Biodiesel Production from Corn Oil via Enzymatic Catalysis with

School of Engineering (ISEP), Polytechnic Institute of Porto (IPP), s/n, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal. Energy Fuel...
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Biodiesel Production from Corn Oil via Enzymatic Catalysis with Ethanol Teresa M. Mata,*,†,‡ Igor R.B.G. Sousa,§ Sara S. Vieira,§ and Nídia S. Caetano§,‡ †

Faculty of Engineering, University of Porto (FEUP), Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal LEPAELaboratory for Process, Environmental, and Energy Engineering, Faculty of Engineering, University of Porto (FEUP), Portugal § School of Engineering (ISEP), Polytechnic Institute of Porto (IPP), s/n, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal ‡

ABSTRACT: This work presents experimental results on alkali and enzymatic catalysis of corn oil into biodiesel with an optimization of operating conditions and further experiments on enzyme reuse. A comparison of the alkali-catalyzed methanolysis and ethanolysis of corn oil is done, followed by the study of the enzymatic-catalyzed ethanolysis using the alcohol at different concentrations (ethanol absolute, 96%, and 70%, v/v). Results show that the best operating conditions for biodiesel production using absolute ethanol (containing no water) as reagent are an oil/alcohol molar ratio of 1:6, a catalyst/oil weight percentage of 2.8 wt %, a reaction time of 12 h, and a reaction temperature of 35 °C. For these conditions it was possible to obtain a reaction yield of 98.95 wt % with a fatty acid ethyl esters (FAEE) content of 69.2 wt %, with linoleate (C18:2) and oleate (C18:1) being the most significant esters (with relative percentages of 42.97 wt % and 22.54 wt %, respectively). Regarding the evaluation of the enzyme activity loss during reaction, it was concluded that under these conditions it is possible to reuse the enzyme four times after which there was a significant loss of the biodiesel quality according to the EN 14214:2009 standard.

1. INTRODUCTION The global biodiesel production has significantly increased over the last decades, and it is expected to increase even more in future. The need to reduce dependence on imported fossil fuels, with the associated carbon dioxide emissions, and to provide better opportunities for farmers in poor regions has been the main driver for using biodiesel1 and the search for different biofuels feedstocks and production technologies.2−7 The most effective and widely used method for biodiesel production industrially is the alkali-catalyzed transesterification of triglycerides (main components of vegetable oils and fats). However, after the recent technological developments in this area, the enzymatic pathway aroused a great interest in the scientific community, in an attempt to improve the reaction conditions, making them viable and available for industrial applications.8−13 Despite the drawbacks that presently still exist and that are mainly economic, the enzymatic process, once optimized, can present very interesting advantages over the chemical process, namely, the ease of the catalyst separation (if enzymes are supported on some material) and the possibility of obtaining products with higher purity and of using hydrated ethanol in the reaction.12−14 As main disadvantages, one can refer to the relatively long reaction times and the still high cost of enzymes.10 Among the various available enzymes, lipases have shown to effectively catalyze the reactions not only of hydrolysis but also of esterification and transesterification.15 Lipases can perform well in the presence of water (of about 4−5 wt %) for the ethanolysis of triglycerides,14 and the re-utilization of immobilized lipases is also envisaged as a way to reduce operating costs of biodiesel production.14,16 © 2012 American Chemical Society

Biodiesel is normally produced using methanol as reagent because it is cheaper and promotes faster reaction rates.17,18 However, methanol has some disadvantages for handling; it is hazardous to health and the environment, and it is difficult to produce from renewable raw materials.19 Methanol is normally derived from crude oil, which makes the resulting biodiesel not completely of renewable origin. Alternatively, ethanol can be used, since it is not so dangerous for human health and the environment and it can be obtained from renewable sources (e.g., sugar cane or lignocellulosic materials). Ethanol has lower reactivity (as a transesterification agent) in comparison with methanol, but it surpasses the need of employing anhydrous materials and low acidic vegetable oils, and minimizes the soap and emulsion formation problems.20 The reasons highlighted above justify the study of the FAEE’s production by enzymatic catalysis with ethanol as reagent. To the purpose of this work, the effects of different operating conditions (oil/alcohol molar ratio, catalyst/oil weight percentage, reaction time, and reaction temperature) are evaluated in order to find the best ones. Also, the resulting biodiesel is characterized according to the EN 14214:2009 standard that regulates the quality of FAME (fatty acid methyl esters), since currently, it does not exist in Europe as a FAEE specific standard.

2. MATERIALS AND METHODS 2.1. Vegetable Oil Characterization. The corn oil used in this experimental work was bought from a local commercial retailer. The Received: February 24, 2012 Revised: April 17, 2012 Published: April 18, 2012 3034

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2.3. Biodiesel Production through Enzyme-Catalyzed Transesterification. Around 200 g of oil was weighed to a screw cap Pyrex bottle, with 500 mL of capacity and the bottle was placed in a thermostatic bath to heat the oil to the reaction temperature. The required volume of alcohol was measured in a hood, and a certain mass of enzyme was weighed, in accordance with the respective trial, and they were added to the oil, together with a magnetic stir bar. Then, the transesterification reaction took place for a certain period of time in a stirring bath (at 60 rpm) at the desired temperature depending on the trial conditions. After the reaction ended, the flask was removed from the bath and the mixture was filtered to recover the enzyme. The filtrate was placed in a separating funnel, and about 75 g of glycerin (72% purity, Normapur) was added, and the mixture was left to stand for 15 min. The denser phase (glycerol) was removed from the bottom of the separating funnel to a previously weighed beaker in order to determine the mass of the recovered crude glycerol. The lighter phase (biodiesel) was purified with a cation-exchange resin (Lewatit GF 202); thus, biodiesel was passed through a column (5 cm in diameter and 30 cm in length) packed with a 15 cm length of ion-exchange resin that retained the impurities (water, ions of K, and glycerol), at a mean flow rate of 2 bed volumes/h (or about 236 cm3/h). Lewatif GF 202 is a macroporous cation-exchange (acidic) resin (R). The R beads are uniform, 0.65 mm in diameter, with a density of 1.24 g/mL and a bulk density of 0.740 g/mL. Biodiesel was then subjected to distillation at 80 °C to recover the excess alcohol. Finally, the purified and neutralized biodiesel was placed in a beaker, and about 2 g of diatomaceous earth was added to remove any water remaining in biodiesel; the mixture was stirred for about 15 min, after which the biodiesel was left to stand and then vacuum filtered through cellulose membranes (47 mm diameter, 1.2 μm pore) to remove the diatomaceous earth. The purified biodiesel was stored in dark glass flasks for its subsequent characterization. 2.4. Biodiesel Characterization. The most important quality parameters of biodiesel were evaluated according to the EN 14214:2009 standard, which defines the quality requirements of FAME. Thus, biodiesel was characterized for the following properties: acid value, iodine value, kinematic viscosity at 40 °C, density at 20 °C, water content, flash point, copper corrosion, cold filter plugging point (CFPP), fatty acid ethyl esters (FAEE) content, and higher heating value (HHV). These properties were determined by applying the standard methods described in Table 1. For determining the FAEE content, the GC analysis was performed using a gas chromatograph (DANI GC 1000 DPC) equipped with an AT-WAX (Heliflex Capillary, Alltech) column (30 m, 0.32 mm internal diameter, and 0.25 μm film thickness). The injector temperature was set to 250 °C, while the flame ionization detector (FID) temperature was set to 255 °C and the oven temperature to 195 °C. The carrier gas used was nitrogen, at a flow rate of 2 mL/min. Injection was made in a split mode, using a split flow rate of 50 mL/ min (split ratio of 1:25), and the volume injected was 0.1 μL. 2.5. Chemicals and Enzyme Used for Biodiesel Production. For biodiesel production, the following chemicals and substances were used: potassium hydroxide (KOH p.a., Pronalab), absolute methanol (containing no water, Pronalab), absolute ethanol (containing no water, Panreac), hydrated ethanol (96% v/v and 70%, v/v, both from Panreac), glycerin (72% purity, Normapur), phosphoric acid (H3PO4, 85% purity, Panreac), diatomaceous earth (Panreac). It is tested in this work the enzyme Lipozyme TL IM by Novozymes as catalyst. This is a low-cost immobilized on granulated silica lipase from Thermomyces lanuginosa that has important industrial applications in the synthesis of detergent,21 resolution of chiral alcohol22,23 and preparation of biodiesel.24,25 Particles are 0.3−1.0 mm diameter, light brown and have a declared activity of 250 IUN/g (interesterification units/g).26

oil was characterized for the following properties: acid value, iodine value, kinematic viscosity at 40 °C, density at 15 °C, and higher heating value (HHV). These properties were determined by applying the standard methods described in Table 1.

Table 1. Standard Methods Applied for the Corn Oil Characterization and Determining the Biodiesel Quality Parameters param. acid value iodine value kinematic viscosity density water content flash point copper corrosion CFPP FAEE content HHV

method applied titrimetric method, ISO 14104:2011 standard titrimetric method with Wijs reagent, EN14111:2009 standard glass capillary viscometers Cannon-Fenske Series 200, ISO 3104:1994 hydrometer method, EN ISO 3675:1998 standard Karl Fischer coulometric titration, NP EN ISO 12937:2003 standard rapid equilibrium closed cup method, ISO 3679:2004 standard copper strip test, ISO 2160:1998 standard standardized filtration equipment, EN 116:2002 standard gas chromatography (GC), EN 14103:2010 standard oxygen bomb calorimeter, ASTM method D240-87

2.2. Biodiesel Production through Alkali-Catalyzed Transesterification. Around 300 g of oil was weighed into a screw cap Pyrex bottle, with 500 mL of capacity, and then placed in a thermostatic bath to heat the oil up to the reaction temperature (60 or 75 °C for methanol or ethanol, respectively). Then, around 3 g of KOH (p.a., Pronalab) catalyst was measured and dissolved in a closed cup in the required volume of alcohol (absolute methanol, Pronalab, or absolute ethanol, Panreac, depending on the trial), slightly warming the mixture and with some stirring. This operation was performed in a hood, during about 5 min (approximate dissolution time) for methanol or 10 min for ethanol. Then, this alcohol/catalyst mixture was added to the previously preheated oil (in the 500 mL screw cap bottle) and shaken vigorously to promote contact between oil, alcohol, and catalyst. Then, this bottle was placed in the thermostatic bath where the reaction took place for 2 h at the desired temperature (60 or 75 °C for the production of methyl or ethyl biodiesel, respectively) and at 60 rpm of stirring speed. After the reaction ended, the flask was removed from the bath and the mixture was placed in a separating funnel. When ethanol was used, about 75 g of glycerin (72% purity, Normapur) was added, and the mixture was then allowed to settle for 15 min for the phase separation to occur. If methanol was the alcohol used, this operation was not needed and it proceeded directly to phase separation (glycerol from biodiesel). The denser phase (glycerol) was removed from the bottom of the separating funnel to a previously weighed beaker in order to determine the mass of the recovered crude glycerol. The lighter phase (biodiesel) was subjected to a distillation step at 80 °C to recover the excess alcohol still existing and then placed again in the separating funnel, where it was neutralized with hot water acidified with about 10 drops of concentrated phosphoric acid. The washing procedure was repeated several times using only hot water until a clear phase was obtained with a neutral pH. When necessary, a few more drops of phosphoric acid were added to the intermediate washing water steps to help neutralize the pH. Finally, the purified and neutralized biodiesel was placed in a beaker, and about 2 g of diatomaceous earth was added to remove any water remaining in biodiesel; the mixture was stirred for about 15 min, after which the biodiesel was left to stand and then vacuum filtered through cellulose membranes (4−7 μm) to remove the diatomaceous earth. The purified biodiesel was stored in dark glass flasks for its subsequent characterization.

3. RESULTS AND DISCUSSION 3.1. Vegetable Oil Characterization. To characterize the vegetable oil used in this work for biodiesel production, some 3035

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content can result in a lower solubility of oil in the alcohol, with a consequent drop in the ethyl esters yield.31 The range of higher heating values determined in this study for corn oil comprises the value (39.5 MJ/kg) reported by Balat29 for this oil. 3.2. Biodiesel Obtained by Alkali-Catalyzed Transesterification. With the purpose of evaluating the advantages or disadvantages of using absolute methanol and absolute ethanol as reagents, with KOH as catalyst, biodiesel was produced in this work, through the conventional process of alkali-catalyzed transesterification. Also, this establishes a basis for comparison with the enzymatic catalysis to be performed in this study. Therefore, for the alkali-catalyzed transesterification a fixed oil/alcohol molar ratio of 1:6, a catalyst/oil weight percentage of 1.0 wt % and a reaction time of 2 h for reaction temperatures of 60 °C (for absolute methanol) and 75 °C (for absolute ethanol) were used. Results are shown in Table 3. As shown in Table 3, the reaction yields for methanol are higher than for ethanol as reagent (with average values of 83.5% and 71.1%, respectively). This is in accordance with expectations, because when ethanol is used reaction rates are generally slower than for methanol.17,18 The yield of the ester phase can be further increased by performing the separation (of the glycerol and ester phases) at about 90 °C instead of 25 °C. However, this also increases the concentration of free glycerol and alkaline metals in the ester phase.32 One way to improve the ethyl ester yield is by using alcohol mixtures of methanol/ ethanol instead of the pure ethanol.33 The type of catalyst also influences the ethyl ester yield, as it was reported by Kucek et al.34 where they obtained a higher ethyl ester yield (of 97.2%) with 0.3 wt % NaOH than with 1.0 wt % KOH under the same reaction conditions of 70 °C and with an oil/ethanol molar ratio of 1:12. On the other hand, one can shorten the reaction times of the triglycerides’ ethanolysis through the use of cosolvents.35 The water content is above the maximum standard limit (of 500 mg/kg) for both alcohols, and it is much higher when absolute ethanol is used than with absolute methanol. The biodiesel density is within the range defined in the EN 14214:2009 standard, regardless of the type of alcohol used.

of the most important physicochemical parameters were determined experimentally, as shown in Table 2. Table 2. Experimental Characterization of the Corn Oil unit

valuea

kg/m mm2/s mg water/kg oil mg KOH/g oil g iodine/100 g oil MJ/kg oil g/mol

919 ± 0 36.27 ± 0.01 749 ± 14 0.26 ± 0.02 127 ± 1 39.8 ± 0.2 865.4 ± 8

param. density at 20 °C kinematic viscosity at 40 °C water content acid value iodine value HHV avg molecular mass

3

a

Experiments were performed in triplicate and data are expressed as mean ± SD (standard deviation).

Regarding the organoleptic parameters, the corn oil showed a yellowish color, clear, with no deposit, odor like vegetable oil, and liquid texture at room temperature (of about 20 °C). The values obtained experimentally for the oil density and iodine value are within the expected ranges of the NP-946 standard for corn oil (915−924 kg/m3 and 103−128 g/100 g oil, respectively). The iodine value indicates the unsaturation degree of the oil. According to Moretto and Fett,27 the density of triacylglycerides is lower as its molecular mass is lower and its degree of unsaturation is higher. Also, the acid value obtained experimentally for corn oil is below the maximum limit of this standard (0.60 mg KOH/g). The acid value is one main indicator of the vegetable oil quality. Also, high oil acidity can neutralize an alkali-catalyst during transesterification, making it necessary to use a much higher amount of catalyst to perform the reaction efficiently. According to Dantas et al.,28 the high free fatty acids content influences the hydrolysis and oxidation of biodiesel. Regarding the kinematic viscosity, the value obtained experimentally is close to the one indicated by Balat29 for corn oil, that is, 34.9 mm2/s (measured at 38 °C). This oil has a low water content (of 749 mg/kg oil), although it is normally recommended to keep it below 0.06% (w/w), making it suitable for the transesterification reactions.30 High water

Table 3. Characterization of Biodiesel Obtained by Alkali-Catalyzed Transesterification with Absolute Methanol and Absolute Ethanol as Reagents and KOH as Catalyst trials

a

param.

1a

2a

3a

4a

5a

6a

EN 14214 limits

type of alcohol (absolute) oil/alcohol molar ratio reaction time (h) reaction temp. (°C) catalyst/oil weight percentage (wt %) reaction yield (wt %) density at 15 °C (kg/m3) kinematic viscosity at 40 °C (mm2/s) water content (mg/kg) iodine value (g iodine/100 g biodiesel) acid value (mg KOH/g biodiesel) group I metals (Na+ + K+) (mg/kg) copper strip corrosion (3 h at 50 °C) avg flash point (°C) CFPP (°C)

methanol 1:6 2 60 1 83.0 881 4.67 1020 126 0.30 14.1 1A >150 −8

methanol 1:6 2 60 1 76.4 880 4.43 824 122 0.28 13.1 1A >150 −7

methanol 1:6 2 60 1 91.1 884 5.04 960 123 0.49 13.3 1A >150 −7

ethanol 1:6 2 75 1 77.9 875 5.68 1415 120 2.08 11.8 1A >150 +4

ethanol 1:6 2 75 1 58.8 877 6.27 1655 118 1.12 11.8 1A >150 +4

ethanol 1:6 2 75 1 76.6 886 7.72 1890 116 1.28 11.8 1A >150 +4

860−900 3.50−5.00 ≤500 ≤120 ≤0.50 ≤5.0 class 1 ≥101 ≤+5a

Limit for temperate climates. 3036

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Table 4. Characterization of Biodiesel Obtained by Enzyme-Catalyzed Transesterification with Ethanol (Absolute, 96%, and 70%) as Reagent and the Lipozyme TL IM as Catalyst trials param.

1b

2b

3b

4b

5b

6b

EN 14214 limits

type of ethanol alcohol used oil/alcohol molar ratio reaction time (h) reaction temp. (°C) catalyst/oil weight percentage (wt %) reaction yield (wt %) density at 15 °C (kg/m3) kinematic viscosity at 40 °C (mm2/s) water content (mg/kg) iodine value (g iodine/100 g biodiesel) acid value (mg KOH/g biodiesel)

absolute 1:3 24 35 2.8 91.0 888 8.23 1367 85 0.93

96% 1:3 24 35 2.8 94.0 890 8.34 2299 88 5.23

70% 1:3 24 35 2.8 74.8 892 12.97 3679 86 19.99

absolute 1:3 48 35 2.8 88.0 892 8.81 1960 120 1.16

96% 1:3 48 35 2.8 85.0 890 7.26 2437 88 4.86

70% 1:3 48 35 2.8 71.0 897 11.53 2721 89 20.51

860−900 3.50−5.00 ≤500 ≤120 ≤0.50

Concerning the CFPP, the values for biodiesel are −7 °C and −8 °C for absolute methanol, which falls within class C (−5 °C), and +4 °C for absolute ethanol, which falls within class A (+ 5 °C). Therefore, regardless of the alcohol used for the biofuel production, it can be used in temperate countries, such as Portugal. 3.3. Biodiesel Obtained by Enzyme-Catalyzed Transesterification with Absolute and Hydrated Ethanol. After comparing the alkali-catalyzed methanolysis and ethanolysis of corn oil, the enzymatic-catalyzed ethanolysis was studied using the alcohol at different concentrations (ethanol absolute, 96%, and 70%, v/v) and the enzyme Lipozyme TL IM as catalyst. In this process, only the reaction time was varied (of 24 and 48 h) and the remaining operating conditions were kept constant, oil/alcohol molar ratio (1:3), catalyst/oil weight percentage (2.8 wt %) and reaction temperature (35 °C). Results are shown in Table 4. As shown in Table 4, the use of a higher reaction time (48 h) does not improve conversion of oil into biodiesel, as the reaction yield is slightly higher for 24 h of reaction time than for 48 h, regardless of the ethanol concentration used in the process, possibly due to the reaction reversibility. On the other hand, for the 24 h reaction time the highest reaction yields (of 91 and 94 wt %) are obtained when using absolute ethanol and ethanol 96% (v/v), respectively. The biodiesel density is within the standard limits regardless of the ethanol concentration. The kinematic viscosity values are out of the standard limits and are higher when ethanol 70% (v/ v) is used, probably due to the low conversion of oil into biodiesel. Also, the water content is much above the maximum standard limit (of 500 mg/kg) for all the samples, indicating the need to use a more efficient drying process as the presence of ethanol facilitates water dissolution in the biodiesel phase. Moreover, as expected, biodiesel produced using ethanol 70% (v/v) shows higher water content. The iodine value of biodiesel is below the maximum standard limit (120 g iodine/100 g biodiesel) in all samples. The opposite is observed for the acid value that is out of the standard limit in all samples, probably due to the presence of unreacted FFA, which is more notable for the lower yield reactions. On the other hand, by reducing the ethanol purity, the acid value becomes substantially worse. One may conclude that, generally, the use of ethanol 70% (v/ v) (with higher water content) worsens the biodiesel properties, such as the kinematic viscosity, water content, and acid value. Moreover, for absolute ethanol and for ethanol 96%

The iodine value of biodiesel produced using absolute ethanol is below the maximum standard limit (120 g iodine/ 100 g biodiesel) which does not happen when absolute methanol is used (with average values of 124 and 118 g iodine/ 100 g biodiesel, respectively). The opposite is observed for the acid value (with average values of 0.36 and 1.49 mg KOH/g for methanol and ethanol, respectively), being above the maximum standard limit (0.50 mg KOH/g biodiesel) for the ethanol process. The high acidity affects the fuel thermal stability in the combustion chamber, and the presence of free fatty acids in biodiesel has a corrosive effect on the metal components of the engine.31 The value of alkaline metals concentration is above the maximum standard limit (5.0 mg/kg) for both alcohols, and it is generally higher for the absolute methanol process than for the absolute ethanol (average values of 13.5 and 11.8 mg/kg, respectively). These metal effects are related to the possibility of formation of metallic soaps or abrasive solids that can clog filters and injectors in the vehicle’s engine. However, the concentration of alkaline metals (potassium and sodium) in the ester phase can be reduced through the addition of an optimal amount of water, which also accelerates the separation of the emulsion after the transesterification.36 This is similar to the water washing of biodiesel often used as a purification process after phase separation. The kinematic viscosity is generally higher for ethanol than for methanol (with average values of 6.56 and 4.71 mm2/s, respectively) and for the primer alcohol it is above the standard maximum limit (of 5.00 mm2/s), which may pose some difficulties during biodiesel combustion in vehicle engine and may damage the injection system and fuel pump. Another physical parameter evaluated is the corrosiveness to copper, as corrosion affects the metallic materials (copper alloys and other metals) in contact with the fuel, particularly the engine components and the storage and maintenance equipment. In this case all the biodiesel samples are noncorrosive as the copper strip corrosion tests showed that they are within class 1 according to the EN 14214:2009 standard. All the biodiesel samples have a flash point higher than 150 °C, which is above the minimum standard limit of 101 °C. If the flash point was below this limit, it would be more susceptible to volatilization at lower temperatures and consequently to premature combustion and higher detrition of the engine components. This also means that the excess methanol was completely removed from the biodiesel phase. 3037

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Table 5. Characterization of Biodiesel Obtained by Enzyme-Catalyzed Transesterification with Absolute Ethanol as Reagent and the Lipozyme TL IM as Catalyst trials param.

1c

2c

3c

4c

5c

6c

7c

EN 14214 limits

type of ethanol alcohol used oil/alcohol molar ratio reaction time (h) reaction temp. (°C) enzyme catalyst/oil (wt %) reaction yield (wt %) density at 15 °C (kg/m3) kinematic viscosity at 40 °C (mm2/s) water content (mg/kg) iodine value (g iodine/100 g biodiesel) acid value (mg KOH/g biodiesel) group I metals (Na+ + K+) (mg/kg) copper strip corrosion (3 h at 50 °C) avg flash point (°C) CFPP (°C)

absolute 1:3 8 35 2.8 73.5 889 12.57 558 102 0.98 11.8 1A >150 +4

absolute 1:3 12 35 2.8 95.3 890 8.95 962 104 0.90 11.8 1A >150 +4

absolute 1:6 12 35 2.8 98.9 890 9.15 1966 92 0.60 11.8 1A >150 +4

absolute 1:9 12 35 2.8 94.6 890 13.18 1637 99 0.52 11.8 1A >150 +4

absolute 1:6 12 35 2.3 84.0 892 10.45 388 96 0.54 11.8 1A >150 +4

absolute 1:6 12 35 3.3 87.6 890 8.09 8297 102 0.76 11.8 1A >150 +4

absolute 1:3 12 45 2.8 83.6 892 9.03 6844 102 1.09 11.8 1A >150 +4

860−900 3.50−5.00 ≤500 ≤120 ≤0.50 ≤5.0 class 1 ≥101 ≤+5*

1:12 ratio) the extra consumption of alcohol is not compensated by the increase in reaction yield and complicates the separation of esters and the recovery of the alcohol and glycerin, which would impair performance in ethyl esters. By comparing the catalyst/oil weight percentages of 2.3, 2.8, and 3.3 wt %,9 keeping constant the other operating conditions, the obtained reaction yields were 84.0, 98.9, and 87.6 wt %, respectively (of trials 5c, 3c, and 6c). Therefore, one may conclude that an excess of enzyme (3.3 wt %) does not promote an increase in the conversion efficiency of oil into biodiesel (maybe because at higher enzyme loads the equilibrium is reached faster), but on the other hand, the use of lower amount of enzyme (2.3 wt %) may be not sufficient to reach a higher conversion at sufficiently fast reaction rates. Comparing the effect of the reaction temperature (35 and 45 °C), keeping constant the other operating conditions, (trials 7c and 2c) results show that at 35 °C higher reaction yield (95.3 wt %) was obtained than at 45 °C (83.6 wt %). The reason could be a protein denaturation at the higher temperature with consequent loss of the enzyme activity, since for Lipozyme TL IM the optimal temperature range of operation is between 30 and 40 °C, according to the producer instructions. Also, Rodrigues et al.40 reported an optimal temperature in the range 30−35 °C for the lipases tested. Therefore, for the highest reaction yield (trial 3c), the best operating conditions to perform the transesterification reaction are an oil/alcohol molar ratio of 1:6, a catalyst/oil mass ratio 2.8 wt %, a reaction time of 12 h, and a reaction temperature of 35 °C, for which the obtained reaction yield was 98.9 wt %. Concerning the quality of the produced biodiesel, Table 5 shows that regardless of the operating conditions density is within the standard limits. All the kinematic viscosity values are out of the standard limits and increase with the oil/alcohol molar ratio from 1:3 to 1:6 and 1:9, being 8.95, 9.15, and 13.18 mm2/s, respectively (trials 2c, 3c and 4c). Also, the viscosity decreases as the catalyst/oil mass ratio increases from 2.3, 2.8, and 3.3 wt % being respectively 10.45, 9.15, and 8.09 mm2/s (trials 5c, 3c and 6c). The water content is much above the maximum standard limit except for trial 5a, meaning that a better drying procedure should be used to remove water. The iodine value of biodiesel is below the maximum standard limit in all samples. The acid value is out of the standard limit in

(v/v), higher reaction yields are obtained, and using absolute ethanol resulted in biodiesel with better quality characteristics. For this reason, in the third part of this work absolute ethanol was chosen to proceed to the following tests. 3.4. Biodiesel Obtained by Enzyme-Catalyzed Transesterification Using Absolute Ethanol. Following the results obtained in section 3.3, FAEE were then produced by enzymatic transesterification using absolute ethanol as reagent and the Lipozyme TL IM as catalyst. The parameters varied in this part of the work were the oil/alcohol molar ratio (1:3, 1:6, and 1:9), the catalyst/oil weight percentage (2.3, 2.8, and 3.3 wt %), the reaction time (8 and 12 h), and the reaction temperature (35 and 45 °C). Results are shown in Table 5, including some of the most important quality parameters for the corresponding biodiesel characterization. As shown in Table 5, higher reaction yields are obtained for 12 h than for 8 h reaction time (trials 2c and 1c, respectively). Also as shown in Table 4 (trials 1b and 4b), the reaction yield obtained for 24 h or 48 h reaction time is lower than for 12 h, suggesting that 12 h should be sufficient to perform the reaction and to favor conversion of oil into biodiesel. Comparing the effect of the oil/alcohol molar ratios (1:3, 1:6, and 1:9), keeping constant the other operating conditions, the obtained reaction yields were respectively 95.3, 98.9, and 94.6 wt % (trials 2c, 3c, and 4c). Therefore, one may conclude that the oil/alcohol molar ratio 1:6 gives the best reaction yield. An oil/alcohol molar ratio above 1:6 (of 1:3) does not contain enough excess alcohol for the oil conversion into biodiesel and, if below 1:6 (of 1:9) it also does not improve the reaction yield although the transesterification of vegetable oils is kinetically favored when an excess of alcohol is used in relation to triglycerides.37 A possible explanation is that a too low oil/ alcohol molar ratio leads to yield losses due to enzyme denaturation.38 Also, it interferes with the separation of glycerol due to its increased solubility in ethanol and the presence of glycerol in the reaction medium favors the formation of triglycerides, and thus, the reaction shifts in favor of the reactants. A similar conclusion was reached in the Encinar et al.39 study, showing that an oil/alcohol molar ratio of 1:9 was quite appropriate to perform transesterification with NaOH at 1% by weight of the oil, as above this ratio (e.g., for 1:6 ratio) the reaction could be incomplete, and below this ratio (e.g., for 3038

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Table 6. Characterization of Biodiesel Obtained after Successive Reuses of Lipozyme TL IM Using Absolute Ethanol as Reagent enzyme reuses param.

1d

2d

3d

4d

5d

EN 14214 limits

type of ethanol alcohol used oil/alcohol molar ratio reaction time (h) reaction temp. (°C) enzyme catalyst/oil (wt %) HHV MJ/kg reaction yield (wt %) FAEE content (wt %) density at 15 °C (kg/m3) kinematic viscosity at 40 °C (mm2/s) water content (mg/kg) iodine value (g iodine/100 g biodiesel) acid value (mg KOH/g biodiesel) copper strip corrosion (3 h at 50 °C) avg flash point (°C) CFPP (°C)

absolute 1:6 12 35 2.8 40.4 93.7 70.4 890.0 10.54 1956 91 0.64 1A >150 +4

absolute 1:6 12 35 2.8 40.2 88.4 67.4 890.7 20.31 1679 92 0.68 1A >150 +4

absolute 1:6 12 35 2.8 40.0 84.1 66.9 894.0 24.18 1567 92 1.05 1A >150 +4

absolute 1:6 12 35 2.8 40.0 80.1 66.3 899.3 26.54 1233 112 1.25 1A >150 +4

absolute 1:6 12 35 2.8 39.8 70.8 56.8 908.0 27.79 870 124 1.60 1A >150 +4

≥96.5 860−900 3.50−5.00 ≤500 ≤120 ≤0.50 class 1 ≥101 ≤+5*

The kinematic viscosity of all biodiesel samples is above the maximum standard limit and the successive enzyme reuses promoted an increase in the viscosity from 10.54 to 27.79 mm2/s. This is in accordance with the expected, since with the subsequent reuse of the enzyme the reaction yield decreased, and thus, lower oil conversion was attained. The water content of all the samples is above the standard limit. However, it is clear that the subsequent reuse of the enzyme promoted a decrease in the water content, from 1956 to 870 mg/kg. The iodine value was within the standard limit, except for the fourth enzyme reuse sample, which was slightly above (124 g iodine/100 g biodiesel). The acid value of all the biodiesel samples is out of the standard limit, and the successive enzyme reuses favored its increase. All the biodiesel samples are noncorrosive as the copper strip corrosion tests showed that they are within class 1, according to the standard, regardless of the enzyme reuses. The flash point was within the standard limits for all biodiesel samples, regardless of the enzyme reuses. 3.6. Biodiesel Characterization by Gas Chromatography. The esters profile is probably the most important parameter influencing the properties of a biodiesel because different esters can have very different physical and chemical properties. Important fuel properties that can be estimated by the esters profile of a biodiesel are the cetane number, oxidative stability, cold flow and kinematic viscosity. Generally, the oxidative stability of fatty esters decreases with increasing unsaturation, as it also happens with the cetane number (measurement of the fuel combustion quality during compression ignition) that decreases with increasing unsaturation and shorter chain lengths.41 Also, for some properties, relatively small amounts of minor components (e.g., linolenate and some contaminants) can have a significant effect on the fuel properties (especially on the oxidative stability and cold flow). Therefore, for the best operating conditions described in section 3.4, the esters profile of the resulting biodiesel was characterized by gas chromatography. This way the esters content, the relative percentage of each ester in the mixture, and the molecular mass of biodiesel (Table 7) were determined.

all samples, but it is just slightly above the maximum limit in trials 4c and 5c (0.52 and 0.54 mg KOH/g biodiesel, respectively). Also, the acid value decreased with the oil/ alcohol molar ratio from 1:3 to 1:6 and 1:9, being 0.90, 0.60, and 0.52, respectively (trials 2c, 3c and 4c), and increased with the catalyst/oil mass ratio from 2.3, 2.8, and 3.3 wt %, being respectively 0.54, 0.60, and 0.76 mm2/s (trials 5c, 3c, and 6c). The value of alkaline metals concentration (Na + K) in biodiesel is above the maximum standard limit (5.0 mg/kg) with an average value of 11.8 mg/kg. All the biodiesel samples had a flash point higher than 150 °C which is above the minimum standard limit of 101 °C and all the biodiesel samples are noncorrosive as the copper strip corrosion tests showed that they are within class 1 according to the standard. Concerning the CFPP, the value of +4 °C falls within class A (+ 5 °C), meaning that the biodiesel can be used in temperate countries, such as Portugal. In summary, one can conclude that the best biodiesel quality was obtained by enzyme-catalyzed transesterification at an oil/ alcohol molar ratio of 1:6, a catalyst/oil mass weight percentage of 2.8 wt %, a reaction time of 12 h, and a reaction temperature of 35 °C, as concluded previously for the best reaction yield (trial 3c). 3.5. Enzyme Reuse. Biodiesel was produced by enzymatic catalysis under the best operating conditions determined in section 3.4, making the reuse of Lipozyme TL IM successively four times after its first utilization. Results are shown in Table 6. Initially, the reaction yield was 93.7%, and after reusing the enzyme four times, the reaction yield decreased to 70.8% (trial 5d). A possible explanation is because some amount of enzyme was lost during its successive reuses, filtration, washing, and drying. In this sense, Shah and Gupta14 found that lipases (Pseudomonas cepacia immobilized on Celite) can be reused four times without any significant loss of activity. The ethyl esters content of all the biodiesel samples were below the minimum standard limit and decreased with the successive enzyme reuses, confirming that lower reaction extent was being successively attained. In terms of the biodiesel density, all samples are within the standard limits, with the exception of the one obtained with the fourth enzyme reutilization (908 kg/m3) that is slightly above the maximum limit. 3039

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highest reaction yield (99.0 wt %) are an oil/alcohol molar ratio of 1:6, a catalyst/oil mass ratio of 2.8 wt %, a reaction time of 12 h, and a reaction temperature of 35 °C. These results obtained under enzymatic catalysis with Lipozym TL IM are better than when alkaline catalysis (with KOH) was used, either using ethanol or using methanol. Under the best operating conditions determined in this work, it was possible to reuse the enzyme four times, after which, there was a significant loss of the biodiesel quality.

Table 7. FAEE Content, Esters Characterization, and Molecular Mass of Biodiesel Obtained by Enzyme-Catalyzed Transesterification with Absolute Ethanol param. FAEE content (wt %) (EN 14214 limit: ≥ 96.5%) relative percentage of FAEE (wt %) myristate (C14:0) palmitate (C16:0) stearate (C18:0) oleate (C18:1) linoleate (C18:2) linolenate (C18:3) arachidate (C20:0) behenate (C22:0) total (wt %) avg molecular mass of FAEE (g/mol)

value 69.2 7.64 15.60 4.61 22.54 42.97 4.65 1.06 0.95 100.0 301.7

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: + 351 22 508 1467. Fax: + 351 22 508 1449. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

As shown in Table 7, the FAEE obtained from corn oil are mainly composed of unsaturated esters (70.15%) and just 29.85% are saturated esters. This high degree of unsaturation of the biodiesel molecules was also expected because of the high iodine value obtained for corn oil (127 g iodine/100 g oil) and the corresponding biodiesel samples (ranging from 85 to 126 g iodine/100 g biodiesel). This property is also a direct indication of the worse oxidative stability of fuel obtained from corn oil; that is, the biodiesel produced from this has lower stability to the oxidation, as compared to methyl esters from animal fats.5 Among the unsaturated esters, special attention should be given to linolenate (C18:3), as the EN 14214:2009 standard establishes the maximum limit of 12% (w/w) for this ester, which is verified in this case (4.65 wt %). This is because linolenate has a lower cetane number than the other esters (i.e., a lower tendency to ignite and more difficulty to combust) and also a lower oxidative stability that will influence the final biodiesel properties. This may pose some restrictions to its use as fuel in vehicle engines, requiring the use of additives to improve (increase) the cetane number and oxidative stability of biodiesel.42 From the identified FAEE, linoleate (C18:2) followed by oleate (C18:1) are the most significant (with 42.97% and 22.54%, respectively), which follows the compositions found by Balat.29 The average molecular mass of FAEE was also determined that is 301.7 g/mol.

REFERENCES

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4. CONCLUSIONS This work studied the production of FAEE from corn oil by enzymatic catalysis with Lipozyme TL IM from Novozymes. First, to establish a basis for comparison, the alkali-catalyzed reaction was performed using absolute methanol and absolute ethanol as reagents. Second, the enzymatic catalysis was performed using absolute and hydrated ethanol (96% and 70%, v/v) and by testing different experimental conditions of oil/alcohol molar ratios (1:3, 1:6 and 1:9), catalyst/oil weight percentages (2.3, 2.8, and 3.3 wt %), reaction times (8, 12, 24, and 48 h), and reaction temperatures (35 and 45 °C) to determine the best operating conditions. Finally, for the best operating conditions, successively reused enzymes were tested and the resulting biodiesel characterized. Results show that although some of the quality parameters analyzed are out of the EN 14214:2009 standard limits (namely, the kinematic viscosity, water content, acid value, and group I metals) the best operating conditions for a good biodiesel quality (with 69.2 wt % of FAEE content) and the 3040

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