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Research ArticleReceived: 16 January 2011 Revised: 12 April 2011 Accepted: 12 April 2011 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.2652

Microalgae biodiesel via in situ methanolysisR.M. Carvalho Junior,a• J.V.C. Vargas,a∗ L.P. Ramos,b C.E.B. MarinoaTS1

and J.C.L. Torrensc

Abstract

BACKGROUND: Microalgae have recently been considered a potential feedstock for biodiesel production, since they do notcompete with agricultural land, unlike oil crops. However, the production processes must be energetically and economicallyviable. Therefore, an in situ methanolysis process is proposed for biodiesel production directly from microalgae biomass, toavoid the need for the separation and extraction steps.

RESULTS: Biodiesel was obtained using methanol as the methylation reactant for the transesterification reaction andhydrochloric acid as the catalyst precursor, at 80 ◦C for 2 h of reaction. A mass return of 23.07 ± 2.76% (m/m) was obtained.Spectrometry in the infrared region showed that the product had equivalent bands of axial deformation of C O, C–O and C–H,i.e. an ester. Tests showed the chromatographic profile of fatty acids in the sample. A process energetic efficiency value of 1.17was obtained for microalgae derived biodiesel, which is higher than from soybean and sunflower, reportedly 1.06 and 1.12.

CONCLUSIONS: Industrial sustainability results from low energetic, economic and environmental losses. The microalgae in situmethanolysis process showed greater fuel available energy than energy consumption, therefore is energetically sustainable.Economic and environmental issues should still be addressed.c© 2011 Society of Chemical Industry

Keywords: biodiesel; microalgae; in situ methanolysis

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NOTATIONAEI peak area corresponding to methyl heptade-

canoateC gas chromatography mass spectrometer peak

percentage, Equation (2)CEI concentration, in milligrams per milliliter, of the

methyl heptadecanoate solutionCN cetane number of the biodiesel, Equation (5)CNME cetane number of individual methyl estersCFPP cold filter plugging point, Equation (6)DU degree of unsaturation, Equation (3)Ebiogas energy content of biogasEoil energy content of algal oilE energy (kJ)hv required specific enthalpy change to vaporize water

produced during fuel combustion (kJ kg−1)HHV higher heating value (kJ mol−1), Equation (10)I current (A)IV Iodine value [gIodine (100 g)−1

sample], Equation (8)LCSF long chain satured factor, Equation (4)LHV lower heating value (kJ mol−1), Equation (12)m mass (g)M molecular weight (g mol−1)n number of molsN total number of compoundsPoil surface productivity of the oil (kg ha−1 year−1)Pbiomass surface productivity of the dead microalgae

(kg ha−1 year−1)Pbiogas surface productivity of the biogas (m3 ha−1 year−1)

qh sensible specific enthalpy change to heat water upto the saturation point (kJ kg−1)

qv water specific enthalpy of vaporization (kJ kg−1)SV saponification value (mgKOH g−1

sample), Equation (9)t reaction time (h)U internal energy (kJ)V voltage (V)VEI volume, in milliliters, of the methyl heptadecanoate

solutionXME weight percentage of each methyl esterYB mass yield of monoesters (%)YE total annual energy yield (GJ ha−1 year−1)Yn net energy yield (GJ ha−1 year−1)

Greek letters�t duration of process stage (s)

∗ Correspondence to: J.V.C. Vargas, Setor de Tecnologia, Programa de PosGraduacao em Engenharia em Ciencia dos Materiais, Universidade Federaldo Parana, CP 19011, Curitiba, PR, 81531-990, Brazil.E-mail: jvargas@demec.ufpr.br

a Setor de Tecnologia, Programa de Pos Graduacao em Engenharia em Cienciados Materiais, Universidade Federal do Parana, CP 19011, Curitiba, PR, 81531-990, Brazil

b Departamento de Quımica, Universidade Federal do Parana, CP 19011,Curitiba, PR, 81531-990, Brazil

c Nucleo de Pesquisa e Desenvolvimento de Energia Auto Sustentavel,Universidade Federal do Parana, CP 19011, Curitiba, PR, 81531-990, Brazil

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η energetic efficiency, Equation (7)∑A total peak area from the methyl ester in C14 to that

in C24 : 1

SubscriptsB biodiesel monoesters obtained by the reaction in

the in situ methanolysisDBB dry microalgae biomass to biodieselH2O waters sampleT total consumption during the reaction steps

INTRODUCTIONAccording to the Intergovernmental Panel on Climate Change(IPCC) data, the CO2 concentration in the atmosphere hasincreased from 280 ppm to 379 ppm.1 To ease the buildupof these gases, photosynthetic organisms can play a majorrole in carbon dynamics. The photosynthesis performed bychlorophyllous organisms converts solar energy into chemicalenergy, with subsequent formation of organic matter (biomass)and release of oxygen through the metabolism of water andcarbon dioxide removed from the medium2, as follows:

6CO2(g) + 6H2O(l) + light energy → C6H12O6(l) + 6O2(g) (1)

It is recognized by the scientific community that forestryprojects are able to mitigate the greenhouse effect by fixatingthe carbon in plant biomass3 as shown in Table 1, which isfollowed by other benefits such as rehabilitation of degradedareas and biodiversity conservation. However, when tilling the soilfor planting, large amounts of CO2 are released to the atmosphere,which might cancel the beneficial effect of CO2 absorption andits incorporation into the biomass of plants throughout their life.4

Thus, the investigation of other photosynthetic organisms with thecapacity to absorb CO2 from the atmosphere may help to minimizethe effects of reforestation, with regard to carbon sequestration.Microalgae are one of these organisms.

Microalgae are mainly responsible for the absorption ofatmospheric CO2 in the oceans.5 As Fig. 1 illustrates, microalgae

Table 1. Forest carbon sequestration3

Species

Productivitycarbon (ton ha−1

year−1)

CO2 equivalent(ton ha−1

year−1)

Swietenia macrophylla King 5.60 20.55

Hymenaea courbaril L. 5.60 20.55

Anadenanthera colubrina 8.60 29.36

Cedrela fissilis Vell. 5.60 20.55

Pinus taeda 10.34 37.95

Havea sp. 5.17 18.97

Acacia mangium 10.55 38.72

Apuleia leiocarpa 12 44.04

Inga sp. 8.00 29.36

have the capacity to absorb up to 15 times more CO2 thanthe rainforests. A portion of the CO2 absorbed by microalgae istransferred to the ocean floor in a process known as a ‘biologicalpump’.6 Thus, microalgae cultivation for carbon sequestrationcould help to reduce the buildup of greenhouse gases in theatmosphere.

In addition to the potential biotechnological applications,7 – 19

studies show that biofuels obtained from microalgae can be usedas an alternative to fossil fuels.20 – 24 The main advantages ofmicroalgae with respect to higher plants are greater biomassproduction per area, possibility of using industrial waste incultivation, greater capacity, and easier CO2 biofixation tosynthesize lipids. Thus microalgae with 30% lipids by dry weightcan produce 58 700 L ha−1 of oil in raceway ponds, and isconsidered a potential source for biodiesel production, with betterproductivity than conventional crops such as corn (172 L ha−1),soybeans (446 L ha−1), canola (1,190 L ha−1) and palm (5,950 Lha−1)20.

Microalgae biomass is considered one of the most promisingfeedstocks for biodiesel, with the potential to meet the goal ofreplacing diesel oil. Chisti20 reported that the use of microalgaefor biodiesel production of 50% biodiesel for transportation in the

CO

2 (t

on h

a-1 y

ear-1

)

Species

Figure 1. Comparison of carbon sequestration by microalgae forests.3

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USA would require the use of 2.5% of the agricultural territory ofthe country, or about 10 times less than the land required for palmoil production for the same use.

The production of biodiesel from microalgae oil has beenreported in the literature using the conventional route,21,22

which involves the extraction of lipids from the microalgaebiomass, followed by its conversion into biodiesel and glycerol.Thus experiments were conducted with three types of solventfor extracting lipids from microalgae (l-butanol, ethanol, and2-propanol) The most efficient solvent for extraction was l-butanol, followed by 2-propanol and then ethanol. Regardingthe yield of methyl esters of fatty acids, these were obtainedby transesterification using 0.6 mol L−1 of hydrochloric acidin methanol for 1 h at 70 ◦C, reaching 68% conversion ofmonoalkylated ester.21

Aiming to increase biofuel production, an integrated methodwas studied to produce biodiesel from microalgae oil usingChlorella protothecoides, with the lipids extracted by n-hexaneas in a soxhlet extractor. The best yield of methyl esters derivedfrom fatty acid were obtained with 56 : 1 molar ratio (alcohol : oil),temperature 30 ◦C and reaction time 4 h.22

Other authors have also achieved success with such heteroge-neous production systems. Umdu et al.23 assessed the effect ofMgO and CaO supported on Al2O3 as a catalyst for the transesteri-fication of oil from microalgae, obtaining 97.5% conversion, usinga molar ratio of 30 : 1 (alcohol : oil) at 50 ◦C and 4 h reaction, using2% catalyst (relative to the mass of oil) CaO supported on Al2O3.

More recently an in situ reaction for biodiesel production frommicroalgae was studied. According to Ehimen et al.24 the bestresults were found when 60 mL methanol was used with 15 gof microalgae biomass, at a temperature of 60 ◦C, reaching 92%conversion with 1 h reaction time and constant stirring.

Several studies have demonstrated at laboratory scale thepossibility of producing biodiesel from microalgae. However, theindustrial engineering aspects, equipment and processes are yetto be properly addressed. Scaling up of microalgae biodieselproduction results in thermodynamic losses that drastically affectthe plant energy balance. From the operational point of view,economic or environmental losses are detrimental to the entireproduction chain.

In this context, the present work focuses on the productionof biodiesel from microalgae by in situ methanolysis, in orderto evaluate the process energetic efficiency at laboratory scale,but with the objective of assessing the potential processthermodynamic losses in future industrial scale installations.

MATERIALS AND METHODSThe experiments used the microalgae Nannochloropsis oculata,which was cultivated by the Group of Integrated Aquacultureand Environmental Studies at the Federal University of Parana(UFPR – GIA). The microalgae were cultivated in an autotrophicculture with a Guillard F/2 medium,25 and natural photoperiod.After growth was stopped, the microalgae were flocculated anddried using the ‘spray drying’ method.

Production of fatty acid methyl esters (FAME)The synthesis of microalgae fatty acid monoesters was initiallycarried out with 2 g of microalgae biomass in a Soxhlet apparatus,using 204 mL of a methanol : HCl : chloroform reactant solventin a 10 : 1:1 volumetric ratio. The reaction was carried out at

Monoesters

Reaction co-products

Microalgae

Figure 2. Products stratification after the centrifugation stage of in situ

Col

orFi

gure

-Onl

ine

only

methanolysis.

80 ◦C for 2 h under constant magnetic stirring. Once the reactionprocedure was completed, the system was cooled down to roomtemperature and 2 mL of water (HPLC grade) were added tothe reaction media, to retain polar substances and to help theseparation of phases ester-gliceride, along with 200 mL of a 4 : 1(v/v) hexane : chloroform mixture in order to keep the estersin organic phase. The addition of such solvents is because cellbreaking is expected before lipid extraction,26 i.e. the contact withnonpolar solvents could weaken the association between lipidsand cell structure. Although the use of these chemicals mightlimit interest in in situ methanolysis, an investigation of possiblesignificant gains in the overall energy balance of the procedurecompared with conventional methanolysis could compensate forthis.

After thorough mixing, the resulting material was centrifugedat 14 000 rpm for 10 min and the organic phase transferredto a round bottom flask. The resulting microalgae fatty esterswere recovered after evaporation of the extraction solvents.Reaction co-products and the residual microalgal biomass werediscarded as shown in Fig. 2. In general, microalgae co-productsare composed of proteins, pigments (primarily chlorophyll) andcarbohydrates.27 Such compounds were not characterized in theresidual microalgae biomass because this was not part of thepresent research.

A parametric study of the reaction conditions was also carriedout using a 22 central composite design in which the two mainreaction variables were involved, time and methanol : HCl ratio.The maximum and minimum levels (−1; +1) for time were 2 and4 h, and for the methanol : HCl ratio, these levels were 2 : 1 and 20 : 1(v/v), respectively. To check the experimental error and performthe analysis of variance (ANOVA), a center point (3 h and a 6 : 1methanol : HCl volumetric ratio) was carried out in three replicates.The experimental data were treated computationally to obtain theresponse surface and the primary and secondary effects of theselected reaction variables, having the alkyl monoesters massyield (YB) as the response factor.

Chemical characterization of the productFourier transform infrared spectroscopy (FTIR)FTIR analyses were performed with an FTIR spectrometer man-ufactured by Bomem. The spectra were obtained in the range

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4000–400 cm−1 with resolution of 4 cm−1 and 64 scans perspectrum. For the analysis, approximately 20 mL of the sam-ple, supported by chips of 100 mg KBr, were used. These analysesdefined the main structural characteristics of the samples.

Technique for gas chromatography/mass spectrometry (GC-MS)A gas chromatograph Shimadzu model 17A GC with split/splitlessinjector, equipped with a capillary column filled with CarbowaxPolyethylene Glycol – 30 m (length) × 0.25 mm (internal diameter)× 0.25 µm (film thickness), was employed. The injector operated insplit mode with a rate of 50 mL min−1, with block temperature of250 ◦C. The oven was programmed to work in the isothermal modeat 210 ◦C. The temperature of the detector (mass spectrometric)was 250 ◦C and carrier gas He was maintained at a constantflow of 1 mL min−1. The identification process followed after theseparation by high resolution gas chromatography using a massspectrometer Shimadzu model GCMS-QP 5050, where the massspectrum was compared with data from the internal library ofthe National Institute of Standards and Technology – NIST, whichwere available in the equipment data bank. This made possiblethe assessment of the fatty profile of the sample obtained inthe synthesis. The quantification of the sample followed DIN EN14 013, using the internal standard methyl heptadecanoate andthe following equation to identify the corresponding percentageof each peak:

C =(∑

A)

− AEI

AEI× CEI × VEI

ms × 10−3 × 100% (2)

Properties of the fuel monoesters obtainedBiodiesel fuel properties depend on the fuel’s fatty acid esterprofile, which is determined by the composition of the parent oiland the techniques employed for extraction and production ofthe oil. Thus, analyzing the composition of the esters obtainedin the in situ transesterification it is possible to assess some ofthe properties needed in order to characterize it as biodiesel(according to current norms). Recent studies show that the amountof saturated, monounsaturated and polyunsaturated esters that abiodiesel presents may be used in order to estimate its properties.

In the search for a simple methodology to predict the cetanenumber (CN) and cold filter plugging point (CFPP) of differentbiodiesel fuels, Ramos et al.28 used two empirical parameters.The first, the degree of unsaturation (DU), takes into accountthe amount of monounsaturated and polyunsaturated fatty acidesters, while the second, the long chain saturated factor (LCSF),takes into account the composition of saturated fatty acid esters,putting more weight on the long chain esters. Both parameters,DU and LCSF, are defined as follows:

DU = (monounsaturated, wt %) + 2(polyunsaturated, wt%)

(3)

LCSF = 0.1 × C16(wt%) + 0.5 × C18(wt%) + C20(wt%)

+ 1.5 × C22(wt%) + 2 × C24(wt%) (4)

The cetane number (CN) is a widely used parameter for dieseland biodiesel quality, once it is related to the ignition delay,combustion quality and pollutant emissions.29 The experimentalprocedure required to determine the cetane number is expensiveand impractical, which motivated the development of variousprediction methods, e.g. Freedman and Bagby30 correlated the

CN of methyl esters with different physical properties. The greaterpotential of the prediction methods, however, is to correlate theCN directly to the composition of the parent oil. For that, thefollowing correlation, formulated by Clements,31 was used in thiswork for the estimation of the biodiesel cetane number as aweighed average of all methyl ester cetane numbers present inthe fuel, as follows:

CN =N∑

i=1

(XME × CNME)i (5)

where N is the total number of compounds found in the biodiesel.Based on the linear regression of experimentally obtained data

for CFPP (R2 = 0.996), Ramos et al.28 proposed the followingequation:

CFPP = 3.1417 × LCSF − 16.477 (6)

Calculation of energetic efficiencyThe thermodynamic (or energetic) efficiency of the process isdefined as the ratio between the energetic content of thegenerated product and the energy consumed in the biodieselproduction processes. Through the calculation of such a quantity,it is possible to quantify the efficiency of obtaining biodiesel fromdry microalgae biomass by in situ methanolysis, as follows:

ηDBB = nB × LHV

ET(7)

The efficiency calculation starts with estimation of the iodinevalue, saponification number, higher and lower heating values.Based on the fatty ester profile obtained, it is possible to estimatethe iodine value, according to the AOCS method Cd 1c-8532 asfollows:

IV = (%C16 : 1 × 0.9976) + (%C18 : 1 × 0.8986)

+ (%C18 : 2 × 1.810) + (%C22 : 1 × 0.7497) (8)

Using data from the average molecular weight obtained withthe gas cromatograph coupled with mass spectrometer (GCMS)analysis it is possible to calculate the saponification number bythe method AOCS Cd 3a-94,33 as follows:

SV = 3 × 56.1 × 1000

[(Ms × 3) + 92.09] − (3 × 18)(9)

Next, the higher (HHV)34 and lower heating (LHV) values aredetermined as follows:

HHV =(

618 000

SV− 0.08 × IV − 430

)× 4.18 (10)

In order to determine the LHV it is necessary to take intoaccount the mass of water produced in the combustion reaction.By definition, LHV is the HHV minus the required energy to vaporizethe mass of water produced, which is determined through thecombustion chemical equation.

Therefore, the required energy to vaporize the mass of wateris the sum of the required sensible specific enthalpy change toheat the water produced to its saturation temperature at the

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pressure at which the reaction occurs, and the specific enthalpy ofvaporization at such conditions, which is calculated as follows:

hv = qh + qv (11)

LHV = HHV − hv × mH2O × 10−3 (12)

where qh is the required sensible specific enthalpy change toincrease water temperature up to the saturation point and qv thewater specific enthalpy of vaporization at the reaction conditions.

The global energetic consumption is the sum of all the processenergy requirements, i.e. electrical energy and solvent internalenergies, and is given by

ET =3∑

i=1

Ei +3∑

i=1

Ui (13)

where3∑

i=1Ei is the sum of the electrical energy consumed, and

3∑i=1

Ui the sum of the internal energies of the solvents consumed

in the three process stages (i = 1 – in situ methanolysis; 2 – rotaryevaporation, and 3 – centrifugation).

Since the process of obtaining biodiesel monoesters wasconducted in a closed system, i.e. recovering the solvents usedusing negligible energy compared with E1, E2, and E3, the totalconsumed energy consumed in the process is given by

ET∼=

3∑i=1

Ei (14)

The electrical energy consumed in each process stage iscalculated by

Ei = Vi × Ii × �ti × 10−3 (15)

where Vi, Ii and �ti are the voltage, current and duration of eachprocess stage (i = 1 – in situ methanolysis; 2 – rotary evaporation,and 3 – centrifugation).

RESULTSFourier transform infrared spectroscopy (FTIR)In order to characterize the material obtained, tests wereconducted using FTIR spectroscopy. The results were consistentwith the chemical properties of esters since analysis of the FTIRspectrum shown in Fig. 3 identified the characteristic bands ofthe links that are part of the organizational structure of fattymonoesters35 through the transmittance (vertical axis), clearlycharacterizing the axial strain of ester C O (1741 cm−1) and C–O(1168–1243 cm−1), in addition to axial and angular deformationof C–H in the regions of 2921, 1461, 1377 and 719 cm−1, which arethe measured wavenumbers (horizontal axis). All samples showedsimilar FTIR profiles.

ChromatographyBecause quantification of the chromatographic test was performedwith internal standardization, it was necessary to use a mass spec-trometer to investigate the peaks related to the samples, aiming tofind their composition. After obtaining the chromatogram reveal-ing the presence of 10 compounds, which are shown in Table 2,each peak was compared with data from the NIST library, availablein the chromatograph data bank, which led to the identificationof nine methyl monoesters, and only one substance was not iden-tified. Based on these results, which confirmed the presence ofalkyl monoesters, and based on the fatty profile, it was possible toestimate some parameters of interest, such as molecular weight,iodine values, saponification numbers, higher and lower heatingvalue and energy efficiency, and predict two important fuel prop-erties, the cetane number and cold filter plugging point. Figure 4

4000 3500 3000 2500 2000 1500 1000 500

50

100

150

200

250

300

350

400

450

500

550

600

650

700

Tra

nsm

ittan

ce (

%)

Wavenumber (cm-1)

Figure 3. Monoesters infrared spectrum.

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Table 2. Resulting fatty acid profile of the sample obtained fromin situ methanolysis

Fatty Acid

Molecularweight

(g mol−1)Percentage

of each peak

1- Methyl laurate 200.30 0.58

2- Methyl Miristate 228.37 6.76

3- Unidentified 0.24

4- Methyl palmitate 256.42 28.18

5- Methyl palmitoleate 254.41 22.61

6- Methyl stearate 284.48 1.03

7- Methyl oleate 282.46 6.19

8- Methyl linoleate 280.45 2.71

9- Methyl Behenate 340.58 4.05

10- Methyl Erucicolate 338.57 27.65

Average molecular weight (MB) 274.00 g mol−1 100.00

Fat

ty a

cid

prof

ile (

%)

∗ (obtained in this work)

Figure 4. Fatty acid composition of some algae and Nannochloropsis

Col

orFi

gure

-Onl

ine

only

oculata∗ .

shows a comparison of the fatty acids composition of N. oculataobtained in this work with literature data36 for other species.

Mass yield of the biodiesel (YB) synthesisIn order to quantify the mass yield of biodiesel (YB), 11 experimentswere performed, resulting in an average yield of 23.07 ± 2.76%(m/m), with 0.4614 g monoesters obtained from 2 g of microalgalbiomass. Such product in situ transesterification values wereconsidered satisfactory since the reaction was processed in asingle step without the need for extraction and purification ofthe oil.

The in situ methanolysis of microalgae biomass was investigatedthrough a 22 central composite design in which the response factorwas the alkyl monoesters mass yield (YB) and the selected reactionvariables were the reaction time (h) and the MeOH : HCl volumetricratio. The statistical significance of the effects of each experimentalvariable and of the possible interactions among them was alsoevaluated as shown in Fig. 5. According to the Pareto chart, thevariable MeOH : HCl had a positive and significant effect on the alkylmonoesters mass yield (YB), which was estimated from the Student-t distribution. This means that an increase of the ratio MeOH : HClcould lead to higher monoesters mass yield. The reaction timeand the interaction between reaction time and ratio MeOH : HClwere not significant at the 95% confidence level of the Student-t

Figure 5. Pareto chart of effects of reaction parameters on the biodiesel

Col

orFi

gure

-Onl

ine

only

mass yield of in situ methanolysis.

YB

(%

)

MeOH:HCl t (h)

< 30%< 20%< 10%

Figure 6. Effect of the ratio MeOH : HCl and reaction time on the resultingC

olor

Figu

re-O

nlin

eon

lybiodiesel mass yield (YB) of in situ methanolysis.

distribution. Therefore, the analysis leads to the conclusion thatthe most important variable in the process is the ratio MeOH : HCl.The software Statistica 8.037 was applied to produce theseresults. ANOVA was performed with the experimental data anda quadratic model was built to generate the response surfaceshown in Fig. 6. In this case, the ANOVA results demonstratedthat both the response surface and estimated effects were validbecause the quadratic model was able to predict 97.37% of theexperimental data. It should be noted that the response surfaceof Fig. 6 was not able to show the optimal condition for the in situtransesterification of microalgae biomass. However, the intentionwas not to optimize the system but only to investigate the maineffects of the reaction variables on the response factor.

Figure 6 is a plot of experimental data showing the combinedeffects of volume ratio MeOH: HCl and reaction time on the massyield of monoesters (YB). Figure 6 shows a fitted surface for the

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YB = 30 %

20 %

10 % MeO

H:H

Cl

t (h)

Figure 7. Contour isovalues of the resulting biodiesel mass yield (YB) with

Col

orFi

gure

-Onl

ine

only

respect to the ratio MeOH: HCl and reaction time of in situ methanolysis.

experimentally obtained mass yield of monoesters. As the ratioMeOH : HCl increases the mass yield of the process increases. Itis also observed that for all values of the ratio MeOH : HCl thereseems to be an optimal reaction time (between 3 and 4 h) so thatthe mass yield of monoesters is maximized. This behavior is alsoobserved in Fig. 7, which shows the contour plot isovalues of themass yield of monoesters. Therefore, the results of Figs 6 and 7demonstrate that the ratio MeOH: HCl has the most importanteffect on the resulting mass yield of monoesters. This couldbe expected since MeOH is one of the reactants in the in situmethanolysis.

Properties of the fuel monoesters obtainedUsing Equation (5), with the CNME values reported in theliterature3,30,38 – 42 as shown in Table 3, and on the biodiesel methylesters composition, it is possible to estimate the cetane numberof the fuel obtained.

Given the high amount of saturated esters found in the samples,a high CN value was expected, estimated at 68.2. Therefore,regarding the cetane number, the fuel complies with both ASTMD 6751–08 and UNE EN-14 214 : 2003 norms, which state thatthe diesel cetane number must be greater than 47 and 51,respectively.

The high amount of saturated esters also affects the cold-flowproperties of a biodiesel, inducing poor performances at lowtemperatures. The biodiesel obtained has a long chain saturatedfactor of 9.4, calculated using Equation (4), which is much higherthan those obtained with most common feedstocks such assoybean and palm (3.4 and 7.728), i.e. larger amounts of long chainsaturated esters are present in the microalgae derived biodiesel.The length of the ester’s chain is related to the fuel’s meltingpoint, and the saturated esters have melting points above roomtemperature. Therefore, at low temperatures, such esters tend tocrystallize, and might interrupt the flow of fuel, causing engine

malfunction due to problems in the fuel distribution system, andincreased pollutant emissions.

Using Equation (6), it was possible to estimate the CFPP of themicroalgae biodiesel obtained by in situ transesterification in thisstudy at 13.7 ◦C. Such an elevated result indicates that this fuelwould not perform properly at low temperatures, and would beviable only in hot climates with elevated temperatures duringthe entire year. Some techniques for reducing the CFPP exist,such as winterizing, but its economic viability still remains to beclarified.

The microalgae fatty acid profile depends greatly on thespecies and cultivation conditions. Thus, optimized cultivationconditions could lower the CFPP, improve the cold flow fuelperformance, and allow the fuel to be used in low temperatureconditions.

Calculation of energetic efficiencyFirst, the iodine value is calculated using Equation (8), so thatIV = 53.75 gIodine (100 g)−1

sample. Next, the saponification value is

calculated with Equation (9) resulting in SV = 195.68 mgKOH g−1s .

Next, the higher (HHV) and lower heating (LHV) values aredetermined according to the previously described methodology.The first result is HHV = 11 385.99 kJ mol−1. The combustionchemical equation was assumed to be similar to the combustionof methyl oleate which has a molecular weight of 280 g mol−1,since the monoesters obtained had an average molecular weightof 274 g mol−1 calculated as shown in Table 2, which is given by:

C19H36O2(l) + 27O2(g) → 19CO2(g) + 18H2O(l) (16)

so that the mass of water produced per mol of fuel is

mH2O(g) = MH2O (g mol−1) × nH2O (mol) = 324 g (17)

Therefore, the required energy to vaporize the mass of wateris the sum of the sensible specific enthalpy change to heat thewater to its saturation temperature at atmospheric pressure (1bar), i.e. 100 ◦C, and the specific enthalpy of vaporization at suchconditions, which was calculated as follows:

hv = 4.18 × (100 − 20) + 2257.2 = 2591.6 kJ kg−1 (18)

LHV = HHV − hv × mH2O × 10−3 = 10 546.31 kJ mol−1 (19)

where the ambient temperature was considered to be 20 ◦C, thespecific heat of water 4.18 kcal kg−1, and the water enthalpy ofvaporization 2257.2 kJ kg−1.

The global energetic consumption to process 2 g of drymicroalgae biomass by the in situ methanolysis was calculatedaccording to Equations (13) to (15). For that, Vi, Ii and �ti , i.e. thevoltage, current and duration of each process stage (i = 1 – in situmethanolysis; 2 – rotary evaporation, and 3 – centrifugation) werecarefully measured in the laboratory experiments. The result wasET = 12.96 kJ.

Table 3. Fatty acid profile of the sample from in situ methanolysis, and corresponding cetane numbers given in the literature

Me 12 : 0 Me 14 : 0 Me 16 : 0 Me 18 : 0 Me 22 : 0 Me 16 : 1 Me 18 : 1 Me 22 : 1 Me 18 : 2

61.436 66.239 74.539 86.939 10028 51340 58.939 763 38.240

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Finally, the energetic efficiency of the process to obtain biodieselthrough in situ methanolysis was then calculated as follows:

ηDBB = nB × LHV

ET=

0.4614

274× 10 546.31

12.96= 1.37 (20)

Based on the values obtained, it was observed that the energyoutput of the system, i.e. the energy released by the product isgreater than the energy consumed in the process (energy input).Therefore, it is possible to conclude that the process as describedis sustainable in terms of the energy balance, especially when thevalue obtained is compared with biodiesel derived from soybeanand sunflower reported as 1.06 and 1.12, respectively,43 or whencompared with ethanol, which was reported to be 1.1.44 However,Equation (20) does not consider the energy spent on spray dryingthe algae biomass, which is included in the calculation in thenext section. Due to the lack of information in the technicalliterature on microalgae derived biodiesel conventional methods,in situ methanolysis using microalgae was compared with soybeanand sunflower derived biodiesel obtained through conventionalmethanolysis.

Estimates for net energy yield considering the productionof biogas from residual microalgae biomassThe prospect of large-scale production of microalgae for appli-cations in biofuels is motivated by the high productivity thatsuch microorganisms can reportedly achieve. In contrast, a largeamount of biomass is generated after the biodiesel productionprocess. From the perspective of large-scale cultivation, the pos-sibility of obtaining energy from the residual biomass and thefertilizers used for cultivation should be considered, thus leadingto a more realistic total energetic efficiency.

Anaerobic digestion is a fundamental process that has thepotential to contribute to the solution of the problem of wastefrom the microalgae biodiesel production process and, in this way,to reduce energy costs.45

The total energy balance of the microalgae derived biodieselprocess could be even more positive than ethanol when consider-ing the co-generation of energy through biogas production fromthe residual microalgae biomass, as reported by Chisti.45 Thus,applying Chisti’s methodology45 to the values found in this study,the total annual energy yield can be estimated from the potentialtotal energy generation from microalgae as follows:

YE = Eoil × Poil + Ebiogas × Pbiogas (21)

In order to estimate Poil, the photobioreactor yield is consideredequal to 0.048 kg m−2 day−1,20 and a mass efficiency equal to 0.9(90%). Additionally, Xoil is defined as the fraction of oil containedin the biomass, which in this case is assumed to be the percentageof biodiesel produced in this work as reported previously in thetext, plus 10% of glycerin from the total contained in the structureof triglycerides,46,47 so that Xoil = 0.33. Therefore Poil is calculatedas follows:

Pbiomass = 0.048 kg m−2 day−1 × 104 m2 ha−1 × 365 day year−1

× 0.9 = 157 680 kg ha−1 year−1 (22)

Poil = Xoil × Pbiomass = 0.33 × 157 680

= 52 034.4 kg ha−1 year−1 (23)

Next, in order to estimate Pbiogas, which is the biogas yield perarea, it is assumed that the biogas yield is Ybiogas = 0.5 m3 kg−1,40.

Thus,

Pbiogas = (1 − Xoil) × Pbiomass × Ybiogas

= (1 − 0.33) × 157 680 kg ha−1 year−1 × 0.5 m3 kg−1

= 52 822.8 kg ha−1 year−1 (24)

Applying the calculated values to Equation (21), the total energyproduced per year results as follows:

YE = Eoil × Poil + Ebiogas × Pbiogas

YE = 37.9 × 10−3 GJ kg−1 × 52 034.4 kg ha−1 year−1

+ 25 × 10−3 GJ m−3 × 52 822.8 m3 ha−1 year−1

= 3292.67 GJ ha−1 year−1 (25)

where the Eoil and Ebiogas values are taken as reported by Chisti.43

Using the calculated values, it is possible to estimate the netenergy yield (Yn) contained in the microalgae biomass as follows:

Yn = YE ×(

1 − 1

ηDBB

)= 3292.67 ×

(1 − 1

1.37

)

= 889.26 GJ ha−1 year−1 (26)

However, Equation (26) does not account for the energyconsumed in drying the microalgae. In the project that suportedthe present work,48 microalgae were dried by spray drying.Therefore, considering that this work used 2 g of microalgae forthe in situ methanolysis, and assuming a 33% water content in thefiltered microalgae biomass, i.e. 1 g of water, 2.257 kJ (enthalpyof vaporization of 1 g of water at 1 bar) to dry the microalgaebiomass. Therefore, incorporating that value in calculating theenergetic efficiency, the result is

ηDBB =0.4614

274× 10 546.31

12.96 + 2.257= 1.17 (27)

Recalculating the net energy yield as described previously withEquation (26), the result is Yn = 478.42 GJ ha−1 year−1. Based onthis result, the actual energy balance of the microalgae derivedbiodiesel was 2.9 times higher than the energy balance of sugarcane, considering ethanol and bagasse as energy products, whichwas reported as equal to 163.9 GJ ha−1 year−1.45,49

CONCLUSIONAn in situ methanolysis methodology for biodiesel productionfrom microalgae has been investigated experimentally and testedusing the species Nannochloropsis oculata. In the proposedmethodology, there is no need for biomass separation andoil extraction before the transesterification reaction. Therefore,energy consumption is reduced compared with classical biodieselproduction processes. The biodiesel mass yield was characterizedthrough spectrometry in the infrared region demonstratingthat alkyl monoesters were indeed obtained, i.e. biodiesel. Inorder to assess the biofuel energy yield and performance, thethermophysical properties were estimated, including the cetanenumber, cold filter plugging point and lower heating value. Thethermodynamic efficiency of the process was calculated, and it wasdemonstrated that through in situ methanolysis the microalgaederived biodiesel energy balance was approximately three times

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higher than the energy balance of sugar cane, considering ethanoland bagasse as energy products, and more efficient than soybeanand sunflower derived biodiesel.

The key conclusion of this study is that the in situ methanolysismethodology for biodiesel production from microalgae improvesthe state-of-the-art by the possible elimination of well knownmajor bottlenecks in microalgae derived biodiesel processes, suchas drying, separation and oil extraction. In fact, the productionof 17% more energy in the form of biodiesel than the energyconsumed in the process stages shows that there is potential forthe future implementation of industrial scale biodiesel productionfrom microalgae, since the process was shown to be energeticallysustainable. Therefore, a low energy consumption methodology ismade available with potential for microalgae derived biodieselindustrial production from any microalgae species. However,future work needs to be conducted in order to assess theeconomical and environmental feasibility of the process, includingthe effects of the energy consumed in cultivation and biomasscollection.

ACKNOWLEDGEMENTSThe authors acknowledge with gratitude the support of the Brazil-ian National Council of Scientific and Technological Development,CNPq.

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