Enzymatic Synthesis of Biodiesel Using Immobilized Lipase on a Non

Article pubs.acs.org/EF

Enzymatic Synthesis of Biodiesel Using Immobilized Lipase on a Non-commercial Support T. A. Costa-Silva,*,† A. K. F. Carvalho,‡ C. R. F. Souza,† H. F. De Castro,‡ S. Said,† and W. P. Oliveira† †

School of Pharmaceutical Sciences of Ribeirão Preto, Laboratory of Pharmaceutical Processes (LAPROFAR), University of São Paulo, Ribeirão Preto, Brazil ‡ Engineering School of Lorena, University of São Paulo, Lorena, São Paulo, Brazil ABSTRACT: In this work was produced an immobilized biocatalyst from Cercospora kikuchii lipase with beneficial catalytic activity and stability. The immobilized derivative was used in the ethanolysis of coconut oil. This is a great option for the Brazilian renewable fuel production, because both coconut oil and ethanol are promptly available in the country, at a reasonably low cost. For this, lipase was immobilized on rice husk (5.5 mgprotein g−1support) by covalent binding using spouted bed drying (gas flow rate, 0.66 m3 min−1; temperature, 100 °C; and solution flow rate, 5.5 g min−1). In a typical batch run, the immobilized derivative was added at a vegetable oil/ethanol molar ratio of 1:12 using tert-butanol as the solvent. The feasibility of immobilization and drying processes was demonstrated toward biodiesel production, attaining a high ester content (97.1%) and low levels of acylglycerides (0.9%) after 72 h. Further properties, such as viscosity (4.56 mm cm−2) and density (888 kg m−3), are in agreement with specifications required by the ASTM D6751 to be used as alternative biofuel.

1. INTRODUCTION Biodiesel is important energy source alternative to conventional diesel as a result of rises in petroleum prices and environmental deterioration (with carbon dioxide emission responsible for the greenhouse effect). This alternative fuel, although coming from renewable sources, is routinely obtained by a conventional chemical route (homogeneous catalysis, basic or acidic). Nevertheless, some drawbacks are connected with this chemical process, such as hardship in glycerol retrieval, high energy cost, the catalyst must be separated from the biodiesel, and effluent treatment.1 As a result of these disadvantages, heterogeneous catalyst systems employing immobilized lipases have obtained great interest over the last few years. Lipase-catalyzed transesterification has become more attractive as a result of some advantages, such as high selectivity (separation and purification processes are facilitated), mild operative conditions (lower energy cost), and environmentally friendly.2,3 In this context, the enzyme immobilization technique plays a beneficial role within applied enzymology.2 The main motive is the possibility to retain and recover the biocatalysts, allowing for the enzyme separation from final product, thereby enabling the achievement of continuous processes.4,5 Several distinct methods for the lipase immobilization have been established by some researchers, including adsorption (on the basis of electrostatic interactions), covalent binding (defined by covalent bond formation between some reactive groups of the matrix and those of the enzyme surface), encapsulation, and entrapment. The main factors that could affect the final enzyme properties are the type of immobilization process and the enzyme characteristics. However, the choice of the support material is another important element influencing the final properties of immobilized derivatives produced.2,6 Despite the fact that one support matrix adequate for all enzymes and their specific industrial applications does not exist, all matrices that are to be considered as an enzyme carrier should satisfy some © 2016 American Chemical Society

operational and quality requirements: presence of functional groups with a high affinity for enzymes, structural stability and rigidity, grand internal surface, capacity of regeneration, nontoxicity, and biodegradability.7 The majority of available commercial supports (silica-based supports and synthetic polymers) is very expensive. In this context, several studies have focused on evaluation of cheaper carriers, such as CaCO3,8 rice husk,9−11 chitin,12 and chitosan.13 The study of enzymes in organic media has expanded dramatically in the past decade, increasing economic exploitation and benefits of these molecules. It has allowed for the efficient use of lipases beyond the traditional hydrolysis system, for example, in esterification and transesterification reactions.14 The low water content is a common feature in all of these media, and it is a special element affecting the catalytic property of a biocatalyst (especially lipase) in a non-aqueous medium. Examples of hurtful effects of water are as follows: hindering the enzyme−substrate interaction and diffusion barrier formation (mainly for hydrophobic substrates) and increasing the hydrolysis reaction rate.14 Consequently, the water content impact on biodiesel conversion should be monitored because the effects are so large. The enzyme immobilization process is generally carried out in the presence of water. Different drying techniques (freeze drying, air drying, or spray drying) are used to dry the immobilized derivatives obtained before the industrial application (especially in organic media). However, several scientific reports have been shown that the dehydration step can induce a severe loss of enzyme activity as a result of heat-induced structural changes. Therefore, the development of alternative processes for dehydrating the immobilized enzyme becomes highly relevant Received: February 1, 2016 Revised: May 10, 2016 Published: June 3, 2016 4820

DOI: 10.1021/acs.energyfuels.6b00208 Energy Fuels 2016, 30, 4820−4824

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according to Costa-Silva et al.17 Teflon was used as seed particles during the drying step (material with a mean diameter of 5.45 mm and density of 2160 kg m−3). Table 1 shows the operating conditions of

to ensure the quality parameters for dried enzyme products (mainly retention of activity and enzyme stability). Herein, we used spouted bed for drying and immobilization of the enzyme in a single step. The study for cheap carriers has motivated our research group to select materials that can be recognized (at the molecular level) as solid surfaces by enzymes.10 Therefore, the aim of this work was to obtain an immobilized derivative of lipase from endophytic fungus Cercospora kikuchii with beneficial catalytic activity and stability. The enzyme was covalently immobilized onto rice husk (non-commercial matrix) using a spouted-bed drying technique. This byproduct presents some advantages as a lipase immobilization support: biodegradability (eco-friendly), abundance, inexpensiveness, easy to handling, and nontoxicity. The characterization of immobilized lipase was carried out (biochemical properties and stability), and it was evaluated for biodiesel production.

Table 1. Spouted-Bed Parameters Set for Drying of the Enzyme−Support System spouted-bed drying parameters inlet gas temperature, Tgi (°C) drying gas flow rate, Wg (m3 min−1) feed system position mass feed flow rate, Ws (g min−1) static bed height, H0 (cm) mass of inert material, Mi (g)

100.0 0.66 top spray 5.5 5.5 255.0

the dryer equipment used for immobilized derivative dehydration. First, the spouting air (0.66 m3 min−1) was heated at 100 °C. Therefore, feeding (flow rate of 5.5 g min−1) of the drying solution (lipase + rice husk, 5.5 mgprotein g−1support) into the spouted-bed column was started. The following methods were used to evaluate the dryer performance and product properties. 2.5.1. Enzymatic Activity. The lipase activity assay was carried out according to section 2.2. The activity retention (RAE, %) was determined according to eq 1.

2. MATERIALS AND METHODS 2.1. Lipase Production. The endophytic fungus C. kikuchii was used for lipase production using soybean oil as a unique carbon source. Lipase production and purification were carried out according to Costa-Silva et al. and used for the immobilization process.11 2.2. Determination of Hydrolytic Activity. Hydrolytic activities of lipases (soluble and immobilized forms) were determined using olive oil as a substrate.15 A total of 1 international unit (IU) of activity was defined as the amount of enzyme that liberates 1 μmol of free fatty acid per minute under the assay conditions. The protein content was carried out according to the method of Bradford.16 2.3. Support Preparation. Agricultural byproduct, rice husk, in nature was supplied by local farmers. This support matrix was then milled (particle sizes of 50−150 mesh), washed with distilled water, and oven-dried at 60 °C before their use in the immobilization process.17 Cellulose microcrystalline (MC-102, Blanver, São Paulo, Brazil) was also used as a support. Figure 1 shows the agricultural byproduct powder used as a support for enzyme immobilization.

RAE (%) = 100 ×

immobilized enzyme activity (units/mg) soluble enzyme activity (units/mg)

(1) 2.5.2. Product Stability. The residual enzyme activity of the dried powder obtained (immobilized derivatives) was assessed after 6 months (stored at 5 °C). 2.5.3. Operational Stability (Reuse). RAE for immobilized lipase after 5 cycles of reaction was carryout using olive oil emulsion as the substrate, according to the methodology described by Soares et al.15 2.5.4. Water Content (Xp). The water content of the final product was determined according to the method of Norenã et al.18 and the World Health Organization (WHO).19 2.5.5. Shape and Surface Characteristics. Analysis of immobilized derivative morphology was performed by scanning electron microscopy (SEM, model EVO-50, Zeiss, Cambridge, U.K.), with 5000.11 2.5.6. Immobilized Derivative Production. The product recovery (RE) was defined as determined according to the methodology described by Costa-Silva et al.17 2.6. Biodiesel Synthesis and Analysis. Immobilized lipase from C. kikuchii was used for biodiesel production. Batch reactions were performed in a jacketed cylindrical glass reactor (60 mm high × 40 mm internal diameter and 50 mL capacity, coupled with a reflux condenser) containing 30 g of substrate consisting of coconut oil/ anhydrous ethanol at a molar ratio of 1:12 in the presence of tertbutanol as a solvent. The mixtures were incubated with the immobilized derivatives at proportions of 20% (w/w) in relation to the total weight of reactants involved in the reaction media. The experiments were carried out at 50 °C for a maximum period of 120 h under a constant magnetic agitation of 150 rpm.3 For the time course studies, an aliquot of reaction medium was taken at various time intervals and analyzed by gas chromatography following previous established conditions.20,21 The theoretical ester concentrations were calculated by taking into account the coconut fatty acid composition and its initial weight mass in the substrate, and the transesterification yield (%) was defined as the ratio between the produced and theoretical ester concentrations.20,21 At the end of the reaction, the immobilized lipase was recovered by centrifugation (1550g) and the collected samples were purified according to the methodology described by Carvalho et al.21 and then evaporated by a rotary evaporator at 80 °C to eliminate the residual solvents. The purified biodiesel samples were dried by adding sodium sulfate, and absolute viscosity values were determined by a LVDVII Brookfield viscometer (Brookfield Viscometers, Ltd., U.K.) at

Figure 1. Agricultural byproducts used as a support for lipase immobilization: (A) rice husk and (B) microcrystalline cellulose (control). 2.4. Support Activation Procedures and Lipase Immobilization. The supports were activated using glutaraldehyde prior to the covalent immobilization method. Activation of supports was carried out by adding 5 g of rice husk powder in 100 mL of glutaraldehyde solution (0.5, 1.5, and 2.5%, v/v) at room temperature for 1 h. The activated byproduct was recovered by filtration, washed to expunge the surplus of activating agent, and dehydrated for 24 h at 60 °C. C. kikuchii lipase was covalently immobilized in the activated supports in the presence of polyethylene glycol (PEG, 1.5%, p/v) as a stabilizing agent.17 Then, the immobilized derivative solution was submitted to spouted-bed drying. 2.5. Drying of Immobilized Derivatives. Immobilized derivatives were dried using a homemade conical−cylindrical spouted bed 4821

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Energy & Fuels Table 2. Results of Dryer Performance and Immobilized Derivative Propertiesa support rice husk

microcrystalline cellulose

GL (%) 0.5 1.5 2.5 0.5 1.5 2.5

Tgo (°C) 78.5 79.8 78.8 80.8 79.3 81.8

± ± ± ± ± ±

0.3 0.7 1.1 0.5 0.9 0.3

Tin (°C) 60.1 59.5 60.4 59.4 60.1 60.8

± ± ± ± ± ±

0.5 0.1 0.2 1.3 0.9 0.2

RE (%) 76.0 74.6 75.0 78.3 77.0 79.8

± ± ± ± ± ±

1.2 0.9 1.0 0.2 0.6 0.8

RAE (%) 140.3 177.1 131.9 130.3 170.8 129.0

± ± ± ± ± ±

0.5 0.7 0.9 0.3 0.4 1.1

Mc (%) 4.78 4.30 4.58 3.91 3.18 3.36

± ± ± ± ± ±

0.27 0.31 0.40 0.17 0.87 0.24

aw 0.22 0.23 0.27 0.18 0.14 0.16

± ± ± ± ± ±

0.03 0.02 0.05 0.01 0.03 0.04

a

GL, glutaraldehyde concentration; RAE, residual enzyme activity; RE, process yield; Tgo, outlet drying gas temperature; Tin, bed temperature; Mc, moisture content (%); and aw, water activity. The responses were calculated from triplicate analyses.

40 °C using a 1.0 mL aliquot of the sample. The biodiesel density was determined by a DMA 35N EX digital densimeter (Anton Paar) at 20 °C using 2.0 mL of the sample.21 Analysis of acylglycerides was performed using an Agilent 1200 series liquid chromatograph (Agilent Technologies, Santa Clara, CA).22

in solution (free form), the final lipase activity was only 12%. These results show the efficiency of the immobilization/drying process to ensure the enzyme stability (positive effects). The moisture content (Mc) in dried powders varied between 3.18 and 4.78%, and the water activity (aw) was in the range of 0.16−0.23. The low water content values are relevant because the dehydration could ensure a protection to the biological activity of these molecules. It was proven by the final activity obtained after the storage period (6 months): rice husk (residual activity of 86%) and microcrystalline cellulose (residual activity of 82%). Herein, the operational stability of C. kikuchii immobilized lipase was carried out under batch recycles using olive oil as the substrate.15 The result showed that the immobilized derivative prepared by the immobilization/drying process retained an average of 85.7% of the initial activity after 5 reuse cycles. The morphology of the immobilized derivatives was analyzed by micrographs obtained by SEM, as shown in Figure 2. From

3. RESULTS AND DISCUSSION Dry enzyme formulations should stay stable for a long period, with the active structure of lipase remaining, even under adverse circumstances (mainly during potting, transport, or storage period). However, the characteristics of enzymes are so specific (size, active site, kinetic model for enzyme activity, permissible loss of biological activity, application, etc.) that the drying process of each product should be considered on an individual basis. Lyophilization (freeze drying) is one of the most frequent techniques for enzyme dehydration but is a relatively costly and time-consuming process. Spouted-bed drying, in contrast, can produce a dried powder in a unique step process. This atomization drying method is based in the feed of liquid formulation into a bed containing inert bodies (spouted by hot gas), leading the enzyme to immediately dry into solid particles. This drying technique has been considered as an preferential process to freeze drying and spray drying in an attempt to generate high-quality solid products at a relatively inexpensive cost.23,24 Several enzyme-engineering methods now focus on the immobilization technique because of the enhanced operational stability and economic advantages.8−11 With the high price of some commercial supports taken into account, studies have been undertaken by several groups to obtain inexpensive matrices.10,25 Herein, the agricultural byproduct was valued as an inexpensive support for the production of C. kikuchii lipase immobilized derivatives using a cross-linking method and spouted-bed drying. The objective was to collaborate toward searching for an alternative cheap matrix and low-cost dehydration process capable of promoting optimum lipase performance and stabilization. Table 2 shows the impact of the matrix and activating agent concentration on the outlet drying gas temperature (Tgo), temperature inside the spouted-bed dryer (Tin), process yield (RE), and residual enzyme activity (RAE) of the product. The spouted bed showed high efficiency of immobilized enzyme derivative production. The yields of immobilized lipase derivative production fall in the range of 74.6−79.8%, depending upon the support and the used glutaraldehyde concentration. Among all preparations evaluated, those containing 1.5% glutaraldehyde showed the best result because they increased in all assays at least 70% of initial activity after spouted-bed drying. The immobilized derivative stability, obtained using of 1.5% glutaraldehyde, was determined during the storage period. The residual enzyme activity of immobilized derivatives evaluated was an average of 75%, while for enzyme

Figure 2. Scanning electron photomicrographs of the immobilized enzyme derivatives: (A) rice husk and (B) microcrystalline cellulose.

SEM, it was possible to verify the morphological differences between the particle obtained by the use of rice husk or microcrystalline cellulose as the support. Both supports are formed by microparticles having distinct arrangements and unequal size. For rice husk, several fissures were found, which may aid the lipase fixation on the byproduct surface. The microparticle size ranged from 1 to 11 μm in all immobilized derivatives obtained. Cellulose microcrystalline was used as a model support for the immobilization step because it is a purer material compared to rice husk and could be used to explain (less interference) the property modification in the enzyme structure. However, the 4822

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yield (98.1%) was attained at 72 h of reaction, and no reversible reaction was observed until 120 h. A correlation between the ethyl ester composition and fatty acids present in the coconut oil was observed (Table 3), especially the ethyl laurate and

results for residual enzyme activity (RAE) were very similar, and further experiments, mainly industrial application, were performed using the byproduct support. The biodiesel production may appear as a simple industrial process, but the process can suffer from some drawbacks, which could be solved before enzyme-catalyzed transesterification. Some of the biodiesel process drawbacks are discussed below and the way that can become economically viable to compete with chemical catalysts. One manner of minimize the problems is the enzyme immobilization process, especially covalent coupling.2 The use of immobilized enzymes by covalent coupling is attractive from an industrial point of view because it improves its stability under distorting conditions, such as the temperature, pH, and organic solvents. Moreover, the immobilization technique facilitates biocatalyst recovery (therefore, enzyme reuse) and improves the product quality; thus, it is beneficial for economic aspects.2,5,6 Herein, biodiesel synthesis was carried out using immobilized C. kikuchii lipase by covalent attachment on rice husk activated with 1.5% glutaraldehyde and coconut oil as feedstock. The advantage of using this crop is due to its high abundance in Brazil, especially northeast Brazil. Besides, in Brazil, coconut oil is a residue of the production-grated coconut and coconut milk; this fact could avoid the competition of coconut use in the food area.26 Finally, the coconut oil characteristics (high amount of lauric acid of 47.1%) support their use for biodiesel production, because the predominance of short carbon chain fatty acids allows for a more effective interaction (low stearic effect) between ethanol and the biocatalyst in transesterification reactions.26−28 The environmental factors were taken into account, both the possible lack of mineral fuels and its impact on health and the environment itself. Thus, ethanol was used to obtain the biodiesel production from fully renewable sources. From this perspective, the biodiesel production by enzymatic transesterification (using lipase immobilized into eco-friendly supports) from coconut oil and ethanol stands as a promising way of renewable energy utilization in our perspective.29 Figure 3 displays the dynamic behavior of the fatty acid ethyl ester (FAEE) formation and the corresponded FAEE yield (%), along with the experiment duration (120 h). The highest FAEE

Table 3. Ethyl Ester Profile and Yield Obtained in the Biodiesel Production from Coconut Oila Using Lipase from C. kikuchii Immobilized onto Rice Husk (72 h) ethyl esters

wt %

ethyl caprylate C8:0 ethyl capronate C10:0 ethyl laurate C12:0 ethyl myristate C14:0 ethyl palmitate C16:0 ethyl stearate C18:0 ethyl oleate C18:1 ethyl linoleate C18:2 total esters (wt %) yield (%)

6.5 ± 0.01 4.8 ± 0.02 25.5 ± 0.24 11.8 ± 0.05 3.7 ± 0.04 1.6 ± 0.05 3.5 ± 0.21 0.65 ± 0.05 57.6 ± 0.02 98.1 ± 0.02

a

Coconut oil fatty acid composition: 8.9 wt % caprylic acid, 6.2 wt % capric acid, 47.1 wt % lauric acid, 18.8 wt % mirystic acid, 7.9 wt % palmitic acid, 2.6 wt % stearic acid, 6.2 wt % oleic acid, and 1.6 wt % linoleic acid.

myristate esters, which are the fatty acids present at the highest proportion in this feedstock. This suggests that the biocatalyst has higher specificity to catalyze the cleavage of saturated shortchain fatty acids, such as those found in the coconut oil. This behavior agrees with that observed by Carvalho et al. when assessing the specificity of Mucor circinelloides lipase in the transesterification of non-edible vegetable oils, such as lauric oils (coconut and macaw palm oils).28 The high FAEE yield of 98.1% of the coconut oil into biodiesel was confirmed by high-performance liquid chromatography (HPLC) analysis (Table 4). The amount of monoTable 4. Properties of the Purified Biodiesel Obtained from Transesterification of Coconut Oil Using Ethanol as an Acyl Acceptor parameter ester content (%) monoacylglycerides (%) diacylglycerides (%) triacylglycerides (%) kinematic viscosity at 40 °C (mm2 s−1) density at 20 °C (kg m−3) water content (%)

97.07 ± 0.16 0.4 ± 0.09 0.5 ± 0.06 0 4.5 ± 0.1 888 ± 0.2 0.02 ± 0.003

and diacylglycerides found were 0.4 and 0.5%, respectively, with the absence of triacylglycerides. These results are in accordance with the ASTM standard for biodiesel (ASTM D6751) that establishes values up to 1.0% for mono- and diacylglycerols.30 Properties, such as kinematic viscosity and density, are parameters widely used to qualify biodiesel. In the present study, the kinematic viscosity obtained for biodiesel was within the limit established by the standard (1.9−6.0 mm2 s−1). Operational stability of immobilized lipase was also analyzed in the transesterification reactions under consecutive batch runs. It was verified that, after five consecutive batches, the FAEE yield decreased from 98.1 to 96.9%. This slight reduction may be associated with the loss of the biocatalyst amount in the

Figure 3. Profile for alkyl ester formation (wt %) and FAEE yield (%) in the ethanolysis reaction of coconut oil catalyzed by lipase from C. kikuchii immobilized onto rice husk: (■) C8, (□) C10, (▲) C12, (△) C14, (◆) C16, (◇) C18, (◀) C18:1, (▶) C18:2, (○) total ethyl esters, and (●) FAEE yield. 4823

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(9) Tantrakulsiri, J.; Jeyashoke, N.; Krisanangkura, K. J. Am. Oil Chem. Soc. 1997, 74, 173−175. (10) de Castro, H. F.; de Lima, R.; Roberto, C. I. Biotechnol. Prog. 2001, 17, 1061−1064. (11) Costa-Silva, T. A.; Souza, C. R. F.; Oliveira, W. P. J; Said, S. Braz. J. Chem. Eng. 2014, 31, 849−858. (12) Gomes, F. M.; Pereira, E. B.; de Castro, H. F. Biomacromolecules 2004, 5, 17−23. (13) Pereira, E. B.; Zanin, G. M.; Castro, H. F. Braz. J. Chem. Eng. 2003, 20, 343−355. (14) Adlercreutz, P. Chem. Soc. Rev. 2013, 42, 6406−6436. (15) Soares, C. M. F.; De Castro, H. F.; De Moraes, F. F.; Zanin, G. M. Appl. Biochem. Biotechnol. 1999, 79, 745−758. (16) Bradford, M. M. Anal. Biochem. 1976, 72, 156−171. (17) Costa-Silva, T. A.; Cognette, R. C.; Souza, C. R. F.; Said, S.; Oliveira, W. P. Drying Technol. 2013, 31, 1756−1763. (18) World Health Organization (WHO). Quality Control Methods for Medical Plants Materials; WHO: Geneva, Switzerland, 1998; pp 235−389. (19) Norenã, C. Z.; Hubinger, M. D.; Menegalli, F. C. Bol. Soc. Bras. Cienc. Tecnol. Aliment. 1996, 30, 91−96. (20) Urioste, D.; Castro, M. B. A.; Biaggio, F. C.; Castro, H. F. Quim. Nova 2008, 31, 407−412. (21) Carvalho, A. K. F.; Da Rós, P. C. M.; Teixeira, L. F.; Andrade, G. S. S.; Zanin, G. M.; De Castro, H. F. Ind. Crops Prod. 2013, 50, 485− 493. (22) Andrade, G. S. S.; Carvalho, A. K. F.; Romero, C. M.; Oliveira, P. C.; De Castro, H. F. Bioprocess Biosyst. Eng. 2014, 37, 2539−2548. (23) Cunha, R. L.; Maialle, K. G.; Menegalli, F. C. Powder Technol. 2000, 107, 234−242. (24) Freire, J. T.; Ferreira, M. C.; Freire, F. B.; Nascimento, B. S. Drying Technol. 2012, 30, 330−341. (25) Brígida, A. I. S.; Pinheiro, A. D. T.; Ferreira, A. L. O; Gonçalves, L. R. B. Appl. Biochem. Biotechnol. 2008, 146, 173−187. Smith, G. P. Textbook of Organic Chemistry; American Chemical Society (ACS): Washington, D.C., 1972; pp 128−132. (26) Martins, C. R.; Jesus Júnior, L. A. Textbook of production and marketing of coconut in Brazil against the international trade. Panorama 2014, 51−123. (27) Bouaid, A.; Martínez, M.; Aracil, J. Bioresour. Technol. 2010, 101 (11), 4006−4012. (28) Carvalho, A. K. F.; Faria, E. L. P.; Rivaldi, J. D.; Andrade, G. S. S.; Oliveira, P. C.; Castro, H. F. Ind. Crops Prod. 2015, 67, 287−294. (29) Lourinho, G.; Brito, P. Rev. Environ. Sci. Bio/Technol. 2015, 14, 287−316. (30) Knothe, G.; Gerpen, J. V.; Krahl, J.; Ramos, L. P. Manual do Biodiesel, 1st ed.; Edgard Blucher: São Paulo, Brazil, 2006. (31) Costa-Silva, T. A.; Souza, C. R. F.; Said, S.; Oliveira, W. P. Afr. J. Biotechnol. 2015, 14, 3019−3026.

successive recycles rather than the enzyme desorption from the support. Protein engineering and the further improvement of enzyme immobilized derivatives and reaction medium have high potential to engender more efficient industrial application (bioconversions) in the next few years. Several research workers have used natural materials (eco-friendly supports), such as olive pomace, rice straw, coconut fibers, and chitin, obtaining great results improving enzyme properties (stability and specificity), industrial application, and final product features.10,25,31 The results attained in the current study are promising and reveal the potential of immobilization of a microbial lipase from endophytic fungus on a new and cheap support for biodiesel production from oil coconut. The immobilized lipase is original and has potential to become a cost-effective alternative for the commercial lipases currently in the market, although the activity of the immobilized preparation requires improvement in terms of the specific activity and stability.

4. CONCLUSION This work demonstrates that a spouted bed can be successfully used for dehydration and immobilization of enzymes (thermally sensitive material) as a result of the high residual enzyme activity obtained after the drying step. Besides, the use of rice husk as an inexpensive support matrix and coconut oil as a cheap feedstock (renewable features) makes this research a promising one for industrial applications. The transesterification of coconut oil achieving ethyl ester contents should be >96% (according to the standard specifications for renewable diesel). Therefore, the immobilized derivative produced can be punctuated as an alternative choice to the traditional chemical route to mediate enzymatic transesterification of vegetable oils for biodiesel synthesis.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +55-11-964244550. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the State of São Paulo Research Foundation (FAPESP). T.A. Costa-Silva received a Ph.D. fellowship from FAPESP (Grant # 2011/00743-8).

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REFERENCES

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DOI: 10.1021/acs.energyfuels.6b00208 Energy Fuels 2016, 30, 4820−4824