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Kinetics of ultrasound-assisted enzymatic biodiesel production from Macauba coconut oil
Renewable Energy 76 (2015) 388e393
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Renewable Energy journal homepage: www.elsevier.com/locate/renene
Kinetics of ultrasound-assisted enzymatic biodiesel production from Macauba coconut oil Simone Michelin a, Frederico M. Penha a, Melania M. Sychoski a, Robison P. Scherer a, rio c, M. Di Luccio c, De bora de Oliveira c, Helen Treichel b, Alexsandra Vale c, * J. Vladimir Oliveira a b c
Department of Food Engineering, URI, Av. Sete de Setembro, 1621, Erechim, 99700-000, RS, Brazil Federal University of Fronteira Sul e Campus de Erechim, 99700-000, Erechim, RS, Brazil polis, 88040-900, SC, Brazil Department of Chemical and Food Engineering, UFSC, Floriano
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2014 Accepted 20 November 2014 Available online
This work reports the production of fatty acid ethyl esters (FAEE) by means of Macauba (Acrocomia aculeata) coconut oil (MCO) solvent-free enzymatic transesterification reactions using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. An experimental design was used to evaluate the effects of temperature (40e70 C), enzyme (5e20 wt%) concentration, oil to ethanol molar ratio (1:3e1:10) and output irradiation power (40e70 % of the maximum supply value) on the reaction yield. Besides, a kinetic study varying the enzyme concentration was also carried out. Results show that ultrasound-assisted lipase-catalyzed transesterification of MCO with ethanol in solvent-free system might be a potential alternative route to conventional alkali-catalyzed and/or traditional enzymatic methods, as reaction yields around 70 wt% were obtained at mild irradiation power supply (~ 132 W), and temperature (65 C) in a short reaction time, 30 min. Reutilization of enzyme showed that it may be advantageously employed up to 5 reuse cycles. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ultrasound Biodiesel Lipase Acrocomia aculeate Convertibility
1. Introduction Macauba (Acrocomia aculeata) is a palm species native of tropical forests and can be found from southern Mexico and the West Indies until Brazil and also in Paraguay, Bolivia and Argentina [1]. The oil produced from Macauba has no tradition as food product and presents several industrial and energetic applications, which demonstrate great potential for biodiesel production [2e5]. These latter characteristics granted importance to the Macauba coconut oil (MCO) as alternative feedstock to produce biodiesel, especially in a country like Brazil where biodiesel is mainly produced from soybean oil (75e80%). Considering that one-third of soybean production in Brazil is destined to biodiesel production and compared to soybean oil productivity, 400e700 kg of oil per hectare per year, MCO with a productivity of around 6000 kg of oil per hectare per year appears as a very promising alternative source [6,7].
* Corresponding author. Tel.: þ55 48 37212508; fax: þ55 48 37219687. E-mail address: [email protected] (J.V. Oliveira). http://dx.doi.org/10.1016/j.renene.2014.11.067 0960-1481/© 2014 Elsevier Ltd. All rights reserved.
The use of cheaper raw materials for biodiesel production is a key point towards economic competitiveness of this biofuel. In that sense, the raw vegetable oil should be studied to determine its potential with respect to treated and even refined oils. Raw vegetable oils generally present compounds such as antioxidants, phospholipids, free fatty acid (FFA), pigments, etc., that can affect ester synthesis in some extent and the presence of water, which demands investigation on the use of rectified alcohols. However, there is a clear lack of technical reports in scientific literature involving the use of MCO as feedstock for biodiesel production. The few published works available have studied the production of biodiesel from MCO using basic and acid catalysts [8] and enzymatic catalysis under microwave irradiation [9]. To the best of our knowledge this is the first report on biodiesel production from MCO through enzymatic transesterification under ultrasound irradiation. Ultrasound is very effective at dispersing material present in solution. The application of ultrasound, therefore, will contribute to a more homogeneous reaction mixture and facilitate dispersion
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of lipase through substrate media, reducing agglomeration so that the reaction rate does not decrease with the increase of lipase concentration [10]. Moreover, as enzymatic synthesis of biodiesel is much slower than alkali-catalyzed transesterification, ultrasound irradiation appears to be a proper method to increase the reaction rate thus making possible greener biodiesel production through a biological catalyst. Enzyme-catalyzed reactions in solvent-free systems under ultrasound power, in spite of its scientific and industrial relevance, can still be considered a new technology, since just a few studies in this subject can be found in the open literature. The present work attempts to contribute to build a platform for biodiesel production trough investigation of new transesterifications techniques, especially by exploring non-edible raw materials. Here, the main objective is to investigate the production of Macauba coconut oil esters with ethanol (fatty acid ethyl estersFAEE) using a commercial lipase under the influence of ultrasound irradiation, in a solvent-free system, evaluating the effects of reaction conditions on the yield of fatty acid esters produced. In this work it was also introduced a new parameter named “convertibility”, which represents the maximum esters conversion that can be achieved from a raw material if every fatty acid available would form an alcoholic ester. 2. Materials and methods 2.1. Materials The MCO was obtained from the processing unit of Macauba in Montes Claros (Minas Gerais, Brazil). Ethanol (Merck 99.9%), nheptane (Nuclear, 99.5 gg mol1%), lauric acid (Vetec, 98 gg mol1%), n-propanol (Synth, 99.5 gg mol1% of purity), sodium hydroxide (Quimex, 97 gg mol1%) and acetone (Quimex, analytical grade) were used without further treatment. Commercial immobilized lipase, Novozym 435, from Candida antarctica (immobilized on a macroporous anionic resin, 1.4 wt% ria, PR, Brazil) and water) was purchased from Novozymes (Arauca presented an enzyme activity of around 46.9 U g1, determined as the initial rates in esterification reactions between lauric acid and propanol at a molar ratio of 3:1, temperature of 60 C and enzyme concentration of 5 wt% in relation to the substrates. 2.2. MCO characterization Several analyses were performed for the characterization of MCO. The acid value was determined by titration according to method AOCS Cd 3d-63. The water content was determined by Karl Fischer titration, according to method AOCS Ca 2e-84, using a DL 50, Mettler-Toledo titrator. Additionally, some physicochemical parameters were measured at 25 C: density (DMA 4500 density/ specific gravity meter, Anton Paar, Austria), thermal conductivity (Decagon Inc., model KD2) and viscosity (rheometer, Thermohaake VT e 550). Considering that oil was submitted to heating during the extraction and it remained stocked exposed to ambient conditions, as normally treated in the processing units, it was assumed that polymerization could occur to some extent, thus the percentage of polymers was determined by means of size-exclusion HPLC (SEHPLC) according to the method AOCS Cd 22-91. Analysis was performed using an HPLC Shimadzu Prominence 20A (Shimadzu, Corporation, Kyoto, Japan), equipped with an evaporative light scattering detector Shimadzu ELSD-LTII, and a column Nucleogel GPC 100-5, 300 mm 0.5 mm (MachereyeNagel, Düren). The fatty acid composition of the MCO was determined by GC analysis as described below.
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2.3. Apparatus and experimental procedure Enzymatic ultrasound-assisted transesterification reactions were carried out using an ultrasonic water bath (Unique apparatus e temperature accuracy of ±0.5 C) in a round bottom flask of 50 mL capacity closed with a lid. The experimental setup consists of an ultrasonic bath equipped with a transducer having longitudinal vibrations. The ultrasonic unit has an operating frequency of 40 kHz and a maximum rated electrical power output of 132 W. The ultrasonic transducer (surface area of 282.2 cm2) is fitted at the bottom of the bath, horizontally along the length, which has been considered a suitable ultrasonic system [11]. Typically, around 20 g of the oil was charged into the reaction vessel, and then precise amounts of oil to ethanol molar ratio and enzyme (according to the experimental design) were also weighed on a precision scale balance (Ohaus Analytical Standard with 0.0001 g accuracy) and loaded into the reaction vessel, immersed in the ultrasonic bath already stabilized at the pre-established experimental temperature. The flask was then closed and the ultrasonic system turned on. 2.4. Experimental design and kinetic evaluation At first, a fractional 241 experimental design with triplicate runs in the central point with 11 assays, was employed to evaluate the effects of four variables e temperature (40e70 C), enzyme concentration (5e20 wt%, by weight of substrates), oil to ethanol molar ratio (1:3e1:10) and output irradiation power (40e70% of the maximum supply value), keeping the reaction time fixed at 90 min. The software Statistica® 8.0 (Statsoft Inc., USA) was used to assist the design and statistical analysis of the reaction yield. Taking into account the results obtained from the execution of the experimental design, reaction kinetic experiments were performed adopting temperature of 65 C, 100% of total ultrasound power (132 W), while varying enzyme concentration of 5 and 10 wt%, keeping fixed the substrates molar ratio at 1:9. It may be important to emphasize that in all cases, destructive experiments, without sampling, were carried out. 2.5. Analytical methods Samples were first submitted to ethanol evaporation to constant weight in a vacuum oven (338 K, 0.05 MPa) and then diluted with 2 mL of ethanol and 8 mL of n-heptane. After this, 50 mL of solution was transferred to a 1 mL volumetric flask, adding 50 mL of internal standard methyl heptadecanoate at concentration of 5000 mg L1 and filled with n-heptane. After that, 1 mL of solution was injected in triplicate in a gas chromatograph (Shimadzu GC-2010), equipped with FID, auto-injector AOC-20i and a capillary column (Rtx-WAX, 30 m 0.25 mm 0.25 mm), with split ratio of 1:50. Column temperature was programmed from 393 K, holding 1 min, heating to 453 K at 15 K min1, holding 2 min, and to 523 K at 5 K min1, holding 2 min. Synthetic air and nitrogen was used as carrier gas, and the injection and detector temperatures were 523 K. Compounds were quantified upon analysis following the standard UNEEN 14103 [12] and FAEE content was then calculated based on the content of ethyl esters in the analyzed sample. 2.6. Convertibility and conversion efficiency Considering that raw vegetable oils could contain several compounds non-convertible to alkyl esters, the maximum conversion achievable from these oils may not correspond to 100%. Thus, in order to determine this maximum conversion, it was defined here the “convertibility” parameter, and for this purpose a sample of the MCO was treated using a quantitative conversion method (based on
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the use of BF3 as catalyst) as described below. Around 40e90 mg of oil was placed inside a screw cap tube, where 1.5 mL of methanolic solution of NaOH 0.5 N were aggregated, air inside the tube was replaced by N2, it was hermetically closed and heated at 373 K for 10 min, maintaining the reaction mixture under magnetic stirring. After 1 min cooling, 2 mL of methanolic solution of BF3 14% were added and then heated at 373 K for 5 min. Afterward, 2 mL of nhexane solution of methyl heptadecanoate with a concentration of 5 mg/mL was added as internal standard. Finally, 5 mL of a saturated NaCl solution was added and after mixture decantation the superior phase was separated, dehydrated with sodium sulfate anhydrous and centrifuged. This last solution was analyzed by gas chromatography (GC) in a Shimadzu GC-14B chromatography equipped with flame ionization detector (FID) and a capillary column SGE BPX70. The temperature program started at 433 K and then it was raised at a 4 K min1 rate until 503 K, holding that temperature for 10 min. By this method all the fatty acids were converted to their corresponding alkyl esters, independently of the form that these fatty acids are found in the sample. The fatty acid composition of the starting MCO was determined using this same procedure, but adding 2 mL of pure n-hexane instead of the solution of internal standard. The convertibility was determined from the area percentage obtained from the GC analysis, using the methyl heptadecanoate peak as the internal standard, as follows:
P Convertibilityðwt%Þ ¼
A AIE CIE VIE 100 AIE m
Alkyl ester yield 100 Convertibility
Fatty acid
Content (wt%)
Lauric acid (12:0) Myristic acid (14:0) Palmitic acid (16:0) Palmitoleic acid (16:1) Estearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linolenic acid (18:3)
0.7 0.3 22.2 4.2 1.9 58.0 9.7 0.6
analysis, is presented in Table 1 where it can be noticed the predominance of oleic acid (58.0 wt%), which is similar to the fatty acid profile found by Fortes and Baugh [13]. The acid value, water content (wt%) and polymer content of MCO were determined as 88.9 mgKOH/g oil, 0.64 and of 6.1%, respectively. The acid value found in this work is in agreement with those reported by Rodrigues [14] and Gonzalez [15], up to 90 mgKOH/g oil. Some physicochemical parameters were measured at 25 C: density (0.9273 ± 6.84.104 g cm3), thermal conductivity (0.115 ± 5.103 W m1 C1) and viscosity (61.75 mPa s). The convertibility of MCO was determined to be 80.1%, thus oil contained 19.9 wt% of compounds that would not contribute to the ester content, independently of the efficiency of the conversion process used.
(1)
P where: A is the total area in the GC chromatogram, AIE, CIE and VIE denote, respectively, the area, concentration (mg/mL), and volume (mL) of the internal standard, and m is the weight of MCO. In order to evaluate the real efficiency of the process it is convenient to refer the alkyl ester yield to the convertibility, thus with this purpose the “Conversion efficiency” is defined as:
Conversion efficiencyðwt%Þ ¼
Table 1 Fatty acid composition of the Macauba oil.
(2)
Hence, if the process efficiency is high the Alkyl ester yield would achieve a level near the Convertibility and the Conversion efficiency would reach a value near 100%. 2.7. Reuse of biocatalyst To evaluate the reuse of Novozym 435 it was adopted the experimental condition of oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme concentration (by weight of substrates), reaction time at 30 min and at full ultrasound irradiation power amplitude (100%). Enzyme recuperation at the end of each reaction was performed by filtration in filter paper followed by two 10 mL n-hexane washings under moderate vacuum, then submitted to rotary evaporation (Fisatom, model 550) and dried in oven (JP 101, J. Prolab) at 40 C for 1 h. Recuperated enzyme was then kept in desiccator for 24 h prior to measurement of its activity and reutilization. The residual relative esterification activity was defined as the ratio of (Final activity/Original activity) 100 and determined after each cycle of utilization of the enzyme. 3. Results and discussion 3.1. MCO characterization The chemical composition of major fatty acids present in the MCO used in this study, determined by gas chromatography
3.2. Production of fatty acid ethyl esters (FAEE) Table 2 shows the results of the experimental design and as it can be seen reaction yields in the range of 35e50% were reached. It can be observed from Fig. 1 that with a confidence level of 95%, the enzyme content was the only significant variable affecting positively the transesterification yields, which agrees with the recent result found by Batistella et al. [16], in studying biodiesel enzymatic production in ultrasound system. Taking into account such fact, the next step toward improving reaction yield efficiency was to conduct a kinetic study keeping fixed the oil to ethanol molar ratio at 1:9, temperature of 65 C and at the maximum output irradiation, 132 W, while varying the enzyme content, 1, 5, 10 and 20 wt%, also performing destructive, independent, experiments at 1, 3, 5, 7, 10, 15, 30, 60, 90, 120, 180, 240 and 360 min. Results for the reactions kinetics are depicted in Fig. 2, where it can be noticed that a raise in enzyme content led to an increase in initial reaction rates. It can also be noted that for all the enzyme contents tested, the maximum yield is reached at 1 h reaction and
Table 2 Matrix of the experimental design (coded values) with answers in terms of FAEE yield (reaction time fixed at 90 min).a Run
T
O:E
[E]
IA
FAEE yield
1 2 3 4 5 6 7 8 9 10 11
(1) 40 (1) 70 (1) 40 (1) 70 (1) 40 (1) 70 (1) 40 (1) 70 (0) 55 (0) 55 (0) 55
(1) 1:3 (1) 1:3 (1) 1:10 (1) 1:10 (1) 1:3 (1) 1:3 (1) 1:10 (1) 1:10 (0) 1:6.5 (0) 1:6.5 (0) 1:6.5
(1) 5 (1) 5 (1) 5 (1) 5 (1) 20 (1) 20 (1) 20 (1) 20 (0)12.5 (0)12.5 (0)12.5
(1) 40 (1) 100 (1) 100 (1) 40 (1) 100 (1) 40 (1) 40 (1) 100 (0) 70 (0) 70 (0) 70
38.6 38.5 42.0 35.6 47.1 45.0 48.7 42.8 46.8 47.4 48.8
a Temperature ( C), T; Oil:ethanol molar ratio, O:E; Enzyme concentration (wt%), [E]; Irradiation amplitude (%), IA; FAEE yield (wt%).
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maximum reaction conversion of 45% for 25 wt% enzyme concentration, 1:9 of oil to alcohol molar ratio and temperature of 30 C in 15 min. According to those authors, mass transfer problems happened due to inefficient agitation of the reaction content and thus better results were found for lower enzyme concentrations, which in turns prevented catalyst agglomeration.
3.3. Enzyme reuse and ultrasound influence on FAEE yield
Fig. 1. Pareto chart for the fractional 241 experimental design of MCO transesterifications using Novozym 435 under ultrasound irradiation for a reaction time of 90 min (see Table 1). Experimental conditions: Temperature ( C), T; Oil:ethanol molar ratio, O:E; Enzyme concentration (wt%), [E]; Irradiation amplitude (%), IA; FAEE yield (wt%).
then a nearly asymptotic behavior is noticed at larger times for all conditions. Hence, from a practical standpoint, the reaction might be interrupted to meet economic aspects - small gains after a certain time. Fig. 2 also shows that reaction yields up around 70% were achieved in only 30 min for the highest enzyme content, which on the basis of the convertibility parameter means in fact conversion efficiency values as high as 90% [17,18]. It is worth noting the good reproducibility of the experimental results found for the enzyme content of 20 wt%, hence pointing the reliability of the experimental information. Data scattering observed in this figure may be explained in terms of experimental errors associated, and the fact that destructive experiments were carried out without sampling, which may be viewed as an important internal consistence test of the results. Recently, Nogueira et al. [9], in investigating the effect of microwave irradiation on the transesterification of Macauba oil using Novozym 435 as catalyst and ethanol as substrate, reached a
Fig. 2. Kinetics of MCO transesterification with ethanol using Novozym 435 as catalyst. Experimental condition: oil to ethanol molar ratio of 1:9, temperature of 65 C, 100% ultrasonic power at varying enzyme contents.
Undoubtedly, enzyme reuse is a key issue for enzymatic process feasibility and accordingly several reaction cycles for Novozym 435 were attempted under ultrasound irradiation. Before performing such assays however, the influence of ultrasound utilization was studied and Fig. 3 shows the reaction kinetics with and in the absence of ultrasound irradiation. As can be seen from this figure, much better results can be obtained using ultrasound power supply, especially for longer times. Afterward, enzyme reuse was tested at 100% ultrasound irradiation amplitude, oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme concentration, and reaction time 30 min Fig. 4 presents such results in terms of FAEE yield and also enzyme activity as a function of cycle number. It can be seen that a monotonically enzyme activity loss coupled with a similar decrease in FAEE yield is noticed as number of reuse raises. According to Wang et al. [19] the cost involved in enzyme immobilization together with cycle utilization should be taken into account in the global economic analysis of an industrial biodiesel plant. It has been argued by Rahman et al. [20] that lipases may lose its activity in non-aqueous medium and selectivity and stability may also be affected. Repeated cycles of Novozym 435 were adopted by Hajar et al. [21] in the methanolysis of canola oil in solvent-free system and the authors noted that after 5 cycles and 432 h reaction, reaction conversion was kept at 97%, but, unfortunately, enzyme activity was not reported in such research work. Of course, enzyme stability is a relevant parameter and further studies should be conducted to keep enzyme activity for practical purposes. As a matter of fact, one problem arising from enzyme reuse in transesterification reactions is the low solubility of glycerol, in the product mixture. This has a strong negative effect, expressed as a blocking effect, on enzyme activity. This effect has been attributed to the deposition of glycerol on the enzyme surface.
Fig. 3. Effect of ultrasound use on enzymatic biodiesel production from MCO in solvent-free system. Reaction conditions: Novozym 435 content of 20 wt%, oil to ethanol molar ratio of 1:9, at 65 C.
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interesting and also potentially economic if a well-balanced reaction system is considered.
Acknowledgments The authors thank CNPq (process 490061) for the financial support of this work and scholarships.
References
Fig. 4. Reuse of lipase in the transesterification of MCO with ethanol using Novozym 435 as catalyst. Experimental condition: oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme, reaction time at 30 min, ultrasound irradiation amplitude (100%).
It is noticed that the ultrasound waves can interfere with the characteristics of the enzyme, changing its structure as a response to the dynamic perturbation caused by the ultrasound [22]. Just a few studies about the effect of the ultrasound under the enzyme activities are related in the literature and contradictory results of activation/deactivation have been presented. Different from the traditional inactivation by the high temperatures, the sonication process does not affect the active sites of the enzyme. This observation has already been demonstrated for a-amylase, peroxidase, laccase and alkaline-phosphatase [23,24]. However, to date, the true potential of ultrasound is not well known, due to the lack of knowledge of the exact molecular effects of the ultrasound on enzymes and living cells. Specifically for the enzymes, the ultrasound has shown increases on stability and catalytic activity of the enzymes. Nevertheless, a better comprehension of the effect of the ultrasound on the enzyme properties is necessary to develop more efficient catalysts with higher activities and stabilities [25]. 4. Conclusions This work confirms the good efficiency of ultrasound-assisted system to convert Macauba oil into alcoholic esters using a commercial lipase as catalyst, hence pointing the promising use of a low-cost, non-edible oil, with very satisfactory field productivity, towards biodiesel production with an eco-friendly, green technique. The convertibility and the conversion efficiency parameters helped to better specify the extent of the esters conversion reaction in the lipase-catalyzed, ultrasound irradiation transesterification process by establishing a “theoretical” maximum esters yield of 80.1% and a “real” conversion efficiency of around 90% for ethanol. Due to the presence of 19.9% of “non-convertible” compounds in the starting oil additional processing steps (e.g. distillation) should be considered in order to achieve higher ester contents in the final product. Although such steps would impact in the overall process costs, it could be absorbed by the relatively low value of the raw material. Of course, further work must be done in order to identify the nature of the non-convertible compounds, which is underway by our research group. Regarding ultrasound influence it was shown that this new technology may be advantageously used for transesterification reactions to provide higher yields in shorter reaction time. Reuse of enzyme by its turn may also be technically
[1] Lorenzi GMAC. Acrocomia aculeata (jacq) lodd. ex mart e arecaceae: bases vel [Dr. thesis]. Brazil (in Portuguese): Federal para o extrativismo sustenta University of Paran a; 2006. [2] Moura EF, Ventrella MC, Motoike SY. Anatomy, histochemistry and ultrastructure of seed and somatic embryo of Acrocomia aculeata (Arecaceae). Sci Agri 2010;67:399e407. ^ncia de macaúba em [3] Motta PEF, Curi N, Oliveira-Filho AT, Gomes JBV. Ocorre ~o com atributos clima ticos, pedolo gicos e vegetacionais. Minas Gerais: relaça Pesqui Agropecu Bras 2002;37:1023e31. [4] Andrade MHC, Vieira AS, Aguiar HF, Chaves JFN, Neves RMPS, Miranda TLS, ~o potencial para et al. Oleo do fruto da palmeira macaúba parte I: uma aplicaça ticos. In: II ENBTEQ e Encontro indústrias de alimentos, f armacos e cosme Brasileiro sobre Tecnologia na Indústria Química/III Semin ario ABIQUIM de Tecnologia. S~ ao Paulo: ABEQ; 2006. [5] Andrade MHC, Vieira AS, Aguiar HF, Chaves JFN, Neves RMPS, Miranda TLS, ~o do et al. Oleo do fruto da palmeira macaúba parte II: processo de extraça leo. In: II ENBTEQ e Encontro Brasileiro sobre Tecnologia na Indústria Quío rio ABIQUIM de Tecnologia. Sa ~o Paulo: ABEQ; 2006. mica/III Semina [6] Caires EF, Ferrari RA, Morgano MA. Produtividade e qualidade da soja em ~o da calagem na superfície em semeadura direta. Bragantia 2003;62: funça 283e90. [7] USDA, Foreign Agricultural Service (FAS). GAIN BR0707 Brazil e 2011 annual oilseeds report, Global Agricultural Information Network (GAIN) on-line, 04/ 04/2011. [8] Rodrigues HS. Production of ethyl and methyl esters by transesterification reaction of the latin american palm macauba e Acrocomia aculeate [Dr. thesis]. ~o Paulo; 2007. Brazil (in Portuguese): University of Sa lix R, et al. [9] Nogueira BM, Carretoni C, Cruz R, Freitas S, Melo-Junior PA, Costa-Fe Microwave activation of enzymatic catalysts for biodiesel production. J Mol Cat B Enzym 2010;67:117e21. [10] Liu Y, Jin Q, . Shan L, . Liu Y, . Shen W, . Wang X. The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system. Ultrason Sonochem 2008;15:402e7. [11] Fiametti KG, Ustra MK, Oliveira D, Corazza ML, Furigo Jr A, Oliveira JV. Kinetics of ultrasound-assisted lipase-catalyzed glycerolysis of olive oil in solvent-free system. Ultrason Sonochem 2012;19:440e51. [12] UNE-EN 14103:2003. Fat and oil derivatives. Fatty Acid Methyl Esters (FAME). Determination of ester and linolenic acid methyl ester contents. Madrid, Spain: AENOR; 2003. [13] Fortes ICP, Baugh PJ. Pyrolysis-GC/MS studies of vegetable oils from Macauba fruit. J Anal Appl Pyrolysis 2004;72:103e11. ~o de e steres etílicos e metílicos, por reaço ~es de trans[14] Rodrigues HS. Obtença leo da palmeira Latino Americana macaúba- Acroesterificaç~ ao, a partir do o comia aculeata [Thesis (Sciences-Chemistry)]. Ribeir~ ao Preto: University of ~o Paulo; 2007. p. 236. Sa ~o contínua de biodiesel por transesterificaç~ leo do [15] Gonzalez SL. Produça ao de o leo de fritura em metanol e em fruto de macaúba (Acrocomia aculeata) e o polis: etanol supercrítico [Doctoral Qualifying (Food Engineering)]. Floriano Federal University of de Santa Catarina; 2011. p. 68. [16] Batistella L, Lerin LA, Brugnerotto P, Danielli AJ, Trentin CM, Popiolski A, et al. Oliveira, Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system. Ultrason Sonochem 2012;19:452e8. [17] Navarro-Díaz JH, Gonzalez SL, Irigaray IB, Vieitez I, Jachmani an I, Hense H, et al. Macauba oil as an alternative feedstock for biodiesel: characterization and ester conversion by the supercritical method. J Supercrit Fluids 2014;93: 130e7. [18] Gonzalez SL, Sychoski MM, Navarro-Díaz HJ, Callejas N, Saibene M, Vieitez I, et al. Continuous catalyst-free production of biodiesel through transesterification of soybean fried oil in supercritical methanol and ethanol. Energy Fuels 2013;27:5253e9. [19] Wang X, Liu X, Zhao C, Ding Y, Xu P. Biodiesel production in packed-bed reactors using lipase-nanoparticle biocomposite. Biores Technol 2011;102: 6352e5. [20] Rahman MBA, Jumbri K, Hanafiah NAMA, Abdulmalek E, Tejo BA, Basri M, et al. Enzymatic esterification of fatty acid esters by tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase. J Mol Catal B Enzym 2012;79:61e5. [21] Hajar M, Shokrollahzadeh S, Vahabzadeh F, Monazzami A. Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa. Enzyme Microb Technol 2009;45:188e94.
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Kinetics of ultrasound-assisted enzymatic biodiesel production from Macauba coconut oil Simone Michelin a, Frederico M. Penha a, Melania M. Sychoski a, Robison P. Scherer a, rio c, M. Di Luccio c, De bora de Oliveira c, Helen Treichel b, Alexsandra Vale c, * J. Vladimir Oliveira a b c
Department of Food Engineering, URI, Av. Sete de Setembro, 1621, Erechim, 99700-000, RS, Brazil Federal University of Fronteira Sul e Campus de Erechim, 99700-000, Erechim, RS, Brazil polis, 88040-900, SC, Brazil Department of Chemical and Food Engineering, UFSC, Floriano
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2014 Accepted 20 November 2014 Available online
This work reports the production of fatty acid ethyl esters (FAEE) by means of Macauba (Acrocomia aculeata) coconut oil (MCO) solvent-free enzymatic transesterification reactions using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. An experimental design was used to evaluate the effects of temperature (40e70 C), enzyme (5e20 wt%) concentration, oil to ethanol molar ratio (1:3e1:10) and output irradiation power (40e70 % of the maximum supply value) on the reaction yield. Besides, a kinetic study varying the enzyme concentration was also carried out. Results show that ultrasound-assisted lipase-catalyzed transesterification of MCO with ethanol in solvent-free system might be a potential alternative route to conventional alkali-catalyzed and/or traditional enzymatic methods, as reaction yields around 70 wt% were obtained at mild irradiation power supply (~ 132 W), and temperature (65 C) in a short reaction time, 30 min. Reutilization of enzyme showed that it may be advantageously employed up to 5 reuse cycles. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ultrasound Biodiesel Lipase Acrocomia aculeate Convertibility
1. Introduction Macauba (Acrocomia aculeata) is a palm species native of tropical forests and can be found from southern Mexico and the West Indies until Brazil and also in Paraguay, Bolivia and Argentina [1]. The oil produced from Macauba has no tradition as food product and presents several industrial and energetic applications, which demonstrate great potential for biodiesel production [2e5]. These latter characteristics granted importance to the Macauba coconut oil (MCO) as alternative feedstock to produce biodiesel, especially in a country like Brazil where biodiesel is mainly produced from soybean oil (75e80%). Considering that one-third of soybean production in Brazil is destined to biodiesel production and compared to soybean oil productivity, 400e700 kg of oil per hectare per year, MCO with a productivity of around 6000 kg of oil per hectare per year appears as a very promising alternative source [6,7].
* Corresponding author. Tel.: þ55 48 37212508; fax: þ55 48 37219687. E-mail address: [email protected] (J.V. Oliveira). http://dx.doi.org/10.1016/j.renene.2014.11.067 0960-1481/© 2014 Elsevier Ltd. All rights reserved.
The use of cheaper raw materials for biodiesel production is a key point towards economic competitiveness of this biofuel. In that sense, the raw vegetable oil should be studied to determine its potential with respect to treated and even refined oils. Raw vegetable oils generally present compounds such as antioxidants, phospholipids, free fatty acid (FFA), pigments, etc., that can affect ester synthesis in some extent and the presence of water, which demands investigation on the use of rectified alcohols. However, there is a clear lack of technical reports in scientific literature involving the use of MCO as feedstock for biodiesel production. The few published works available have studied the production of biodiesel from MCO using basic and acid catalysts [8] and enzymatic catalysis under microwave irradiation [9]. To the best of our knowledge this is the first report on biodiesel production from MCO through enzymatic transesterification under ultrasound irradiation. Ultrasound is very effective at dispersing material present in solution. The application of ultrasound, therefore, will contribute to a more homogeneous reaction mixture and facilitate dispersion
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of lipase through substrate media, reducing agglomeration so that the reaction rate does not decrease with the increase of lipase concentration [10]. Moreover, as enzymatic synthesis of biodiesel is much slower than alkali-catalyzed transesterification, ultrasound irradiation appears to be a proper method to increase the reaction rate thus making possible greener biodiesel production through a biological catalyst. Enzyme-catalyzed reactions in solvent-free systems under ultrasound power, in spite of its scientific and industrial relevance, can still be considered a new technology, since just a few studies in this subject can be found in the open literature. The present work attempts to contribute to build a platform for biodiesel production trough investigation of new transesterifications techniques, especially by exploring non-edible raw materials. Here, the main objective is to investigate the production of Macauba coconut oil esters with ethanol (fatty acid ethyl estersFAEE) using a commercial lipase under the influence of ultrasound irradiation, in a solvent-free system, evaluating the effects of reaction conditions on the yield of fatty acid esters produced. In this work it was also introduced a new parameter named “convertibility”, which represents the maximum esters conversion that can be achieved from a raw material if every fatty acid available would form an alcoholic ester. 2. Materials and methods 2.1. Materials The MCO was obtained from the processing unit of Macauba in Montes Claros (Minas Gerais, Brazil). Ethanol (Merck 99.9%), nheptane (Nuclear, 99.5 gg mol1%), lauric acid (Vetec, 98 gg mol1%), n-propanol (Synth, 99.5 gg mol1% of purity), sodium hydroxide (Quimex, 97 gg mol1%) and acetone (Quimex, analytical grade) were used without further treatment. Commercial immobilized lipase, Novozym 435, from Candida antarctica (immobilized on a macroporous anionic resin, 1.4 wt% ria, PR, Brazil) and water) was purchased from Novozymes (Arauca presented an enzyme activity of around 46.9 U g1, determined as the initial rates in esterification reactions between lauric acid and propanol at a molar ratio of 3:1, temperature of 60 C and enzyme concentration of 5 wt% in relation to the substrates. 2.2. MCO characterization Several analyses were performed for the characterization of MCO. The acid value was determined by titration according to method AOCS Cd 3d-63. The water content was determined by Karl Fischer titration, according to method AOCS Ca 2e-84, using a DL 50, Mettler-Toledo titrator. Additionally, some physicochemical parameters were measured at 25 C: density (DMA 4500 density/ specific gravity meter, Anton Paar, Austria), thermal conductivity (Decagon Inc., model KD2) and viscosity (rheometer, Thermohaake VT e 550). Considering that oil was submitted to heating during the extraction and it remained stocked exposed to ambient conditions, as normally treated in the processing units, it was assumed that polymerization could occur to some extent, thus the percentage of polymers was determined by means of size-exclusion HPLC (SEHPLC) according to the method AOCS Cd 22-91. Analysis was performed using an HPLC Shimadzu Prominence 20A (Shimadzu, Corporation, Kyoto, Japan), equipped with an evaporative light scattering detector Shimadzu ELSD-LTII, and a column Nucleogel GPC 100-5, 300 mm 0.5 mm (MachereyeNagel, Düren). The fatty acid composition of the MCO was determined by GC analysis as described below.
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2.3. Apparatus and experimental procedure Enzymatic ultrasound-assisted transesterification reactions were carried out using an ultrasonic water bath (Unique apparatus e temperature accuracy of ±0.5 C) in a round bottom flask of 50 mL capacity closed with a lid. The experimental setup consists of an ultrasonic bath equipped with a transducer having longitudinal vibrations. The ultrasonic unit has an operating frequency of 40 kHz and a maximum rated electrical power output of 132 W. The ultrasonic transducer (surface area of 282.2 cm2) is fitted at the bottom of the bath, horizontally along the length, which has been considered a suitable ultrasonic system [11]. Typically, around 20 g of the oil was charged into the reaction vessel, and then precise amounts of oil to ethanol molar ratio and enzyme (according to the experimental design) were also weighed on a precision scale balance (Ohaus Analytical Standard with 0.0001 g accuracy) and loaded into the reaction vessel, immersed in the ultrasonic bath already stabilized at the pre-established experimental temperature. The flask was then closed and the ultrasonic system turned on. 2.4. Experimental design and kinetic evaluation At first, a fractional 241 experimental design with triplicate runs in the central point with 11 assays, was employed to evaluate the effects of four variables e temperature (40e70 C), enzyme concentration (5e20 wt%, by weight of substrates), oil to ethanol molar ratio (1:3e1:10) and output irradiation power (40e70% of the maximum supply value), keeping the reaction time fixed at 90 min. The software Statistica® 8.0 (Statsoft Inc., USA) was used to assist the design and statistical analysis of the reaction yield. Taking into account the results obtained from the execution of the experimental design, reaction kinetic experiments were performed adopting temperature of 65 C, 100% of total ultrasound power (132 W), while varying enzyme concentration of 5 and 10 wt%, keeping fixed the substrates molar ratio at 1:9. It may be important to emphasize that in all cases, destructive experiments, without sampling, were carried out. 2.5. Analytical methods Samples were first submitted to ethanol evaporation to constant weight in a vacuum oven (338 K, 0.05 MPa) and then diluted with 2 mL of ethanol and 8 mL of n-heptane. After this, 50 mL of solution was transferred to a 1 mL volumetric flask, adding 50 mL of internal standard methyl heptadecanoate at concentration of 5000 mg L1 and filled with n-heptane. After that, 1 mL of solution was injected in triplicate in a gas chromatograph (Shimadzu GC-2010), equipped with FID, auto-injector AOC-20i and a capillary column (Rtx-WAX, 30 m 0.25 mm 0.25 mm), with split ratio of 1:50. Column temperature was programmed from 393 K, holding 1 min, heating to 453 K at 15 K min1, holding 2 min, and to 523 K at 5 K min1, holding 2 min. Synthetic air and nitrogen was used as carrier gas, and the injection and detector temperatures were 523 K. Compounds were quantified upon analysis following the standard UNEEN 14103 [12] and FAEE content was then calculated based on the content of ethyl esters in the analyzed sample. 2.6. Convertibility and conversion efficiency Considering that raw vegetable oils could contain several compounds non-convertible to alkyl esters, the maximum conversion achievable from these oils may not correspond to 100%. Thus, in order to determine this maximum conversion, it was defined here the “convertibility” parameter, and for this purpose a sample of the MCO was treated using a quantitative conversion method (based on
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the use of BF3 as catalyst) as described below. Around 40e90 mg of oil was placed inside a screw cap tube, where 1.5 mL of methanolic solution of NaOH 0.5 N were aggregated, air inside the tube was replaced by N2, it was hermetically closed and heated at 373 K for 10 min, maintaining the reaction mixture under magnetic stirring. After 1 min cooling, 2 mL of methanolic solution of BF3 14% were added and then heated at 373 K for 5 min. Afterward, 2 mL of nhexane solution of methyl heptadecanoate with a concentration of 5 mg/mL was added as internal standard. Finally, 5 mL of a saturated NaCl solution was added and after mixture decantation the superior phase was separated, dehydrated with sodium sulfate anhydrous and centrifuged. This last solution was analyzed by gas chromatography (GC) in a Shimadzu GC-14B chromatography equipped with flame ionization detector (FID) and a capillary column SGE BPX70. The temperature program started at 433 K and then it was raised at a 4 K min1 rate until 503 K, holding that temperature for 10 min. By this method all the fatty acids were converted to their corresponding alkyl esters, independently of the form that these fatty acids are found in the sample. The fatty acid composition of the starting MCO was determined using this same procedure, but adding 2 mL of pure n-hexane instead of the solution of internal standard. The convertibility was determined from the area percentage obtained from the GC analysis, using the methyl heptadecanoate peak as the internal standard, as follows:
P Convertibilityðwt%Þ ¼
A AIE CIE VIE 100 AIE m
Alkyl ester yield 100 Convertibility
Fatty acid
Content (wt%)
Lauric acid (12:0) Myristic acid (14:0) Palmitic acid (16:0) Palmitoleic acid (16:1) Estearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linolenic acid (18:3)
0.7 0.3 22.2 4.2 1.9 58.0 9.7 0.6
analysis, is presented in Table 1 where it can be noticed the predominance of oleic acid (58.0 wt%), which is similar to the fatty acid profile found by Fortes and Baugh [13]. The acid value, water content (wt%) and polymer content of MCO were determined as 88.9 mgKOH/g oil, 0.64 and of 6.1%, respectively. The acid value found in this work is in agreement with those reported by Rodrigues [14] and Gonzalez [15], up to 90 mgKOH/g oil. Some physicochemical parameters were measured at 25 C: density (0.9273 ± 6.84.104 g cm3), thermal conductivity (0.115 ± 5.103 W m1 C1) and viscosity (61.75 mPa s). The convertibility of MCO was determined to be 80.1%, thus oil contained 19.9 wt% of compounds that would not contribute to the ester content, independently of the efficiency of the conversion process used.
(1)
P where: A is the total area in the GC chromatogram, AIE, CIE and VIE denote, respectively, the area, concentration (mg/mL), and volume (mL) of the internal standard, and m is the weight of MCO. In order to evaluate the real efficiency of the process it is convenient to refer the alkyl ester yield to the convertibility, thus with this purpose the “Conversion efficiency” is defined as:
Conversion efficiencyðwt%Þ ¼
Table 1 Fatty acid composition of the Macauba oil.
(2)
Hence, if the process efficiency is high the Alkyl ester yield would achieve a level near the Convertibility and the Conversion efficiency would reach a value near 100%. 2.7. Reuse of biocatalyst To evaluate the reuse of Novozym 435 it was adopted the experimental condition of oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme concentration (by weight of substrates), reaction time at 30 min and at full ultrasound irradiation power amplitude (100%). Enzyme recuperation at the end of each reaction was performed by filtration in filter paper followed by two 10 mL n-hexane washings under moderate vacuum, then submitted to rotary evaporation (Fisatom, model 550) and dried in oven (JP 101, J. Prolab) at 40 C for 1 h. Recuperated enzyme was then kept in desiccator for 24 h prior to measurement of its activity and reutilization. The residual relative esterification activity was defined as the ratio of (Final activity/Original activity) 100 and determined after each cycle of utilization of the enzyme. 3. Results and discussion 3.1. MCO characterization The chemical composition of major fatty acids present in the MCO used in this study, determined by gas chromatography
3.2. Production of fatty acid ethyl esters (FAEE) Table 2 shows the results of the experimental design and as it can be seen reaction yields in the range of 35e50% were reached. It can be observed from Fig. 1 that with a confidence level of 95%, the enzyme content was the only significant variable affecting positively the transesterification yields, which agrees with the recent result found by Batistella et al. [16], in studying biodiesel enzymatic production in ultrasound system. Taking into account such fact, the next step toward improving reaction yield efficiency was to conduct a kinetic study keeping fixed the oil to ethanol molar ratio at 1:9, temperature of 65 C and at the maximum output irradiation, 132 W, while varying the enzyme content, 1, 5, 10 and 20 wt%, also performing destructive, independent, experiments at 1, 3, 5, 7, 10, 15, 30, 60, 90, 120, 180, 240 and 360 min. Results for the reactions kinetics are depicted in Fig. 2, where it can be noticed that a raise in enzyme content led to an increase in initial reaction rates. It can also be noted that for all the enzyme contents tested, the maximum yield is reached at 1 h reaction and
Table 2 Matrix of the experimental design (coded values) with answers in terms of FAEE yield (reaction time fixed at 90 min).a Run
T
O:E
[E]
IA
FAEE yield
1 2 3 4 5 6 7 8 9 10 11
(1) 40 (1) 70 (1) 40 (1) 70 (1) 40 (1) 70 (1) 40 (1) 70 (0) 55 (0) 55 (0) 55
(1) 1:3 (1) 1:3 (1) 1:10 (1) 1:10 (1) 1:3 (1) 1:3 (1) 1:10 (1) 1:10 (0) 1:6.5 (0) 1:6.5 (0) 1:6.5
(1) 5 (1) 5 (1) 5 (1) 5 (1) 20 (1) 20 (1) 20 (1) 20 (0)12.5 (0)12.5 (0)12.5
(1) 40 (1) 100 (1) 100 (1) 40 (1) 100 (1) 40 (1) 40 (1) 100 (0) 70 (0) 70 (0) 70
38.6 38.5 42.0 35.6 47.1 45.0 48.7 42.8 46.8 47.4 48.8
a Temperature ( C), T; Oil:ethanol molar ratio, O:E; Enzyme concentration (wt%), [E]; Irradiation amplitude (%), IA; FAEE yield (wt%).
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maximum reaction conversion of 45% for 25 wt% enzyme concentration, 1:9 of oil to alcohol molar ratio and temperature of 30 C in 15 min. According to those authors, mass transfer problems happened due to inefficient agitation of the reaction content and thus better results were found for lower enzyme concentrations, which in turns prevented catalyst agglomeration.
3.3. Enzyme reuse and ultrasound influence on FAEE yield
Fig. 1. Pareto chart for the fractional 241 experimental design of MCO transesterifications using Novozym 435 under ultrasound irradiation for a reaction time of 90 min (see Table 1). Experimental conditions: Temperature ( C), T; Oil:ethanol molar ratio, O:E; Enzyme concentration (wt%), [E]; Irradiation amplitude (%), IA; FAEE yield (wt%).
then a nearly asymptotic behavior is noticed at larger times for all conditions. Hence, from a practical standpoint, the reaction might be interrupted to meet economic aspects - small gains after a certain time. Fig. 2 also shows that reaction yields up around 70% were achieved in only 30 min for the highest enzyme content, which on the basis of the convertibility parameter means in fact conversion efficiency values as high as 90% [17,18]. It is worth noting the good reproducibility of the experimental results found for the enzyme content of 20 wt%, hence pointing the reliability of the experimental information. Data scattering observed in this figure may be explained in terms of experimental errors associated, and the fact that destructive experiments were carried out without sampling, which may be viewed as an important internal consistence test of the results. Recently, Nogueira et al. [9], in investigating the effect of microwave irradiation on the transesterification of Macauba oil using Novozym 435 as catalyst and ethanol as substrate, reached a
Fig. 2. Kinetics of MCO transesterification with ethanol using Novozym 435 as catalyst. Experimental condition: oil to ethanol molar ratio of 1:9, temperature of 65 C, 100% ultrasonic power at varying enzyme contents.
Undoubtedly, enzyme reuse is a key issue for enzymatic process feasibility and accordingly several reaction cycles for Novozym 435 were attempted under ultrasound irradiation. Before performing such assays however, the influence of ultrasound utilization was studied and Fig. 3 shows the reaction kinetics with and in the absence of ultrasound irradiation. As can be seen from this figure, much better results can be obtained using ultrasound power supply, especially for longer times. Afterward, enzyme reuse was tested at 100% ultrasound irradiation amplitude, oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme concentration, and reaction time 30 min Fig. 4 presents such results in terms of FAEE yield and also enzyme activity as a function of cycle number. It can be seen that a monotonically enzyme activity loss coupled with a similar decrease in FAEE yield is noticed as number of reuse raises. According to Wang et al. [19] the cost involved in enzyme immobilization together with cycle utilization should be taken into account in the global economic analysis of an industrial biodiesel plant. It has been argued by Rahman et al. [20] that lipases may lose its activity in non-aqueous medium and selectivity and stability may also be affected. Repeated cycles of Novozym 435 were adopted by Hajar et al. [21] in the methanolysis of canola oil in solvent-free system and the authors noted that after 5 cycles and 432 h reaction, reaction conversion was kept at 97%, but, unfortunately, enzyme activity was not reported in such research work. Of course, enzyme stability is a relevant parameter and further studies should be conducted to keep enzyme activity for practical purposes. As a matter of fact, one problem arising from enzyme reuse in transesterification reactions is the low solubility of glycerol, in the product mixture. This has a strong negative effect, expressed as a blocking effect, on enzyme activity. This effect has been attributed to the deposition of glycerol on the enzyme surface.
Fig. 3. Effect of ultrasound use on enzymatic biodiesel production from MCO in solvent-free system. Reaction conditions: Novozym 435 content of 20 wt%, oil to ethanol molar ratio of 1:9, at 65 C.
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interesting and also potentially economic if a well-balanced reaction system is considered.
Acknowledgments The authors thank CNPq (process 490061) for the financial support of this work and scholarships.
References
Fig. 4. Reuse of lipase in the transesterification of MCO with ethanol using Novozym 435 as catalyst. Experimental condition: oil to ethanol molar ratio 1:9, 65 C, 20 wt% enzyme, reaction time at 30 min, ultrasound irradiation amplitude (100%).
It is noticed that the ultrasound waves can interfere with the characteristics of the enzyme, changing its structure as a response to the dynamic perturbation caused by the ultrasound [22]. Just a few studies about the effect of the ultrasound under the enzyme activities are related in the literature and contradictory results of activation/deactivation have been presented. Different from the traditional inactivation by the high temperatures, the sonication process does not affect the active sites of the enzyme. This observation has already been demonstrated for a-amylase, peroxidase, laccase and alkaline-phosphatase [23,24]. However, to date, the true potential of ultrasound is not well known, due to the lack of knowledge of the exact molecular effects of the ultrasound on enzymes and living cells. Specifically for the enzymes, the ultrasound has shown increases on stability and catalytic activity of the enzymes. Nevertheless, a better comprehension of the effect of the ultrasound on the enzyme properties is necessary to develop more efficient catalysts with higher activities and stabilities [25]. 4. Conclusions This work confirms the good efficiency of ultrasound-assisted system to convert Macauba oil into alcoholic esters using a commercial lipase as catalyst, hence pointing the promising use of a low-cost, non-edible oil, with very satisfactory field productivity, towards biodiesel production with an eco-friendly, green technique. The convertibility and the conversion efficiency parameters helped to better specify the extent of the esters conversion reaction in the lipase-catalyzed, ultrasound irradiation transesterification process by establishing a “theoretical” maximum esters yield of 80.1% and a “real” conversion efficiency of around 90% for ethanol. Due to the presence of 19.9% of “non-convertible” compounds in the starting oil additional processing steps (e.g. distillation) should be considered in order to achieve higher ester contents in the final product. Although such steps would impact in the overall process costs, it could be absorbed by the relatively low value of the raw material. Of course, further work must be done in order to identify the nature of the non-convertible compounds, which is underway by our research group. Regarding ultrasound influence it was shown that this new technology may be advantageously used for transesterification reactions to provide higher yields in shorter reaction time. Reuse of enzyme by its turn may also be technically
[1] Lorenzi GMAC. Acrocomia aculeata (jacq) lodd. ex mart e arecaceae: bases vel [Dr. thesis]. Brazil (in Portuguese): Federal para o extrativismo sustenta University of Paran a; 2006. [2] Moura EF, Ventrella MC, Motoike SY. Anatomy, histochemistry and ultrastructure of seed and somatic embryo of Acrocomia aculeata (Arecaceae). Sci Agri 2010;67:399e407. ^ncia de macaúba em [3] Motta PEF, Curi N, Oliveira-Filho AT, Gomes JBV. Ocorre ~o com atributos clima ticos, pedolo gicos e vegetacionais. Minas Gerais: relaça Pesqui Agropecu Bras 2002;37:1023e31. [4] Andrade MHC, Vieira AS, Aguiar HF, Chaves JFN, Neves RMPS, Miranda TLS, ~o potencial para et al. Oleo do fruto da palmeira macaúba parte I: uma aplicaça ticos. In: II ENBTEQ e Encontro indústrias de alimentos, f armacos e cosme Brasileiro sobre Tecnologia na Indústria Química/III Semin ario ABIQUIM de Tecnologia. S~ ao Paulo: ABEQ; 2006. [5] Andrade MHC, Vieira AS, Aguiar HF, Chaves JFN, Neves RMPS, Miranda TLS, ~o do et al. Oleo do fruto da palmeira macaúba parte II: processo de extraça leo. In: II ENBTEQ e Encontro Brasileiro sobre Tecnologia na Indústria Quío rio ABIQUIM de Tecnologia. Sa ~o Paulo: ABEQ; 2006. mica/III Semina [6] Caires EF, Ferrari RA, Morgano MA. Produtividade e qualidade da soja em ~o da calagem na superfície em semeadura direta. Bragantia 2003;62: funça 283e90. [7] USDA, Foreign Agricultural Service (FAS). GAIN BR0707 Brazil e 2011 annual oilseeds report, Global Agricultural Information Network (GAIN) on-line, 04/ 04/2011. [8] Rodrigues HS. Production of ethyl and methyl esters by transesterification reaction of the latin american palm macauba e Acrocomia aculeate [Dr. thesis]. ~o Paulo; 2007. Brazil (in Portuguese): University of Sa lix R, et al. [9] Nogueira BM, Carretoni C, Cruz R, Freitas S, Melo-Junior PA, Costa-Fe Microwave activation of enzymatic catalysts for biodiesel production. J Mol Cat B Enzym 2010;67:117e21. [10] Liu Y, Jin Q, . Shan L, . Liu Y, . Shen W, . Wang X. The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system. Ultrason Sonochem 2008;15:402e7. [11] Fiametti KG, Ustra MK, Oliveira D, Corazza ML, Furigo Jr A, Oliveira JV. Kinetics of ultrasound-assisted lipase-catalyzed glycerolysis of olive oil in solvent-free system. Ultrason Sonochem 2012;19:440e51. [12] UNE-EN 14103:2003. Fat and oil derivatives. Fatty Acid Methyl Esters (FAME). Determination of ester and linolenic acid methyl ester contents. Madrid, Spain: AENOR; 2003. [13] Fortes ICP, Baugh PJ. Pyrolysis-GC/MS studies of vegetable oils from Macauba fruit. J Anal Appl Pyrolysis 2004;72:103e11. ~o de e steres etílicos e metílicos, por reaço ~es de trans[14] Rodrigues HS. Obtença leo da palmeira Latino Americana macaúba- Acroesterificaç~ ao, a partir do o comia aculeata [Thesis (Sciences-Chemistry)]. Ribeir~ ao Preto: University of ~o Paulo; 2007. p. 236. Sa ~o contínua de biodiesel por transesterificaç~ leo do [15] Gonzalez SL. Produça ao de o leo de fritura em metanol e em fruto de macaúba (Acrocomia aculeata) e o polis: etanol supercrítico [Doctoral Qualifying (Food Engineering)]. Floriano Federal University of de Santa Catarina; 2011. p. 68. [16] Batistella L, Lerin LA, Brugnerotto P, Danielli AJ, Trentin CM, Popiolski A, et al. Oliveira, Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system. Ultrason Sonochem 2012;19:452e8. [17] Navarro-Díaz JH, Gonzalez SL, Irigaray IB, Vieitez I, Jachmani an I, Hense H, et al. Macauba oil as an alternative feedstock for biodiesel: characterization and ester conversion by the supercritical method. J Supercrit Fluids 2014;93: 130e7. [18] Gonzalez SL, Sychoski MM, Navarro-Díaz HJ, Callejas N, Saibene M, Vieitez I, et al. Continuous catalyst-free production of biodiesel through transesterification of soybean fried oil in supercritical methanol and ethanol. Energy Fuels 2013;27:5253e9. [19] Wang X, Liu X, Zhao C, Ding Y, Xu P. Biodiesel production in packed-bed reactors using lipase-nanoparticle biocomposite. Biores Technol 2011;102: 6352e5. [20] Rahman MBA, Jumbri K, Hanafiah NAMA, Abdulmalek E, Tejo BA, Basri M, et al. Enzymatic esterification of fatty acid esters by tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase. J Mol Catal B Enzym 2012;79:61e5. [21] Hajar M, Shokrollahzadeh S, Vahabzadeh F, Monazzami A. Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa. Enzyme Microb Technol 2009;45:188e94.
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