Biodiesel production from castor oil in the presence of lipase/ functionalized mesoporous SBA-15

Background Enzyme immobilization is efficient tool to reduce enzymatic processes cost and improve catalyst stability. The enzymatic catalytic transesterification reaction of castor oil was studied for the production of biodiesel. In this research, castor seeds oil was extracted and used along with methanol as substrates for enzymatic transesterification reaction. Porcine pancreatic lipase (PPL) immobilized on calcium alginate (Ca-Alg) and mesoporous SBA-15 supports identified, and its potential was investigated for used as the reaction catalyst. The mesoporous SBA-15 supports with a high specific surface area (519.25 m 2 /g) and the pore diameter (83.2 Å) was suitable for immobilization of the PPL with 4.6 ⨯ 2.6 ⨯ 1.1 nm 3 dimensions. The effect of temperature, biocatalyst concentration, methanol/oil molar ratio and water content on biodiesel conversion were investigated in presents of PPL immobilized on mesoporous SBA-15-OH-NH 2 -GA. The biodiesel conversion in the presence of PPL immobilized on SBA-15-OH-NH 2 -GA and Ca-Alg was calculated 88.6% and 77.65% in optimal conditions respectively. The result indicate that castor plant has a very high potential for use in the future of the biodiesel industry as a substrate for transesterification reaction. The biodiesel production efficiency is higher when the PPL is immobilized on the SBA-15-OH-NH2-GA toward than conditions that immobilized on the Ca-Alg.


Introduction
The ever-increasing need of human to energy resources, more or less, been a great challenged for all countries of the world. The decline in fossil resources and environmental concerns were persuaded the researchers to find new sources of non-fossil energies, especially for the transportation. The biofuels are appropriate alternatives that can be used for transportation applications instead of fossil fuels. Biodiesel is a diesel fuel which produced from renewable sources such as vegetable oils (edible and non-edible oil) or animal fats [1][2][3]. Among the benefits of biodiesel into petroleum diesel can be noted to these cases: (I) renewable; (II) biodegradable; (III) almost contains no sulphur, thus SOx will not be released; (IV) the emission of hydrocarbons, carbon monoxide and soot decreases; (V) it can be used without modifying existing engines [4,5]. Transesterification is the most common process of biodiesel production.
Transesterification reaction has been carried out using short chain alcohols with lipids in the presence of a acid, alkali, and enzymatic catalysts. The most of the world biodiesel is produced from edible oils such as olive oil, sunflower oil, soybean oil, and palm oil [6][7][8][9]. The edible oilseeds are easily available on large scale. However, the extensive use of these foodbased feedstocks can cause serious food shortages. As a result, non-edible oilseeds (e.g., castor oil) have drawn attention for biodiesel production. Also, cultivation these oilseeds leads to consuming of carbon dioxide and reducing greenhouse effect. Ricinus Communis (Castor) seed oil is one of the options being considered for biodiesel production due to suitable annual production and high oil content (40-55% oil) [10,11].
The special properties of lipases make them an effective biocatalyst for many reactions. Lipases have high catalytic activity and specificity in transesterification of triglycerides. One of the most significant limitation for using enzymes as catalyst is relatively high production cost.
Enzyme immobilization is efficient tool to reduce enzymatic processes cost and improve catalyst stability. Enzyme immobilization on substrate surface has been developed through several techniques including Physical adsorption, covalent binding, cross-linking, and encapsulation [12,13].
The various solid supports such as calcium alginate, alumina, ion-exchange resins, and nanoparticles have been applied as support for enzymes immobilization. The covalent bonding of the enzyme to the SBA-15 nanomaterials has been carried out, which have a large intrinsic surface area and thus lead to high enzyme loading and consequently high enzyme activity. It is common to modify nanomaterials with glutaraldehyde, which has a positive effect on the ability of SBA-15 to interaction with enzymes, thereby the catalytic activity of the immobilized enzyme has significantly improved [14,15]. In one research, Candida rugosa lipase was immobilized on SBA-15/chitosan-glutaraldehyde, and its hydrolytic activity measurement has been shown an increase in the stability of the biocatalyst in the reaction medium [16]. In another study , Candida antarctica lipase was immobilized on SBA-15 and ionic liquid modified SBA-15 (CH3-IL-SBA-15-BF4), and the results has indicated an improvement in catalytic activity in the modified SBA-15 [17].
In this research, castor seeds oil was extracted in two steps and used along with methanol as substrates for enzymatic transesterification reaction. The mesoporous SBA-15 was created in a typical synthesis and modified by APTES and GA. The PPL was immobilized on Ca-Alg and SBA-15-OH-NH2-GA carriers, by entrapment and covalent bonding methods, respectively.
The amount of immobilized lipase activity as well as the produced biodiesel from it during transesterification reaction were investigated.

Enzyme supports characterization
The field-emission scanning electron microscope (FE-SEM) was carried out to explore the synthesized Ca-Alg polymeric beads morphology. The spherical shape of Ca-Alg beads were obvious on FE-SEM image ( Figure 1). The FE-SEM micrographs of synthesized mesoporous SBA-15 and SBA-15-OH-NH2-GA are shown in figure 2. According to FE-SEM images, the morphology of both bare and modified SBA-15 are uniform and rod-like particles which illustrates functionalization has no effect on the particle morphology. The functionalization step of SBA-15-OH with APTES ( Figure 3c) was identified by the peak appeared at 3279 cm -1 related to N-H stretching mood, as well as the peaks in the 1579 cm -1 and 689 cm -1 are characteristic of bending NH2. Also, the peak at 2924 cm -1 is attributed to the stretching C-H mood of CH2 groups in the propyl chain, indicated that the amine group is grafted to mesoporous structure. In SBA-15-OH-NH2-GA FT-IR spectrum (Figure 3d), with grafting of GA to SBA-15-OH-NH2, two peaks were appeared at 1648 cm -1 and 1574 cm -1 can be related to the stretching mood of C=O and C=N, respectively. Also a peak was appeared at 2854 cm -1 related to the aldehyde C-H bonds.

PPL immobilization and PPL activity assay
The amount of enzyme loading on supports was evaluated using Bradford's method (table 2).
Since mesoporous SBA-15-OH-NH2-GA was provided more surface area compared with Ca-Alg polymeric beads, enzyme loading on SBA-15-OH-NH2-GA was 3 time higher than Ca-Alg polymeric beads. Hydrolysis and Esterification activity of immobilized PPL on supports were evaluated and results were reported in table 2. As shown in this table, hydrolytic activity of biocatalysts decreased after immobilization on support due to partial conformational change of enzyme during immobilization. But hydrolysis activity of PPL/SBA-15-OH-NH2-GA biocatalyst showed the highest activity which was comparable with free PPL. Grafting of APTES and GA to mesoporous supports creates a wider spherical area for enzyme attachment which facilitate accessibility of substrate to enzyme active sites. The similar trend was observed in esterification reaction in present of both biocatalysts.

2.3.Design of experiments
To obtain the maximum of biodiesel production efficiency and the effect of different parameters on the transesterification reaction, experimental design was performed via Taguchi method. An orthogonal array with 9 runs (L9) including 4 parameters in 3 levels was selected ( using Taguchi design analysis, which is visible in figure 6. As shown, the optimal is the reaction run with parameters at level 3. the statistical analysis (ANOVA) of the results was done and the effect of each parameter on the biodiesel production yield was determined. As shown in Error! Reference source not found., catalyst amount, alcohol to oil molar ratio, the reaction water content, and temperature were the most effective parameters, respectively.
From Taguchi method prediction, the biodiesel production yield at level 3 (the optimum), should be 90.3% and the experimental result was 88.6% which was close to the prediction. For comparison, the optimum condition was used for biodiesel production in the presence of immobilized lipase on Ca-Alg seeds. The yield of biodiesel production was 77.65%, which indicates that mesoporous SBA-15-OH-NH2-GA carriers are better than Ca-Alg seeds for PPL immobilization. All biodiesel production yield values were obtained after purification of the samples by removing the catalyst, glycerol, remained methanol, and hexane.

Conclusions
In this study, the efficiency of oil extraction from castor seeds was determined by the oil expeller machine, followed by the Soxhlet apparatus, at about 60% W/W. Also, the main triglyceride component of castor oil is ricin oleic acid, which is an unsaturated fatty acid with a hydroxyl group, which makes it polarized, so it is suitable for the transesterification reaction in the presence of methanol. The above points indicate that castor plant has a very high potential for use in the future of the biodiesel industry as a substrate for transesterification reaction. The results of the SAXS analysis and N2 adsorption-desorption isotherms conforms that the twodimensional hexagonal structure and highly ordered of mesoporous SBA-15, which this good feature provides functionalization, modification and infiltrate of the reactants through the pore of the SBA-15. The results of the 1H NMR spectra of the produced biodiesel samples in the optimum state indicate that the biodiesel production efficiency is higher when the PPL is immobilized on the SBA-15-OH-NH2-GA toward than conditions that immobilized on the Ca-Alg. This is due to the fact that, due to the decrease in material size, the SBA-15 nanostructure can provide a higher surface for linkage of the PPL, consequently, it will increase the amount of enzyme loading per unit of the mass carrier. Also, the larger surface area for the mass transfer between particles decreases the resistance of the substrate transfer to the particle containing the immobilized enzyme, which in turn leads to an increase in the activity of the immobilized lipase and thereby increase in overall speed of the reaction. Therefore, nano-scale substances are ideal substrate materials for enzymes immobilizing used for biocatalysis reactions. The covalent bond of lipase on the mesoporous SBA-15 has the advantage of a strong interaction between the enzyme and the carrier in the immobilization process, which makes the PPL sturdier in its place during the catalytic process, thus it's less talented for denaturation and also the sustainability of PPL is much increasing in organic solvents. As well as, the immobilization of lipase on Ca-Alg involves the physical entrapment of the enzyme in a small space that it causes problems such as leakage and emit of the enzyme inside the solution, plus a significant amount of infiltrating limitations, which reduces the biodiesel production efficiency than use of the mesoporous SBA-15 as the carrier.

Extraction of oil from castor seeds
The castor oilseed used in this study was collected by Omid industrial oilseed press workshop in Iran. Two steps extraction was applied for Castor oil extraction. In the first step, oil extraction was done via oil expeller pressing machine. The seeds were squeezed under high pressure, either in one step or more steps. This expeller pressing machine was equipped with a temperature control device to avoid any damage to the oil that may affect its properties. Then the oil extracted was collected and filtered. To extract the residual oil from press cakes solventextraction method was used as second step treatment. The oil was separated by the Soxhlet extractor apparatus in the presence of a hexane solvent at 70 °C for 5 h. In the end, hexane solvent was separated using a rotary evaporator (ROTAVAPOR-RE, BUCHI, Switzerland) and the total content of the oil existent in castor seeds was determined by using the Eq. (1).

PPL/Ca-Alg biocatalyst preparation
A 100 mL of Na-Alg solution containing 0.2 g of PPL was added into the 200 mL CaCl2 solution (2 M) dropwise with syringe at 4 °C. Ca-Alg gels were formed instantly through cross-linking by dripping the solution of Na-Alg and PPL into the CaCl2 solutions. As soon as the Na-Alg polysaccharide is placed in a solution of calcium ions, Ca 2+ ions are exchanged with the Na + ions in the polymer. All formed gels were stored at 4 ° C for 2 h to harden then separated from the solution by vacuum filtration and washed with deionized water. Figure 9 is a graphical showing of PPL/Ca-Alg biocatalyst preparation.
where CL is the amount of entrapped lipase per gram of Ca-Alg (mg L/g Ca-Alg), C0 the initial concentration of lipase in the Na-Alg solution (mg L/ml), V0 the Na-Alg solution volume (ml), Cf the concentration of lipase in the filtered solution (mg L/ml), Vf the volume of the filtered solution (ml) and mb the amount of Ca-Alg beads (g) after drying.

Mesoporous SBA-15 synthesis
For mesoporous SBA-15 preparation, 5 g of Pluronic P-123 as a structure-directing agent was dissolved to 25 mL HCl 37% W and 150 mL distilled water. Then stirred at 35 °C for 20 h. P-123 forms the micelle in an aqueous medium. Then, 10.5 g of silica source (TEOS) was added to the medium containing micelles and the resulting solution was stirred at the same temperature for 20 h. The TEOS hydrolysis along with polymerization occurs in an acidic medium and forms a silica network around the template. Finally, the mixture is transferred to a Teflon-lined autoclave for particle growth and crystallization at 100 °C for 24 h. After filtration and washing with distilled water, the powder was dried in the hot air oven at 40 °C and calcined to remove the surfactant (P-123) at 550 °C for 5 h in a muffle furnace.

Activation of mesoporous SBA-15.
The problem of the calcination process is that many active silanol groups at the surface of the

Functionalization of mesoporous SBA-15-OH.
Amine functionalizing of mesoporous was performed by adding 0.5 mL APTES to 1 g of activated SBA-15 nanostructures in 40 mL of anhydrous toluene. The mixture was exposed to reflux under the argon atmosphere for 10 h. Finally, the mesoporous SBA-15-OH-NH2 were collected by filtration, washed with acetone and dried overnight at 50 °C.

Immobilization of PPL on mesoporous SBA-15-OH-NH2-GA
For immobilization of lipase through covalent bonding to mesoporous SBA-15-OH-NH2-GA, 1-1.75 g of carrier was dissolved in 625 mL phosphate buffer with pH=7.5 then 1 mg/ml of PPL solution was added. The mixture was stirred at 25 °C for 24 h. After completion of the reaction, the solution was incubated at 4 °C for a short time.

Hydrolysis activity assay of immobilized PPL
Free and immobilized PPL activity were investigated in the 1,2,3-triacetoxypropane hydrolysis reaction. 2 g triacetin, 50 mL distilled water and 25 mL phosphate buffer (1M, pH=7) was stirred for about 10 min to prepare a triacetin emulsion [32]. When the pH solution was reached to a constant value, a certain amount of free (0.25 g) and immobilized PPL was added to the above mixture. Acetic acid is produced as a processed product that reduces the pH of the environment. The resulting mixture was then continuously titrated with NaOH solution (0.01 M) achieve to a pH of about 12. The consumption volume was recorded exactly and PPL activity was calculated (one unit of lipase activity is defined as the amount of enzyme required to release 1 μmol of fatty acid at 37 °C per minute). The reaction was performed once in the presence of immobilized lipase on Ca-Alg polymeric beads (3 g) and again in the presence of immobilized lipase on mesoporous SBA-15-OH-NH2-GA (0.5 g).

Esterification activity assay of PPL
Esterification activity of biocatalysts were measured in an enzymatic alcoholysis reaction. The The FAMEs production yeild (%) = The consumed fattey acids The primery fatty acids ×100 (5)

Biodiesel production
The reactions were carried out in clear tubular screw cap vial 40 mL containing 2 g castor oil, a certain amount of catalyst (immobilized PPL on mesoporous SBA-15-OH-NH2-GA), different temperatures, different molar ratios of alcohol to oil and variable water content (in the form of phosphate buffer with pH=7). To each one reaction vessels, 5 mL of hexane solution were added. The reactions were carried out in a bath of oil set up on a magnetic stirrer with a speed of 250 rpm. Optimization of the effective parameters on transesterification reaction in order to biodiesel production was performed based on Taguchi experimental design using Minitab software. The above experiments were performed in two repetitions [34]. In order to compare, the optimal conditions of the above experiments were used to produce biodiesel using immobilized PPL on Ca-Alg.

Biodiesel purification
To remove biocatalysts and glycerol from the reaction medium, the product was centrifuged for 20 min at 3500 rpm. Then, biodiesel was collected. A mixture of 2:1 volume ratio of deionized water to biodiesel was stirred at 50 °C for 30 min. Again, the upper layer which contains fatty acid methyl esters and hexane was separated via separatory funnel. After separation, a rotary evaporator was used to remove the hexane solvent and pure biodiesel was obtained.