Process optimization of biodiesel production from ...

Renewable Energy 116 (2018) 755e761

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Process optimization of biodiesel production from Hevea brasiliensis oil using lipase immobilized on spherical silica aerogel A. Arumugam*, D. Thulasidharan, Gautham B. Jegadeesan School of Chemical & Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, 613401, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2017 Received in revised form 22 September 2017 Accepted 7 October 2017 Available online 10 October 2017

In this study, biodiesel was synthesized in an enzymatic transesterification process from Hevea brasiliensis, crude non-edible oil, using lipase immobilized on spherical silica aerogels. Enzymatic transesterification is preferred to chemical methods as it is milder and is more environmentally friendly. Lipase based transesterification of Hevea brasiliensis under optimal conditions provided high FAME (fatty acid methyl esters) yields up to 93%. Response Surface Methodology (RSM) was used to optimize the process for maximum FAME yield. The maximum yield was obtained at a temperature of 35 C, water content of 15% (v/v %) and methanol/oil molar ratio of 8:1. Percent yields of FAME from the transesterification process followed second order model. Even after 10 cycles of reuse, lipase immobilized on spherical silica aerogel showed only 10.7% reduction in percentage yield of FAME. The results from this study demonstrate the viability of economical biodiesel production using waste products as both source and catalyst. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Biodiesel Transesterification Hevea brasiliensis (Rubber seed) oil Lipase Mesoporous silica aerogel Response surface methodology (RSM)

1. Introduction Biodiesel has been generally accepted and defined as a substitute for or an additive to diesel fuel, which is derived from natural sources [1]. Sources for biodiesel production are plenty mostly from oils and fats of plants and animals derived from a renewable lipid feedstock [2]. The main advantages of using this alternative fuel is its renewability, lower gaseous emissions, and its biodegradability. Since all the organic carbon present has a photosynthetic origin, there is no net increase in CO2 levels in the atmosphere [3]. Given the food scarcity in several regions around the world, there is an increased focus on the use of non-edible oils for biodiesel production. Some of the non-edible feed stock available and reported are Barbados nut (Jatropha curcus) [4], Neem (Azadirachta indica) [5], Castor (Ricinus communis) [6], Linseed (Linumus itatissimum) [7], Karanja (Pongamia pinnata) [8] and Pinnai (Calophyllum inophyllum) [9]. The two main challenges to large-scale biodiesel production are: (1) selection of non-edible feedstock with high oil content and their abundance; and (2) synthesis method (chemical or enzymatic). Hevea brasiliensis, commonly known as rubber seed, is one such low

* Corresponding author. E-mail address: [email protected] (A. Arumugam). https://doi.org/10.1016/j.renene.2017.10.021 0960-1481/© 2017 Elsevier Ltd. All rights reserved.

cost non-edible feedstock, which is found in abundance in the Amazon [10]. The Hevea brasiliensis seed contains high oil content (up to 89.4%) and approximately 80.5% of the oil is in the form of unsaturated fatty acids (essentially linoleic and oleic acids). The oil extracted from the seed is blackish brown in colour with an unpleasant aromatic odour [11,12]. Given the high oil content, there has been increased recent focus on using this feedstock for biodiesel production, as summarized in Table 1. As can be seen in Table 1, the focus of most studies was to evaluate the biodiesel yield from rubber seed oil. In most studies, chemical catalysts (homogenous and heterogeneous) are used [13e17]. Use of acid catalysts such as H2SO4 and base catalysts such as KOH [13,18] has shown that Hevea brasiliensis oil transesterification yields 31e99% methyl esters, depending upon the conditions. Dhawane and his coworkers reported almost 90% yields at an optimum molar ratio of 15:1, 55 C, reaction time of 60 min and 3.5% (w/w of oil) catalyst loading. The study also revealed that after three cycles of reuse, there was only 1.4e1.8% reduction in percentage conversion [18]. Another study using methyl propyl sulfonic acid-functionalised MCM-41 as catalyst showed 96% FAME (fatty acid methyl esters) yield at 5% catalyst loading, 120 min reaction time and 153 C reaction temperature [15]. Widayata and his co-workers produced 91% biodiesel yield from rubber seed using H2SO4 catalyst (0.1e1% of catalyst loading, 0.5 (v/v%), 1:1.5e1:3 quantity of methanol/oil (molar), and a reaction time of 120 h [19].

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Table 1 The comparison of biodiesel production from Hevea brasiliensis using various catalyst in the literature with the present work. Catalyst

Operating parameter's

% yield

Work

Lipase from Aspergillus niger

8:1 molar ratio of methanol to oil, 15% water content and 35 C, 430 mg immobilized lipase/g oil, reaction time 7 h. Methanol to oil molar ratio of 3:1e15:1, a catalyst concentration of 0.25e0.25%wt., a reaction temperature of 50e70 C, a reaction time of 30e150 min and speed agitation of 800e1200 rpm Methanol to oil molar ratio of 1:4 and catalyst concentration of 15 (w/w) % of oil after 48 h. Reaction temperature 55 C, Reaction time 60 min, catalyst loading 3.5 wt% and methanol to oil ratio 15:1. Methanol to oil ratio 16:1, Catalyst loading of 14.5 wt% and a reaction time and temperature of 48 h and 129.6 C, Temperature 60 C, reaction time 1 h, and 5 g of carbon based catalyst at varying quantities of catalyst loading (0.5, 2, 3.5, 5 wt%) and methanol to oil ratio (5:1e20:1) 6:1 alcohol to oil ratio, 1% catalyst concentration, 55 C reaction temperature and 67.5 min reaction time. Catalyst concentration in range 0.1e1% (v/v), raw material to methanol molar ratio (1:2), Temperature 60 C, ratio of raw material to methanol in range 1:1.5 e1:3 and reaction time 60 min. Catalyst loading of 5 wt %; methanol to oil molar ratio of 4:1; reaction temperature of 65 C and reaction time of 4h Methanol/oil molar ratio 6:1, stirring speed 1000 rpm, reaction temperature 60 C, 3 wt% NaOH/NaPAA sample with NaOH loading amount of 7.5 mmol/g was used as catalyst Methanol/oil ratio 0.28% v/v, sodium hydroxide of 0.75% w/v, Temperature 51.23 C, Reaction time 82.52 min Methanol/oil molar ratio of 9:1 and 0.5% by weight of sodium hydroxide 1% catalyst loading, 6:1 alcohol to oil ratio, 97.8 ± 0.2 C C reaction temperature and 60 min reaction time.

93

Present work

99.32

A.S. Silitonga et al., 2016 [13]

31

V.V. Vipin et al., 2016 [52]

89.81

Sumit H. Dhawane et al., 2015 [14]

84

S. Karnjanakom et al., 2015 [15]

89.3

Sumit H. Dhawane et al., 2015 [16]

96.8

Junaid Ahmad et al., 2014 [20]

91.05

Widayat et al., 2013 [19]

96.9

Jolius Gimbun et al., 2013 [47]

96

Ru Yang et al., 2013 [48]

97.1

D.F. Melvin Jose et al., 2013 [49]

e

A.S. Ramadhas et al., 2005 [50]

84

O.E. Ikwuagwu et al., 2000 [51]

Acid esterification: KOH Base transesterification: H2SO4

Rhizopus Oryzae Lipase Activated carbon impregnated with pure KOH SO3H-MCM-41

Carbon based KOH impregnated heterogeneous catalyst from flamboyant pods (Delonix regia) Acid esterification: H2SO4 Base transesterification: KOH H2SO4

Limestone based catalyst

Poly (sodium acrylate) supported NaOH

Alkaline esterification: NaOH Base transesterification: NaOH Alkaline esterification: H2SO4 Base transesterification: NaOH NaOH

The other challenge, as noted earlier, is the method of transesterification. Chemical transesterification of non-edible oils to biodiesel is a preferred option because of its flexibility in use, large production rates and capacities, and high yields [20,21]. However, the high costs, post-synthesis environmental issues and high energy requirements warrant the need to search alternate technologies. As noted earlier, most studies on transesterification of Hevea brasiliensis oil used chemical catalytic methods. However, the chemical catalyzed transesterification process requires reaction temperature of at least 60 Ce80 C, high methanol to oil molar ratio of 12:1 to 36:1 and multi stage pre-treatment processes to reduce the free fatty acid content. These drawbacks can be eliminated by enzymatic transesterification, which offer a promising alternative to the chemical methods. Enzymatic transesterification offers the advantages of low temperature and pressure conditions (ambient), high catalyst recyclability and equally good FAME yields. In our previous works [9,21], we have successfully demonstrated that use of immobilized enzyme for transesterification process which is not only viable, but also a better alternate to chemical transesterification. To the best of our knowledge, there has been no work till date on the production of biodiesel using Hevea brasiliensis oil via enzymatic transesterification process. This work is the first of its kind to be reported on biodiesel production from crude Hevea brasiliensis oil (CHBO) catalyzed by immobilized lipase spherical silica aerogel. Immobilized enzymes are used to: (1) reduce the cost of the enzyme; (2) improve catalyst stability and recyclability; and (3) prevent reaction inhibition due to high substrate and product concentrations. Further, this process seeks to improve activity and

selectivity which is challenging for commercialization of lipasecatalyzed biodiesel production. The specific objectives of the study are: (1) optimization of the process parameters such as molar ratio of substrates, water content and temperature on yield; and (2) reusability of the catalyst for efficient biodiesel production. Optimization of the important process variables namely molar ratio of methanol to oil (3:1e8:1), volume of water (5%v/v - 25% v/v) used and reaction temperature (30 C-40 C) maintained were done using Response Surface Methodology (RSM). 2. Materials and methods The lipase (E.C.3.1.1.3) used in the present study is the commercial Lipase (purity 99%, activity 16000 LU/g was purchased from Himedia Pvt. Ltd. The crude Hevea brasiliensis (rubber seed) oil (Average molecular weight, 861.4 g mol-1, specific gravity, 0.919) was obtained from nearby agricultural field in Thanjavur, India. Coal bottom ash is obtained from NLC India Limited (Tamilnadu, India). Sodium hydroxide pellets (99%, Merck), ethanol (95%, Merck), hydrochloric acid (98%, Merck) are obtained from Merck Chemicals Pvt. Ltd. Methanol, dipotassium hydrogen phosphate, copper sulphate pentahydrate, lipase, sodium potassium tartarate, potassium dihydrogen phosphate, sodium carbonate, Folin‘sciocalteau reagent and gum arabic have been purchased from Himedia Laboratories Pvt. Ltd. 2.1. Preparation of silica aerogel and lipase immobilization Silica aerogel microspheres are prepared following the


procedure reported in literature [22]. 0.1 g of spherical aerogel particles was dispersed in 10 mL of potassium phosphate buffered to pH of 7. Precisely weighed lipase (10 mg) was added to the above mixture and stirred for 12 h, maintaining temperature to 30 C. The immobilized lipase was separated by filtration and the percentage immobilization and specific enzyme activity were determined. The enzyme concentration was measured by Lowry method [23]. Specific enzyme activity for immobilized enzyme was estimated by Olive oil emulsion method [24]. The percentage immobilization, specific enzyme activity of the immobilized lipase on silica aerogel was 77% and 14800 U/g.

2.2. Transesterification reaction The transesterification reaction was carried out in a 100 mL screw capped vessel using 20 gm of CHBO, 15% (v/v %) water content, 430 mg immobilized lipase and at a constant temperature of 30 C. Typically, higher water content ensures higher enzymatic activity [25]. However, the water content was maintained at this level to: (1) prevent hydrolysis of the ester linkages at even higher water content, thereby allowing for the forward transesterification reaction [26]; and (2) ensure insignificant mass transfer interferences between the aqueous and oil phase (since enzyme lipase have a unique feature to act at the interface between the aqueous and organic phases). Methanol was added to the reaction vessel in a three step process at 1:8 M ratio of oil/methanol. For example, 2.72 mL of methanol was added to 22.5 mL of oil, which corresponded to a molar ratio of 1:3. To increase the methanol: oil molar ratio to 1:9, 8.15 mL of methanol was added to 22.5 mL of oil, while keeping the amount catalyst at 430 mg. The reaction was carried out in a shaking incubator at 180 oscillations per minute. After a 10 h reaction, 100 mL of sample was taken from the reaction mixture and centrifuged. The upper layer was analysed in GC-MS [27]. All the experiments were done in triplicates.

2.3. Response Surface Methodology (RSM) Various experiments have been conducted to identify the important process variables, their effect on the response variables and the interaction between the variables. Response Surface Method is a statistical experimental design that helps identify optimum process conditions with less number of experimental runs as compared to conventional experimental design. Thus, cost of expensive experimental runs is minimized by RSM [28,29]. The main advantage in using RSM is the speed and reliability of the outcome. Contours are response curves drawn in 2-dimensional plane, keeping other variables fixed [30]. The shape of contour plots of the response helps us to visualize interaction between the variables. A quadratic model was employed to express the response variable in terms of the independent variables. Later the model was solved analytically to obtain the optimum conditions [28]. All the statistical analyses were conducted using Minitab statistics software (Version 16.2.2, Minitab Inc, Pennsylvania, USA). Analysis of variance (ANOVA) was performed on the data to test the effects of the parameters and their interactions. Tukey's multiple tests were performed to determine the differences among the levels of each parameter. The a-level chosen was 0.05. The RSM utilized Taylor first order and second order series with experimental data for optimization. The surface of Taylor expansion curve was determined using RSM and this describes the response. It is of the form (Eq. (1)):

Response ¼ b0 þ

X

757

b i xi þ

X

bii x2 þ

X

bij xi xj

(1)

where,

bi, bij ¼ Regression coefficients x ¼ Process variable

2.4. Catalyst and product characterization The synthesized catalyst matrix was characterized using Scanning Electron Microscope imaging for surface morphology (6701 F, JEOL, Japan). Brunauer-Emmett-Teller (BET) method was used to determine specific surface area of support matrix. Barrett-JoynerHalenda model (BJH) was used to determine pore size distribution. N2 gas adsorption technique was used to determine volume of pores, average pore diameter and surface area of support matrixes. Methyl esters formed during the experiment were examined by Gas ChromatographyeMass Spectroscopy (CLARUS 500, PerkinElmer, USA). Fourier transform infrared spectroscopy (spectrum 100, Perkin Elmer, USA) was used to characterize the functional group attached on mesoporous silica. Quantification of methyl ester content in the reaction mixture was carried out using (0.1 mm 10 cm) gas chromatography capillary column. The column temperature was kept at 180 C for 0.5 min, raised to 300 C at 10 C min1 and maintained at this temperature for 10 min. The temperatures of the injector and detector were set at 245 C and 305 C, respectively. The fuel properties such as density, flash point, fire point, pour point, cloud point, kinematic viscosity, Cetane number, Calorific value of biodiesel obtained from CHBO were measured using ASTM methods. 3. Result and discussion 3.1. Characterization SEM image of silica aerogel (Fig. 1) shows that the synthesized aerogel particles are in the form of solid clusters with size ranging from 22 to 25 nm [31]. The BET surface area, average pore volume and average pore diameter were determined to be 443 m2 g1, 0.19 mL g1 and 2.88 nm respectively (Supplementary Figs. 1a and

Fig. 1. SEM image of Silica aerogel.

758


Fig. 2. FTIR spectra of bare and lipase immobilized silica aerogel material.

1b). Fig. 2 shows the FT-IR spectrum for the bare and lipase immobilized silica aerogel. The presence of a broad band at about 3471 cm1, corresponding to the vibration of SieOH terminal groups. An absorption peak at 1108 cm1 corresponds to asymmetric stretching vibrations of siloxane bond SieOeSi. The FT-IR spectrum of silica aerogel after immobilization confirmed the presence of a number of functional groups in the enzyme structure. Signal at 1661 and 1542 cm1 generated by the NeH bending is the characteristic for the pure enzymes corresponds to amide 1 and amide II bands [32]. Contribution of the CeN stretching vibrations is more likely due to amide III (1426 cm1) bands resulting from NeH bending. Water is critical for enzymes structures, conformation and interaction with solid hydrophilic surfaces. The 2961 cm1 bands may be assigned to eOH bonded to the protein [33]. There was no change in spectrum frequency of surface functional group and disappearance of any peak of silica aerogel, suggesting physical adsorption of the enzyme on the solid matrix. 3.2. Effect of oil-alcohol ratio on reaction (MR) The rate of the reaction depends on the alcohol to oil mole ratio in the reaction mixture. If the alcohol concentration is increased, an increased reaction rate can be observed. But too high methanol concentration leads to deactivation of enzyme. Addition of alcohol in regular intervals to maintain low concentration can improve the enzyme catalysis and will prevent the denaturation. Fig. 3 shows the variation of methanol to oil molar ratio on percentage yield of biodiesel. The yield of biodiesel increases from 3:1 to 6:1 and

Fig. 3. Effect of Methanol to oil molar ratio on immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for Temperature 30 C, 10% v/v water content and reaction time of 10 h.

reaches maximum yield of 93% at 8:1 and then decreases. Increasing trend in the biodiesel yield is due to higher concentration of methanol and decreasing yield might be due to the


759

Table 2 Experimental results based on central composite design.

Fig. 4. Effect of temperature on immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for 8: 1 molar ratio of methanol to oil, 10%v/v water content and reaction time of 10 h.

StdOrder

RunOrder

PtType

Blocks

T

MR

W

Exp % yield

Pred % yield

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 40 30 40 30 40 30 40 30 40 35 35 35 35 35 35 35 35 35 35

4 4 8 8 4 4 8 8 6 6 4 8 6 6 6 6 6 6 6 6

5 5 5 5 25 25 25 25 15 15 15 15 5 25 15 15 15 15 15 15

66.30 59.30 69.10 71.20 64.67 54.28 67.30 66.40 83.21 78.61 86.55 93.44 73.76 71.20 89.20 88.45 89.40 88.12 89.77 88.66

66.40 59.18 68.99 71.07 64.82 54.41 67.44 66.33 82.82 78.65 86.19 93.44 73.88 70.72 89.01 89.01 89.01 89.01 89.01 89.01

Table 3 Estimated Regression Coefficients for yield of FAME.

denaturation of the immobilized lipase [34]. 3.3. Effect of temperature (T) Biodiesel production by enzymatic method requires milder conditions which is less energy intensive when compared to the chemical method. It is well known that rates of most reactions (enzymatic or chemical) tends to increase with temperature, and usually expressed by their Arrhenius constant. In this study also, it was observed that the rate increased with temperature. From Fig. 4, it was observed that the yield of biodiesel is highest (92%) at the 30 C [35]. However, most studies have limited their range of temperature investigated to below 40 C for two reasons: (1) Increasing temperature above 40 C may denature the enzyme, decreasing the rate of reaction; (2) good yields can be obtained at lower temperatures [36]. It was observed that increase in temperature beyond 30 C caused the decline in the yield of biodiesel (Fig. 4). Increasing the temperature from 20 C to 30 C showed a sudden rise in biodiesel yield owing to higher reaction rate. As the temperature increases beyond 35 C the yield of biodiesel reduces due to the loss of the enzyme activity [37]. It has been noted that lower temperature slows down the rate, thereby prolonging the reaction time required to produce a similar yield. Increasing the reaction temperature may lower yield due to the reversible nature of the reaction, and loss of active sites due to enzyme denaturation [38]. 3.4. Optimization parameters for biodiesel production In this study, 3-level-3-factor design was implemented, totally 20 experiments were done in duplicates [39]. The prophase research results showed that the three independent variable parameters such as reaction temperature (T), molar ratios of methanol to oil (MR) and water content (W) have important effects on FAME yield [33,34]. Water content (v/v) was 5e25%; molar ratios of methanol to oil (mol/mol) was 3:1e8:1 and the reaction temperature was between 30 and 40 ( C). The feedback was FAME yield in percentage (FAME). The non-dependent factors, levels, and experimental model are tabulated in Tables 2 and 3 [40,41]. The model predicted was correlated to coefficients of

Term

Coef

SE Coef

T

P

Constant T MR W

89.0349 2.0792 3.6340 1.5810

0.1763 0.1622 0.1622 0.1622

505.067 12.822 22.410 9.750

0.000 0.000 0.000 0.000

T*T MR * MR W*W

8.2784 0.8076 16.7074

0.3092 0.3092 0.3092

26.772 2.612 54.030

0.000 0.026 0.000

T* MR T*W MR *W

2.3238 0.7988 0.0062

0.1813 0.1813 0.1813

12.817 4.406 0.034

0.000 0.001 0.973

S ¼ 0.512786 PRESS ¼ 7.25860. R-Sq ¼ 99.90% R-Sq(pred) ¼ 99.73% R-Sq(adj) ¼ 99.82%.

Table 4 Analysis of Variance for yield. Source

DF

Seq SS

Adj SS

Adj MS

F

P

Regression Linear T MR W Square T*T MR * MR W*W Interaction T* MR T*W MR *W Residual Error Lack-of-Fit Pure Error Total

9 3 1 1 1 3 1 1 1 3 1 1 1 10 5 5 19

2698.97 200.29 43.23 132.06 25.00 2450.38 1587.44 95.31 767.62 48.30 43.20 5.10 0.00 2.63 0.67 1.96 2701.60

2698.97 200.29 43.23 132.06 25.00 2450.38 188.46 1.79 767.62 48.30 43.20 5.10 0.00 2.63 0.67 1.96

299.885 66.762 43.231 132.060 24.996 816.793 188.461 1.794 767.624 16.101 43.199 5.104 0.000 0.263 0.134 0.392

1140.47 253.90 164.41 502.22 95.06 3106.28 716.72 6.82 2919.29 61.23 164.28 19.41 0.00

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.026 0.000 0.000 0.000 0.001 0.973

0.34

0.868

interactions, linear and quadratic effects. The correlation coefficients for each model and variable significance was measured by the probability values are shown in Table 4. All the factors and their square interactions (P < 0.05) except interaction term of methanol

760


to oil molar ratio and water content were significant at the. The best fit for the experimental data (Table 2), expressed by polynomial model for the percentage yield of FAME is as follows:

Percentage yield ¼ 300:46 þ 21:61T 8:74MR þ 5:4W 0:33T2 þ 0:202MR2 0:167 W 2 þ 0:232T MR 0:0159T W þ 0:000313MR W

(2)

The R2 value determines the amount of variability in the observed response values that could be described by the experimental factors and their interactions [41]. Adj-R2 value of 99.82% was observed. As both R2 and Adj-R2 values are high and close to 1.0, a high link between the observed and predicted values can be observed. This proves that the regression model provides exceptional data on the relationship between independent variables and the response validation of the model [42,43]. The optimum level of percentage yield of FAME was 94.33% at methanol to oil molar ratio of 8:1, reaction temperature of 35 C and 15% water content (v/v). 3.5. Reusability studies of immobilized lipase The reusability of lipase immobilized on spherical silica aerogel was investigated by recovering the immobilized particles after each reaction cycle (Fig. 5). Approximately 90% of the activity (in terms of methyl ester formation) was retained after 10 cycles of reaction [44,45]. From Fig. 5 it is evident that the gradual decrease in FAME yield was attributed to both loss of activity of immobilized lipase and loss of enzyme due to leaching. Lipase immobilized on silica aerogel can be reused repeatedly without significant loss in activity in the production of biodiesel from CHBO [46]. 3.6. Fuel properties Fuel properties of crude Hevea brasiliensis oil were determined by ASTM methods. Values of flash point, fire point, pour point and cloud point were 149 ± 0.52 C, 191.6 ± 0.89 C, 4 ± 0.026 C, and 2 ± 0.10 C, respectively. Calorific value and Cetane number (38 C) of the biodiesel produced were 37.9 (MJ/Kg) and 52 respectively. Kinematic viscosity and specific gravity (38 C) of the biodiesel produced from rubber seed oil were 4.97 ± 0.35 (mm2/

Fig. 5. Immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for several cycles at optimum conditions (8: 1 molar ratio of methanol to oil, 10%v/v water content, Temperature 30 C and reaction time of 10 h).

sec) and 0.831 ± 0.02 (g/cm3). Fuel properties of rubber seed Biodiesel were found to be compatible with ASTM Biodiesel Standard (D 6751a) and European Biodiesel Standards (EN 14214). This demonstrates the feasibility of crude Hevea brasiliensis oil biodiesel as fuel. 4. Conclusion In this work, we have demonstrated the production of biodiesel using Hevea brasiliensis, in an immobilized lipase based transesterification process. The optimal condition for methanolysis was 8:1 M ratio of methanol to oil and reaction temperature of 30 C. Under these conditions, 93% yield of methyl ester were obtained. RSM based studies suggested a second order model, with yield dependent on temperature, pH and methanol to oil molar ratio. The fuel produced from the lipase based transestrification process was found compatible with ASTM Biodiesel Standard (D 6751a) and European Biodiesel Standards (EN 14214). Effective use of a waste material as a support material for the enzymatic reaction helps reduce the production cost. The results suggests strongly that enzymatic transesterification of triglycerides offer a more environmentally approach to biodiesel production, with potential for direct use of the product in diesel engines. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.renene.2017.10.021. References [1] Mohammad Hossein Mohammadi Ashnani, Anwar Johari, Haslenda Hashim, Elham Hasani, A source of renewable energy in Malaysia, why biodiesel? Renew. Sustain. Energy Rev. 35 (2014) 244e257. [2] Baharak Sajjadi, Abdul Aziz Abdul Raman, Hamidreza Arandiyan, A comprehensive review on properties of edible and non-edible vegetable oilbased biodiesel: composition, specifications and prediction models, Renew. Sustain. Energy Rev. 63 (2016) 62e92. [3] Ming Li, Yan Zheng, Yixin Chen, Xifeng Zhu, Biodiesel production from waste cooking oil using a heterogeneous catalyst from pyrolyzed rice husk, Bioresour. Technol. 154 (2014) 345e348. [4] Rui Wang, Baoan Song, Wanwei Zhou, Yuping Zhang, Deyu Hu, Pinaki S. Bhadury, Song Yang, A facile and feasible method to evaluate and control the quality of Jatropha curcus L. seed oil for biodiesel feedstock: gas chromatographic fingerprint, Appl. Energ 88 (2011) 2064e2070. [5] Vasanthakumar Sathya Selvabala, Thiruvengadaravi Kadathur Varathachary, Dinesh Kirupha Selvaraj, Vijayalakshmi Ponnusamy, Sivanesan Subramanian, Removal of free fatty acid in Azadirachtaindica (Neem) seed oil using phosphoric acid modified mordenite for biodiesel production, Bioresour. Technol. 101 (2010) 5897e5902. [6] Maryam Ijaz, Khizar Hayat Bahtti, Zahid Anwar, Umar Farooq Dogar, Muhammad Irshad, Production, optimization and quality assessment of biodiesel from Ricinus communis L. oil, J. Radiat. Res. Appl. Sci. 9 (2016) 180e184. [7] Rajeev Kumar, Pankaj Tiwari, Sanjeev Garg, Alkali transesterification of linseed oil for biodiesel production, Fuel 104 (2013) 553e560. [8] Fadjar Goembira, Shiro Saka, Advanced supercritical Methyl acetate method for biodiesel production from Pongamia pinnata oil, Renew. Energ. 83 (2015) 1245e1249. [9] A. Arumugam, V. Ponnusami, Biodiesel production from Calophylluminophyllum oil using lipase producing Rhizopusoryzae cells immobilized within reticulated foams, Renew. Energ. 64 (2014) 276e282. [10] Sergey Blagodatsky, Jianchu Xu, Georg Cadisch, Carbon balance of rubber (Hevea brasiliensis) plantations: a review of uncertainties at plot, landscape and production level, Agric. Ecosyst. Environ 221 (2016) 8e19. [11] Suzana Yusup, Modhar Khan, Basic properties of crude rubber seed oil and crude palm oil blend as a potential feedstock for biodiesel production with enhanced cold flow characteristics, Biomass. Bioenerg. 34 (2010) 1523e1526. [12] S.N.A.M. Hassan, M.A.M. Ishak, K. Ismail, S.N. Ali, M.F. Yusop, Comparison study of rubber seed shell and kernel (Hevea brasiliensis) as raw material for bio-oil production, Energy Procedia 52 (2014) 610e617. [13] A.S. Silitonga, H.H. Masjuki, H. Chyuan, T. Yusaf, F. Kusumo, T.M.I. Mahlia, Synthesis and optimization of Hevea brasiliensis and Ricinus communis as feedstock for biodiesel production: a comparative study, Ind. Crop. Prod. 85 (2016) 274e286. [14] S.H. Dhawane, Tarkeshwar Kumar, Gopinath Halder, Biodiesel synthesis from


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