7.144MR 1 0.516MR2 – 0.1066T*C 0.935 0.889FAME60 min = –155.22 1 3.849T – 0.037T2 1 112.686C – 40.593C2 1 21.692MR – 1.612MR2 0.839 0.753TAG60 min = 56.15 – 1.359T 1 0.014T2 1 0.217C2 – 0.0041MR2 0.778 0.710DAG60 min = 67.79 – 1.26T 1 0.012T2 – 21.796C 1 7.892C2 – 6.057MR 1 0.413MR2 0.898 0.843

Figure 4. Time course of transesterification of Cynara oil, at labo-ratory scale, under previously optimized reaction conditions (52 7C,molar ratio of 6.4 : 1 and 1.4% of catalyst).

sodium methoxide, from the following oils: crude rapeseed oil,crude and refined corn oils, refined and previously neutralizedcrude olive residue oils [20-22, 24]. As in the present study,ester production was fitted to second-order polynomial mod-els represented by convex surfaces, with stationary points(optima) inside the experimental domain, for all the oils test-ed. When rapeseed and corn oils were used, the production ofFAME was significantly affected by the amount of catalystand temperature and not so much by the molar ratio of meth-anol/oil used. According to the models, the predicted opti-mum reaction conditions were (i) 58 7C and 1.4 wt-% of cat-alyst for crude rapeseed oil [20] and (ii) 50 7C, 1.7 or 2.0 wt-% of sodium methoxide when crude and refined corn oils wereused, respectively [21]. Concerning the alcoholysis of refinedolive residue oil, the following conditions were found to max-imize FAME production: a catalyst concentration of 1.6 wt-%,a reaction temperature of 55 7C, and a molar ratio of metha-nol/oil equal to 7 : 1 [24]. When crude olive residue oil wasneutralized prior to use for transesterification with methanol,a total conversion into FAME was predicted by the modelwhen the following conditions were used: 1.9% of catalyst,68 7C and a molar ratio of methanol/oil of 9.4 : 1 [22].

For the production of biodiesel from waste frying oilsusing sodium hydroxide as catalyst, an increase in FAMEyield was observed with an increase in the amount of catalyst

up to 2 wt-%. A further increase of the catalyst concentrationled to a decrease in FAME yields due to the formation of soaps[25]. Also, the use of high amounts of catalyst in the produc-tion of biodiesel from Cynara oil led to the formation of anemulsion with increased viscosity and gel formation [6]. Thebiodiesel produced from cardoon oil was obtained at 60 7C,the highest temperature value tested by Encinar et al. [6],when 1% sodium methoxide and a molar ratio of methanol/oilof about 5 : 1 were used. Soap formation was also promoted attemperatures higher than 60 7C and catalyst (sodium hydrox-ide) loads higher than 1.5% in the methanolysis of sunfloweroil [23]. As determined by RSM, optimum conditions forsunflower oil FAME production were found to be mild tem-peratures (20–50 7C) and a high catalyst concentration (1.3%)[23]. Using RSM, a quadratic polynomial equation was alsofitted to the conversion of several vegetable oils into FAME, at65 7C, where the catalyst concentration was found to be themost effective variable in the reaction [26]. The best resultswere achieved with 1.3% catalyst after 2 h of reaction time.

Concerning the effect of the molar ratio of methanol to oilon the yield of FAME, different optimum values have beenreported. Molar ratios higher than the stoichiometric value(3 mol methanol to 1 mol oil) are beneficial to improve oilconversion to FAME.

According to Sonntag [12], the amount of methanol usedmay be just 1.2 times the stoichiometric amount (3 molmethanol to 1 mol oil), since an excess of alcohol is beneficialto promote FAME formation. However, when this molar ratiowas used in the transesterification of crude rapeseed oil with alow FFA content (0.4%) catalyzed by sodium methoxide, anincomplete conversion to FAME was observed [20]. Yields inFAME higher than 95% were obtained with molar ratios ofmethanol to oil equal to 6 : 1 [14, 20, 27] or higher [22, 24,28]. In fact, the excess methanol has been shown to promotethe forward reaction. However, very large excesses of alcoholmay not increase the ester yield and make the recovery ofFAME and glycerol more difficult and costly [14].

3.3 Production of FAME in a pilot reactor

Two sets of experiments were carried out at pilot scale underthe previously optimized reaction conditions. In the experi-



ments 2 (2a and 2b), 70% (vol/vol) of the total methanol wasadded at the beginning of the reaction, while the remaining30% (vol/vol) was added after 30 min of reaction time and theglycerol was removed after 30 and 60 min of reaction (Section2.2.4). The obtained results are presented in Fig. 5. After90 min of reaction time, 80 and 88% of FAME were obtainedin experiments 1 and 2, respectively. These results indicatethat the fractionated addition of methanol with glycerol

Figure 5. Time course of transesterification of Cynara oil, at pilotscale, under previously optimized reaction conditions (52 7C, molarratio of 6.4 : 1 and 1.4% of catalyst), with the addition of all theamount of methanol at the beginning of the experiment(Exp. 1a, 1b) or fractionated addition of methanol (Exp. 2a, 2b).

removal after 30 min of reaction will increase the amount ofFAME. However, in these pilot reactor experiments, the con-version values observed at laboratory scale were not reached(73% FAME at 30 min against 97% FAME at laboratoryscale). This may be explained by mass transfer limitationsbetween the hydrophilic and the hydrophobic phases, due toan insufficient mixing since the stirring was performed byexternal circulation of the reaction medium.

3.4 FAME separation and characterization

The FAME produced in the pilot reactor were purified aspreviously described (Section 2.2.5) and analyzed accordingto the DIN EN 14214 standard. The results of the analyticalparameters for cardoon biodiesel are presented in Table 5.Except for the IV, the values for all the parameters meet thestandard. The IV of 126 is only slightly higher than the max-imum of 120 allowed by the EU. This fact may be overcomeby blending cardoon biodiesel with a biodiesel of lower IV.Since several biodiesel feedstocks present IV higher than thelegislated limits, the transesterification of blends of differenttypes of vegetable oils in order to achieve the desired proper-ties of the fuel is a current industrial practice. For example, theIV of some oils are: 35–61 for palm oil, 94–120 for rapeseedoil, 117–143 for soybean oil, 110–143 for normal sunfloweroil, and 126–152 for safflower oil [29]. Methanol, free glyc-erol, MAG, DAG and TAG contents were considerably

Table 5. Analytical parameters of Cynara biodiesel.

Parameter Method Result Specification DIN EN 14214 Unit

Min. Max.

Ester content DIN EN 14103 .99 96.5 – wt-%Viscosity at 40 7C DIN EN ISO 3104 4.216 3.5 5.0 mm2/sDensity at 15 7C DIN EN ISO 12185 884.5 860 900 kg/m3

Flash point DIN EN ISO 3679 178 120 – 7CAcid value DIN EN 14104 0.239 – 0.5 mg KOH/gIodine value DIN EN 14111 126 – 120 g I/100gCetane number IP 498 51.6 51.0 – –Sufated ash ISO 3987 ,0.001 – 0.02 wt-%Total contamination DIN EN 12662 1 – 24 mg/kgCopper strip corrosion DIN EN ISO 2160 1 1 corrosion degreeLinolenic acid methyl ester DIN EN 14103 0.1 – 12.0 wt-%Methanol content DIN EN 14110 ,0.01 – 0.20 wt-%Free glycerol ,0.01 – 0.020 wt-%MAG content 0.25 – 0.80 wt-%DAG content DIN EN 14105 0.04 – 0.20 wt-%TAG content ,0.01 – 0.20 wt-%Total glycerol 0.07 – 0.25 wt-%Sulfur content DIN EN ISO 20884 2.1 – 10.0 mg/kgCarbon residue (10%) DIN EN ISO 10370 0.01 – 0.3 wt-%Phosphorus content DIN EN 14107 ,0.5 – 10.0 mg/kgMetals I (Na 1 K) DIN EN 14108/109 ,0.5 – 5.0 mg/kgMetals II (Ca 1 Mg) DIN EN 14538 ,0.5 – 5.0 mg/kg



lower than the maximum allowed values, showing that down-stream processing and purification of the produced Cynarabiodiesel was successful. It is worthy to notice the very lowlevels of sulfur, sulfated ashes, carbon residue and metals (Iand II), indicating that cardoon biodiesel is a high-gradequality biodiesel with low environmental impact.

Acknowledgments

The authors are grateful to Isabel Baptista and Ana Lourenco forhelping with laboratory biodiesel purification and to Dr. TeresaQuilhó for the advice on image analysis. This work was supportedby the project “070-Energy Crops: Possibilities of Large ScaleImplementation in the Atlantic Space”, Program INTERREGIII B “Atlantic Space”.

Conflict of interest statement

The authors have declared no conflict of interest.

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