Production of Methyl Ester from Oedogonium sp. Oil Using

Sep 11, 2012 - In this study, the production of methyl ester from Oedogonium sp. oil was studied using an isolated thermo-, solvent-, and sono-toleran...
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Production of Methyl Ester from Oedogonium sp. Oil Using Immobilized Isolated Novel Bacillus sp. Lipase Ramachandran Sivaramakrishnan and Karuppan Muthukumar* Department of Chemical Engineering, Alagappa College of Technology Campus, Anna University Chennai, Chennai 600 025, India ABSTRACT: In this study, the production of methyl ester from Oedogonium sp. oil was studied using an isolated thermo-, solvent-, and sono-tolerant Bacillus sp. lipase immobilized on celite. The application of ultrasound during the reaction reduced the reaction time significantly. The effect of sonication time, enzyme dosage, water content, methanol/oil molar ratio, and solvent addition on the performance of transesterification was studied. The reaction time required in the presence and absence of ultrasound was 2 and 40 h, respectively. Under optimum conditions, 75 and 82% fatty acid methyl ester (FAME) yields were obtained for normal and ultrasound-assisted transesterification, respectively. The reusability of the immobilized enzyme after five cycles did not show much loss in enzyme activity, and this indicates that the isolated enzyme was not affected as a result of the application of ultrasound.

1. INTRODUCTION The depletion of fossil fuel resources, the increase in ecological awareness, and the remarkable rise in petroleum prices have necessitated the search for fuels from renewable sources. Methyl ester is a natural substitute for diesel fuel, which comes from renewable sources, such as vegetable oils.1 The advantages associated with biodiesel include its biodegradable nature and nontoxic and low-emission profiles. Biodiesel degrades up to 4 times faster than petroleum diesel. The exhaust emits less smoke and particulate levels compared to diesel fuel. The use of biodiesel decreases the emission of greenhouse gases and, in turn, global warming.2 Biodiesel is produced from a variety of vegetable oil sources, such as soybean, sunflower, cottonseed, rapeseed, palm, etc. However, the availability of oil sources for biodiesel production is limited.3 Therefore, it is essential to find alternative raw materials, and algae have the potential to become the major source of biodiesel.4 The growth of dense macroalgal mats is a major problem in rivers and lakes, and it causes eutrophication.5 Oedogonium sp. and Spiroygyra sp. oils were successfully used for the production of biodiesel.6 Various processes developed for the production of biodiesel are mostly based on chemical and enzymatic methods. Using a chemical catalyst poses many problems, such as engine corrosion, soap formation, recovery of glycerol, etc. Enzymatic catalysts overcome these problems and are also good alternatives because of their eco-friendly selective nature. However, enzymatic methods have not been industrialized, because of their low enzyme activity compared to chemical catalysts. Hence, it is essential to explore a suitable method to improve the reaction rate. The activity of Novozym 435 was enhanced by the application of ultrasound.7 In ultrasound-assisted reactions, the formation of cavitation bubbles generates an enormous interfacial area, which accelerates the reaction. The implosive cavitation bubble collapse produces high pressures and high temperatures, which, in turn, intensify the reaction. The high pressure and temperature have a very short lifespan.8 Ultrasound enhanced the porcine pancreatic lipase activity to produce 1,2,3,4-tetrahydro-1-napthol (R-2).9 The application of ultrasound irradiation enhanced the emulsification of immis© 2012 American Chemical Society

cible reaction media, which is very important for biodiesel production.10 Liu et al. studied the lipase-catalyzed hydrolysis of soy oil in the presence of ultrasound and reported a higher reaction rate.11 Another study reported that the rate of transesterification of triolein with several alcohols under ultrasonic irradiation was higher than that of the conventional stirring method.12 In our previous work, we have isolated the thermostable and solvent-tolerant Bacillus sp. lipase for the production of biodiesel.13 The organism was isolated from the oilcontaminated soil and identified as Bacillus sp., and the lipase obtained was purified by ion-exchange chromatography. The purified lipase was lyophilized and stored. The molecular weight of isolated enzyme was found to be 45 kDa, and the enzyme showed a maximum activity at 55 °C and pH 7. The enzyme was highly stable toward various organic solvents. Also, the transesterification efficiency of the isolated lipase was effective, and it could be used as a potential biocatalyst for biodiesel production.13 This work aims to improve the isolated lipase efficiency for methyl ester production by the application of ultrasound. In the present study, a novel thermo-, solvent-, and sono-tolerant lipase immobilized in celite was used to produce methyl ester from Oedogonium sp. oil, in the presence and absence of ultrasound. The influence of the operating parameters, such as enzyme dosage, methanol/oil molar ratio, solvent addition, and water content, were investigated.

2. EXPERIMENTAL SECTION 2.1. Algae. In the future, algae must be the source for oil production; therefore, it is very important for lipase to catalyze those kinds of algae oils. Hence, in this work, algae oil was used for the transesterification studies instead of other plant oils. Oedogonium sp. (macroalgae) was obtained from the Bengal Aquarium, Ramanadhapuram, India. It was washed with distilled water, dewatered by sieving Received: May 6, 2012 Revised: September 7, 2012 Published: September 11, 2012 6387

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through 2 mm mesh cloth, and then washed 3 times with distilled water to remove other contaminants. The washed samples were airdried for approximately 48 h. The dried biomass was ground in a laboratory blender, and the powder obtained was sieved using American Society for Testing and Materials (ASTM) sieves. The particles ranging in size from 0.10 to 0.12 mm were collected and stored in sealed plastic bags at ambient conditions. About 10 g of algae powder was placed in a lab-scale Soxhlet extractor fitted with a 250 mL round-bottom flask and a condenser. The extraction was executed for 15 h with 70 mL of hexane as the solvent. The extract obtained was concentrated in a rotary evaporator, until the complete removal of the solvent was achieved. The oil yield was expressed as percent oil extraction, which is defined as the ratio of the weight of oil extracted by the weight of sample taken. The Oedogonium sp. oil composition is presented in Table 1, and its properties are presented in Table 2.

obtained were subjected to further analysis to determine the ester content. The fuel properties of the methyl ester, such as density, viscosity, sulfur, glyceride, water content, cetane number, flash point, pour point, calorific value, and acid value, were analyzed by ASTM standard methods. 2.5. Lipase Activity. The lipase enzyme activity was measured by titrimetric analysis. The mixture containing 2.5 mL of water, 1 mL of Tris-HCl buffer (200 mM, pH 7), and 3 mL of olive oil (substrate) was added with 1 mL of lipase. The contents were incubated in a shaker at 125 rpm at 55 °C for 30 min. The blank was prepared and stored at 4 °C. After 30 min, 3 mL of 95% ethanol was added to terminate the reaction. Then, 4 mL of 0.9% (w/v) thymolpthalein indicator was added, and the amount of fatty acids released during the reaction was determined by titrating against 50 mM NaOH until the end point was observed. A total of 1 unit of lipase activity was equivalent to 1 μmol of fatty acid released per milliliter under incubation. The activity expressed in units per milliliter of enzyme was calculated using the following equation:

Table 1. Oedogonium sp. Oil Composition compositions

percentage

lauric acid myristic acid palmitic acid stearic acid oleic acid linoleic acid linolenic acid arachidic acid behanic acid eicosapentaenoic acid (EPA) lignoceric acid richinoleic acid

0.699 1.096 16.869 4.629 37.637 30.858 0.649 1.621 3.466 0.291 1.436 0.749

(NaOH)(normality of NaOH)(1000)(2)(df) units = mL of enzyme (1) (1) where NaOH is the difference between the volume of sodium hydroxide used for the test minus the volume of sodium hydroxide used for the blank (mL), 1000 is the conversion factor from milli- to microequivalent, 2 is the conversion factor from 30 min to 1 h, df is the dilution factor, and 1 is the volume of enzyme used (mL). To determine the immobilized enzyme activity, 1 g of the immobilized enzyme was added instead of 1 mL of enzyme solution and the same procedure was followed. The activity of the immobilized lipase was found to be 130 units/g. 2.6. Analytical Methods. The fatty acid composition of the Oedogonium sp. oil and methyl ester content in the reaction mixtures were analyzed by gas chromatography (Sigma), equipped with an AC30 Carbowax column (3 met and 1/8 in.) and a flame ionization detector. Nitrogen was used as a carrier gas, and hydrogen and oxygen were employed for the purpose of ignition. While injecting the samples, the column temperature was kept at 150 °C, raised to 240 °C at 10 °C/min, and maintained for 10 min. The injector and detector port temperatures were set at 250 °C. The ebullition point of the methyl esters and Oedogonium sp. oil was analyzed using a thermogravimetric analysis (TGA) analyzer (Netzsch Technologies). The TGA consists of a precision mass balance, which records the initial instantaneous mass of a sample, and a furnace, which increases the temperature in a linear relationship with time (the temperature ranges between 20 and 1000 °C). Dry nitrogen was flushed over the balance chamber at a flow rate of 40 mL/min, while dry air was used over the sample at a flow rate of 60 mL/min using platinum pans. A total of 10 μL of sample was taken. The thermobalance temperature was equilibrated at 50 °C and then increased to 600 °C at a ramp rate of 10 °C/min. The fatty acid methyl ester (FAME) compositions were determined using high-performance liquid chromatography (HPLC). The HPLC using Shimadzu LC10 with an ultraviolet (UV) detector set at 205 nm and C18 column was used, and the instrument was equipped with a helium degasser, an autosampler, and a quartenary pump module. The mobile phase gradient was from 100% methanol to 50% methanol in equilibrium, with the subsequent quantity of isopropanol/hexane in a 5:4 volume ratio. The total run time was 15 min at a flow rate of 1 mL/min. HPLC calibration was carried out using methyl ester standards. The desired amount of standard was mixed with necessary solvents, and the mixture was injected into HPLC. The concentration of standard was calculated from the area of the peaks detected with respect to the retention time. This was repeated 5 times to calibrate the instrument accurately. After the transesterification reaction, the upper methyl ester layer was collected and further purified by distilled water washing. After washing 3 times with water, methyl ester obtained was treated with sodium sulfate to remove residual water present. The

Table 2. Properties of Oedogonium sp. Oil acid value (mg of KOH/g) water content (vol %) density at 15 °C (g/cm3) viscosity at 40 °C (cSt) sulfur (mass %) flash point (°C) pour point (°C) calorific value (MJ/kg)

8.32 0.05 0.92 39.68 0.12 230 −2 41.05

2.2. Sonolyzer. A bath-type sonolyzer with the operating frequency of 30 kHz was used in the present study. The sonolyzer tank was made of stainless steel, and the bottom of the tank was fitted with an ultrasonic transducer. The timer and temperature indicator were provided with the sonicator system. 2.3. Enzyme. In our previous work, we have isolated the thermostable and solvent-tolerant Bacillus sp. lipase for the production of methyl ester; the organism was isolated from the oil-contaminated soil and identified.13 To immobilize the enzyme, 1 g of celite (SRL, India) was added to 5 mL of solution containing purified Bacillus sp. lipase (260 units/mL), and the mixture was stirred for 1 h at room temperature. The immobilized lipase recovered by centrifugation was dried and stored at 4 °C. 2.4. Transesterification. Transesterification reactions were performed in glass vials in the presence and absence of ultrasound irradiation. The glass vials containing the reactants were placed inside the ultrasonic bath containing water, and then the ultrasound was applied for a desired time. Transesterification in the absence of ultrasound was carried out at 55 °C in an orbital shaker at 150 rpm. The reaction mixture used was under the baseline described by Yu et al.7 and was slightly modified with respect to isolated enzyme behavior to obtain a higher yield of methyl esters. The optimum condition values of the operating parameters were arrived at by varying one parameter at a time, while the rest remained constant. The samples 6388

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final product was filtered through Whatman Filter No. 42, and then 20 μL of sample from the filtrate was injected into HPLC.

3. RESULTS AND DISCUSSION 3.1. Methanol/Oil Molar Ratio. The use of a base or acid catalyst demands a large amount of alcohol for methanolysis, whereas the enzymatic reaction requires normally a 3:1−6:1 molar ratio. The usage of a higher amount of alcohol in enzymatic reactions may inactivate the activity of lipase. The effect of different molar ratios of methanol/oil on the methyl ester yield was studied for both of the cases (in the presence and absence of ultrasound), and the results are shown in Figure 1. The results showed a higher yield for the molar ratio of 3:1

Figure 2. Effect of the t-butanol concentration on the methyl ester yield. Conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/oil molar ratio, 3:1; immobilized lipase, 6%; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations.

Vulfson and Sarney19 studied the effect of ultrasound on subtilisin-catalyzed interesterification with various alcohols and reported that the pretreatment of subtilisin by ultrasound increased the enzyme activity. This effect of ultrasound depended upon the amplitude of ultrasound and water content of the reaction medium and was more distinct in alcohols. The subtilisin was more resistant to the inactivation by ultrasound irradiation in organic solvents than in water.19 As discussed earlier, the strong polarity of methanol strips the water from the active site of the enzyme, which leads to enzyme deactivation. The addition of organic solvent as a cosolvent in the reaction mixture increased the solubility of methanol and limited the stripping of water from the active sites of the enzyme. The addition of the co-solvent also increases the solubility of oil and methanol and controls the concentration of methanol surrounding the enzyme. Therefore, a significant increase in the yield was observed. From the results observed in the present study, 0.75 mL/g of oil was found to be the optimum amount for both ultrasound-assisted and normal transesterification. 3.3. Enzyme Dosage. The effect of enzyme dosage (enzyme immobilized on celite) on ultrasound-assisted transesterification was studied, and the results are shown in Figure 3. The enzyme dosage was varied from 2 to 10% (w/w of oil). Transesterification in the absence of ultrasound showed the highest methyl ester yield at 6% enzyme dosage, and a further

Figure 1. Effect of methanol/oil (mol/mol) on the methyl ester yield. Conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; t-butanol, 0.75 mL/g of oil; immobilized lipase, 6%; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations.

for both of the cases. The increase or decrease in the molar ratio from this value negatively affected the yield. This is due to the fact that the addition of a higher amount of methanol strips the active water from the active site of the enzyme, which deactivates the enzyme.14−16 On the other hand, the addition of methanol less than the stoichiometric requirement will affect the yield. Therefore, the optimum amount should be added to obtain a maximum yield. In ultrasound-assisted transesterification, the main advantage is the reduction of the reaction time. The addition of methanol increased the generation of cavitation bubbles, because of its coalescence inhibiting nature, and this phenomenon intensifies the reaction. Therefore, further studies were carried out with a 3:1 molar ratio for both of the cases. 3.2. Addition of the Solvent. The addition of a co-solvent into the reaction mixture influences the yield of methyl ester in the present study.17 The influence of solvents, such as hexane, tbutanol, and petroleum ether, was tested, and better results were observed with t-butanol. Therefore, detailed studies were carried out with different amounts of t-butanol, and the results are shown in Figure 2. For both of the cases, the methyl ester yield was very low in the solvent-free system (23.5% with ultrasound and 18% without ultrasound), while the addition of solvent increased the yield. The increase in t-butanol addition of up to 0.75 mL/g of oil increased the methyl ester yield, and a further increase in the concentration of t-butanol decreased the yield. This may be due to the dilution of reactants with a large amount of t-butanol present in the system.18 Li et al.18 studied the effect of t-butanol on the methanolysis of rapeseed oil and reported a higher methyl ester yield for the volume ratio of tbutanol/oil of 0.75.

Figure 3. Effect of enzyme dosage on the methyl ester yield. Conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/ oil molar ratio, 3:1; t-butanol, 0.75 mL/g of oil; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations. 6389

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increase in the enzyme dosage decreased the yield. This is due to the fact that the addition of an excess enzyme content decreased the liquid volume required for the reaction to occur. Furthermore, under these conditions, the external mass-transfer resistance becomes the limiting step for the oil transesterification.20 The increase in enzyme dosage increased the viscosity of the mixture, which, in turn, reduced the reaction rate.21 However, in ultrasound-assisted transesterification, the yield was increased with an increase in the enzyme dosage of up to 5%, and a further increase did not affect the yield. The decrease in the yield observed for the reaction in the absence of ultrasound was not observed in this case. This may be due to the effect of the ultrasound, which facilitated the dispersion of lipase and reduced the agglomeration. However, the expected result is that the yield should increase with the enzyme concentration increasing, until the maximum yield was obtained, where the enzyme saturates the interface and then remains constant at any further increase in the enzyme concentration.11 3.4. Effect of the Water Content. Water is not a reactant or a product of the transesterification reaction, but its content influences the enzymatic activity. Water acts as a lubricant for polypeptide chains and helps the necessary mobility of the enzyme to explicate the catalytic action.22 Water plays a vital role in minimizing the solvent-induced conformational rigidity, which is one of the causes for the reduced catalytic activities observed in non-aqueous media.23 Therefore, in the present study, the effect of the water content on the yield of methyl ester was examined, and its range was varied from 0.25 to 1% (v/w of oil). The maximum yield was observed at 0.5% for both of the cases. The increase or decrease in the water content from 0.5% affected the yield, and this indicates that a very little amount of water is required to activate the enzyme (Figure 4).

Figure 5. Effect of the sonication time on the methyl ester yield. Reaction conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; tbutanol, 0.75 mL/g of oil; temperature, 55 °C; and ultrasound, 30 kHz. Data points were the mean of triplicate values, with the error bars showing standard deviations.

to 10% at a 6:1 molar ratio did not show a significant effect on the methyl ester yield. On the other hand, the yield obtained for the 6:1 molar ratio was found to be less compared to the 3:1 molar ratio. This may be due to the presence of excess alcohol, which inactivated the enzyme activity, and this prevailed even with an increase in the enzyme dosage from 6 to 10%. In contrast, the increase in the alcohol content may dilute oil, and this, in turn, reduced the methyl ester yield. The effect of the reaction time on the methyl ester yield in the absence of ultrasound is shown in Figure 6. The methyl ester yield

Figure 6. Effect of the time on the methyl ester yield. Reaction conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/oil molar ratio, 3:1; t-butanol, 0.75 mL/g of oil; immobilized lipase, 6%; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations.

Figure 4. Effect of the water content on the methyl ester yield. Conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/ oil molar ratio, 3:1; t-butanol, 0.75 mL/g of oil; immobilized lipase, 6%; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations.

gradually increased with an increase in the reaction time up to 40 h, and a further increase did not affect the yield. The results showed that the reaction time required by the ultrasoundassisted transesterification (2 h) was 20 times less than the time required by the transesterification in the absence of ultrasound (40 h). This is due to the fact that the microturbulence generated by the cavitation bubbles form a fine emulsion between oil and alcohol, which creates an enormous interfacial area, and this, in turn, accelerates the reaction rate.7 On the other hand, the reaction rate is influenced by the mass-transfer characteristics of the immobilized reaction systems. In this case, reactants have to diffuse from the bulk liquid to the external surface of the immobilized matrix and then diffuse through the film surrounding the particle to reach the surface. In addition, the reactants need to diffuse through the pores to reach the

The decrease in enzyme activity, at a higher water content, could be attributed to the enzyme particle aggregation that might consequently lead to a limited contact of the substrate with the enzyme active site.24 3.5. Effect of the Sonication Time. The influence of the sonication time on methyl ester production was studied, and the results are shown in Figure 5. The methyl ester yield was found to increase with an increase in the sonication time, and the maximum yield was observed at 120 min. A further increase in the sonication time, beyond 120 min, did not improve the yield. Furthermore, the increase in the enzyme content from 6 6390

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enzyme, which is immobilized in the matrix. This entire process involves many resistances. The application of ultrasound enhances the mass transfer in the solid−liquid reactions and reduces the magnitude of the resistances.25 Ultrasound for biological processes is an excellent tool for the improvement of the rate of reaction, particularly in highly viscous and immiscible reaction systems.26 Stavarache et al. investigated the production of biodiesel from vegetable oil using ultrasound and mechanical stirring.27 The results clearly indicated that the application of ultrasound reduced the time required compared to mechanical stirring.27 The effect of ultrasound on enzyme activity may direct activity loss or gain by the behavior of the enzyme involved, and conformational change in the enzyme structure may occur during ultrasonic treatment. An ultrasound wave with a periodic pressure fluctuation can control the enzyme characteristics by altering its structure.28 Ultrasound irradiation below 100 kHz may disturb the domain regions of an enzyme, and therefore, affect its activity. The tolerance of enzyme activity on ultrasound may depend upon the physical location of the enzyme in the cell and the molecular weight of the enzyme. The reports related to the exact effect of the ultrasound on enzyme activities are scarce, and very little has been reported because of the conflicting results of the activation and deactivation of the enzymes upon ultrasound. Unlike heat denaturation, ultrasound waves do not destroy the active site of an enzyme, and it was clearly confirmed for α-amylase, laccase, and alkaline phosphatase.29,30 The deactivation of enzymes as a result of ultrasound was observed while using high ultrasound power of 500 W. This is most likely due to the formation of hydrogen radicals at the protein backbone. This may lead to enzyme aggregation, thereby destroying the active sites and protein stability.31 The ultrasound effect on enzymes depends upon the energy input and irradiation time. The enzyme stability of the irradiated enzyme was increased by redox mediators, such as polyvinyl alcohol, violuric acid, and 2,2-azinobis-3-ethylbenzthiazoline-6-sulfonic acid.29 The optimization of parameters gives the optimum conditions for ultrasound irradiated enzymes, and hence, the enzyme activity was not affected much. In another study, under sonication, the subsilisin in the phosphate buffer showed inactivation at 50% power for 2 h, although no such effect was found in the buffer containing t-amyl alcohol.19 The highintensity ultrasound waves break the cells and denature the enzyme structure, while low-intensity ultrasound modifies cellular metabolism or improves the mass transfer. Therefore, immobilized enzymes are more resistant to thermal denaturation produced by ultrasound than native enzymes.31 In the present study, the isolated novel enzyme showed efficient performance in the presence of ultrasound. Its application for the production of methyl ester was very useful, and the ultrasound-assisted process consumed less time. The energy consumption during the ultrasound-assisted process is also less as a result of its less time consumption. 3.6. Enzyme Reusability. The economics associated with enzymatic reactions necessitates the analysis of the reusability of the enzyme. Hence, in the present study, the reusability of catalysts for methyl ester production was carried out, and the results are shown in Figure 7. The immobilized lipase after each reaction was washed with a phosphate buffer and reused in the subsequent reaction. The results indicate a very marginal decrease in the yield even after five cycles, and the reduction

Figure 7. Reusability of the enzyme. Conditions: with ultrasound, 30 kHz; Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/oil molar ratio, 3:1; t-butanol, 0.75 mL/g of oil; immobilized lipase, 6%; and temperature, 55 °C. Data points were the mean of triplicate values, with the error bars showing standard deviations.

was found to be 10%. Hence, these results suggest that the enzyme used in the present study is a suitable catalyst for methyl ester production, in the presence of ultrasound. 3.7. TGA. TGA is a rapid and economic technique used to determine the boiling point of the transesterification product. In addition, TGA can confirm the occurrence of the transesterification reaction because the boiling points of the oil and esters are very different. The comparison of Oedogonium sp. oil and its methyl ester is presented in Figure 8. The

Figure 8. TGA of methyl ester and Oedogonium sp. oil.

temperature employed was 50−600 °C at a ramp rate of 10 °C/ min. The mass of the Oedogonium sp. oil starts to decrease at approximately 290 °C and continues to decrease until the entire oil sample is vaporized. In the same way, the evaporation of methyl ester starts at approximately 160 °C and continues to decrease until the entire methyl ester sample is completely volatilized at 404 °C. Therefore, Oedogonium sp. oil and its methyl ester start to decrease at 290 and 160 °C, respectively, and the values were in agreement with those by de Oliveira Lima et al.32 3.8. HPLC Analysis. The HPLC analysis was used to analyze the purity of the methyl ester samples. The methyl ester concentration was reported as the total area percentage of the FAME peak area in the HPLC chromatogram (Figure 9). The results showed 96% of FAME and the presence of some traces of glycerol and mono- and diglycerides. Hence, HPLC results also confirmed that the product formed from the transesterification reaction was FAME. The results observed were in agreement with the results reported by Fogila et al.33,34 The FAME peaks occur nearly at the elution time of 4 min. The 6391

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scheme. Ramachandran Sivaramakrishnan is grateful to DST, New Delhi, India, for the award of the DST-PURSE Fellowship.

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Figure 9. HPLC of Oedogonium sp. methyl esters: (1) glycerol and (2) methyl esters. Reaction conditions: Oedogonium sp. oil, 2 g; water content, 0.5%; methanol/oil molar ratio, 3:1; t-butanol, 0.75 mL/g of oil; immobilized lipase, 6%; and temperature, 55 °C.

important fuel properties of the methyl ester were analyzed adopting ASTM standard methods, and the results are presented in Table 3. Table 3. Properties of the Oedogonium sp. Methyl Esters density at 15 °C (g/cm3) viscosity at 40 °C (cSt) sulfur (wt %) free glycerol (wt %) total glycerol (wt %) water content (%, w/w) cetane number flash point (°C) pour point (°C) calorific value acid value (mg of KOH/g)

0.862 4.027 0.01 0.016 0.19 0.038 57 141 −5 39.72 0.174

4. CONCLUSION The present study focuses on the ultrasound-assisted methyl ester production from Oedogonium sp. oil using isolated novel Bacillus sp. lipase. The effect of operating parameters was studied, and at optimum conditions [t-butanol, 0.75 mL/g of oil; methanol/oil molar ratio, 3:1; oil enzyme dosage, 6% (w/ w); oil−water content, 0.5% (v/w); and temperature, 55 °C], the methyl ester yield observed for the transesterification in the presence and absence of ultrasound was 82% (reaction time of 2 h) and 75% (reaction time of 40 h). The isolated enzyme was found to be stable under ultrasound irradiation, and no noticeable loss in lipase activity was observed, after repeated use for five cycles.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone: +91-44-22359188. Fax: +91-44-22352642. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Department of Science and Technology (DST), New Delhi, India, for providing financial support to carry out this research work under PURSE 6392

dx.doi.org/10.1021/ef300769s | Energy Fuels 2012, 26, 6387−6392