Valorization of Waste Cooking Oil into Biodiesel via Bacillus stratosphericus Lipase Amine-Functionalized Mesoporous SBA-15 Nanobiocatalyst

In this study, evaporation-induced self-assembly was applied to prepare amine-functionalized nano-silica (NH 2 -Pr-SBA-15). Tat was simply used to immobilize Bacillus stratosphericus PSP8 lipase (E–NH 2 –Pr-SBA-15), producing a nanobiocatalyst with good stability under vigorous shaking and a maximum lipase activity of 45 ± 2 U/mL. High-resolution X -ray dif-fractometer, Fourier transform infrared spectroscopy, N 2 adsorption-desorption, feld-emission scanning electron, and high-resolution transmission electron microscopic analyses proved the successful SBA-15 functionalization and enzyme immobilization. Response surface methodology based on a 1/2 fraction-three-levels face center composite design was applied to optimize the biodiesel transesterifcation process. Tis expressed efcient percentage conversion (97.85%) and biodiesel yield (97.01%) under relatively mild operating conditions: 3.12 :1 methanol to oil ratio, 3.08 wt.% E–NH 2 –Pr-SBA-15 loading, 48.6 ° C, 3.19 h at a mixing rate of 495.53 rpm. E–NH 2 –Pr-SBA-15 proved to have a long lifetime, operational stability, and reusability.


Introduction
Te increased global population with the highly eminent use of transportation fuels and industrial activities dramatically intensifes greenhouse gas emissions, global warming, and the negative consequences of climate change [1,2]. Consequently, there is a worldwide conduit towards sustainable and green fuels [3], i.e., biodiesel [4], bioethanol [5], biojet [6], and biogas [7], which play a vital role in achieving the three pillars of sustainable development:economy, society, and environment [3].
Nowadays, there is a tremendous worldwide investment in the long-chain fatty acid monoalkyl ester, i.e., biodiesel, which is a natural, eco-friendly, biodegradable, and sustainable alternative fuel to the nonrenewable petro-diesel without the need for any engine modifcations [8]. However, the frst-generation biodiesel from edible feedstock induced many environmental and societal concerns, negatively impacting food security and land use [2,9]. Besides, the main bottlenecks in the biodiesel industry are the high cost of feedstock [4,10], the massive consumption of nonsustainable homogenous catalysts in the transesterifcation process, and water in the washing step [11]. In addition to the possible decrease in biodiesel yield that may occur with the conceivable manifestation of saponifcation and the miscibility of the unreacted catalyst, which hurdles the glycerol recovery [12].
Te valorization of sustainable resources such as waste cooking oil (WCO) into biodiesel is estimated to decrease the total cost of the transesterifcation process by two to three folds [10,13,14] and solve the environmental problems caused by the uncontrolled disposal of WCO [1,15]. Nevertheless, the use of nano-bio-composite heterogeneous catalysts for biodiesel production in the form of nano-immobilized sustainable lipase enzyme has various advantages compared to conventional catalysts, including mild operating conditions and reasonable economics, ease of product separation and recovery, absence of side reactions, and the elimination of washing steps [16][17][18][19]. However, the efciency, reusability, and catalytic stability of the nano-immobilized sustainable lipase and its tolerance to toxic short-chain alcohols are the main apprehensions for its application in the biodiesel industry [20,21]. Terefore, fnding new ways to avoid these negatives is one of the most critical challenges, and there are many suggested published methods for lipase immobilization to sustain its catalytic activity and enhance its stability, involving entrapment, encapsulation, cross-linking, covalent bonding, and physical adsorption [22][23][24][25][26][27].
Tis manuscript aimed to prepare amine-functionalized nano-silica (NH 2 -Pr-SBA-15) for the practical immobilization of Bacillus stratosphericus PSP8 lipase (E-NH 2 -Pr-SBA-15) to ingeniously valorize sustainable WCO into biodiesel. Response surface methodology (RSM) based on the 1/2 fraction-three-level face-centered central composite design (FCCCD) of experiments was applied for statistical optimization of the transesterifcation process to maximize biodiesel yield. Furthermore, the reusability of the E-NH 2 -Pr-SBA-15 for successive transesterifcation processes has been done to prove its stability. Physicochemical characterization of the produced biodiesel (B100) has also been performed to assure the feasibility of the prepared nano E-NH 2 -Pr-SBA-15 in the biodiesel industry.

Preparation of Amine-Functionalized Nano-Silica (NH 2 -
Pr-SBA-15). Te evaporation-induced self-assembly (EISA) method was applied to prepare NH 2 -Pr-SBA-15, according to Zhu et al. [41]. In a 300 mL Tefon beaker and at room temperature, 6 g of Pluronic -P123 was dissolved in a mixture of 0.1 N HCl, 150 mL anhydrous ethanol, and 4 mL mesitylene. After complete dissolution and under gentle stirring, a mixture of 4 : 1 (v/v) tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES, NH 2 (CH 2 ) 3 Si(OC 2 H 5 ) 3 ) was drop-wisely added. Ten, the temperature was elevated to 45°C for 30 min to initiate the hydrolysis of the silica precursor (i.e., the TEOS). After that, the mixture was poured on a Tefon Petri dish and left for two days. Te obtained powder was neutralized with 0.05 N NaOH to remove HCl and break the NH 3 -Cl bond. Ten, the powder was redispersed in a mixture of hexamethyldisilazane and ethanol (4.5 mL:20 mL) and retained in an ultrasonic for 15 min at room temperature to complete the removal of excess P123 and unreacted silica precursor. Finally, the free-surfactant NH 2 -Pr-SBA-15 was washed with ethanol for fve cycles and then vacuum dried overnight at 40°C.

2
International Journal of Chemical Engineering 2.3. Lipase Immobilization. In a 100 mL water/ethanol mixture (80 : 20 v/v), a prescribed amount of lyophilized PSP8 lipase was added to 1 g of the pre-dispersed NH 2 -Pr-SBA-15 in a fnal concentration range of 0.01-1.5% w/w. Ten, it was blended for six h after the adjustment of the pH to pH7 using 0.05 M phosphate bufer. Finally, the immobilized lipase was separated by centrifugation. Figure 1 briefy illustrates the steps of lipase immobilization. Te best lipase loading was demarcated by assessing the immobilized lipase activity according to the technique reported by Castro-Ochoa et al. [42] using p-nitrophenyllaurate (pNPL) as a substrate. A previously prepared standard curve using 10-1000 U/mL of Amano lipase A from Aspergillus Niger (Sigma Aldrich, Darmstadt, Germany) has been used. One unit (U) of lipolytic activity was defned as the amount of lipase that released 1 mol of p-nitrophenol (molar absorption coefcient 4.6 mM −1 cm −1 ) from pNPL within 30 min under the tested conditions.
Te enzyme leaching test of the immobilized lipase amine-functionalized mesoporous SBA-15 was performed according to the technique described by Gao et al. [43] using the preselected optimum enzyme loading ratio. Te immobilized lipase was suspended in 0.05 M phosphate bufer at pH7 at a fnal concentration of 1.25 mg/mL and shaken at 200 rpm. At diferent prescribed time intervals, aliquots of the suspensions were withdrawn, centrifuged, and the residual lipase activities were determined as reported by Castro-Ochoa et al. [42].
A preliminary investigation of the capabilities of the SBA-15, NH 2 -Pr-SBA-15, and immobilized lipase at a preselected optimum loading ratio for biodiesel production has been performed under the following operating conditions:6 : 1 methanol:oil (M : O molar ratio), six wt.% nanobiocatalyst loading, 60 o C, three h, and 400 rpm.

Catalyst Characterization.
Te prepared SBA-15, NH 2 -Pr-SBA-15, and the nano-immobilized enzyme with the preselected optimum enzyme loading ratio E-NH 2 -Pr-SBA-15 were characterized for their crystalline structure using a high-resolution X-ray difractometer (XRD; PANalytical XPERT PRO MPD, EA Almelo, the Netherlands) coupled with Cu kα radiation source (λ � 1.5418Å). Moreover, the functional species of the prepared nanobiocatalyst were identifed via Fourier transform infrared spectroscopy (FTIR; Perkin Elmer Spectrum One, Shelton, CT, USA). Teir specifc surface areas were determined by the Brunauer-Emmett-Teller (BET) technique using a low-temperature N 2 adsorption-desorption isotherm (NOVA3200e, Quantachrome, FL, USA). Te catalyst sample was tested for pore diameter, volume, and size distribution by applying the Barret-Joyner-Halenda (BJH) method. Te morphology and International Journal of Chemical Engineering structure of the as-prepared catalysts were determined by the feld-emission scanning electron microscope (FE-SEM S-4800, Hitachi High-Technologies Corporation, Tokyo, Japan) and high-resolution transmission electron microscope (HR-TEM, JEOL-JEM 2100F, 80-200 kV, Tokyo, Japan).

Statistical Optimization of the Biodiesel Transesterifcation Process.
Response surface methodology (RSM) is based on a 1/2 fraction-three-levels face center composite design (FCCD) of fve experimental factors; M : O (molar ratio, A), nano-immobilized enzyme concentration (wt.%, B), reaction temperature (°C, C), reaction time (h, D), and mixing rate (rpm, E) were employed. A design of 32 batch processes with 16 factorial, ten axial, and six central points was implemented to avoid bias (Table 1). Te transesterifcation batch process was performed according to Ismail et al. [18] in an automized magnetically stirred threenecked 25 mL reactor equipped with a thermometer and a refux condenser set at the prescribed reaction temperature, time, and stirring speed. Te prescribed concentration of immobilized enzyme was added after the M : O mixture reached the prerequisite reaction temperature. Ten the reaction mixture, at the end of each batch, was allowed to separate overnight. Next, pure glycerol and nanobiocatalyst were obtained by centrifugation of the separated lower layer at 10,000 rpm for 10 min. Finally, a rotary evaporator set at 65 o C and 20 kPa was used to recycle the excess unreacted methanol for reusability. Ten, the obtained purifed biodiesel was applied for further physicochemical characterization according to the standard test methods for petroleum products of the American Society for Testing and Materials [44].
Te design Expert 6.0.7 software (Stat-Ease Inc., Minneapolis, USA) was used to design the transesterifcation experiments and inspect the interactive efects of the different physicochemical parameters on the valorization of WCO into biodiesel using the immobilized lipase. It was also applied to statistically optimize the transesterifcation process for maximizing the biodiesel yield, performing the regression and graphical analyses of the obtained data and the statistical analysis of variance (ANOVA) of the predicted regression model equation, estimating the response surface.
Te valorization of WCO into biodiesel was calculated according to Ismail et al. [18] via the depletion of the kinematic viscosity of the oil layer, at 40°C using the following equation: where V BD and V WCO are the viscosity of the produced biodiesel and valorized WCO, respectively. All measurements were in triplicates and the average data were calculated within a standard deviation (StD) range of ±2%, and P < 0.05 is statistically signifcant at α � 0.05 level, 95% confdence interval. Te produced fatty acid methyl esters (FAME, Figure 2) were identifed by gas chromatography equipped with a fame ionization detector (GC/FID model HP Agilent 7890, CA, USA) using a prepared reference mixture of palmitate, stearate, oleate, linoleate, and linolenate (Sigma-Aldrich    International Journal of Chemical Engineering Chemie GmbH, Taufkirchen, Germany), and linolenate was injected as an internal standard [18]. Ten, the biodiesel yield was calculated according to Nassar et al. [2] based on the calculated % purity as follows: where PAR is the ratio of the total FAME peak area to that of the added internal standard.

Reusability of the Prepared Nano-Immobilized PSP8
Lipase. Tat was done according to Karimi (2016) to evaluate the stability of the prepared nano-immobilized lipase [23]. Te transesterifcation reaction of WCO was performed at the predicted optimum conditions. Te nanobiocatalyst was separated from the reaction mixtures at the end of each batch by centrifugation at 10,000 rpm for 10 min, then washed with phosphate bufer (0.2 M, pH7) and tert-butanol (1 : 1 v:v), and fnally lyophilized by a bench-top freeze dryer (Eyela-FDU-1200, Bohemia, NY, USA). Te recovered nanobiocatalyst was then reused in a subsequent transesterifcation reaction at the optimum conditions. Finally, the reusability of immobilized lipase was inspected by repeating the aforementioned steps for eight successive cycles, and the % conversion and biodiesel yield were determined for each transesterifcation cycle.

Statistical Analysis.
Statistical analysis for the obtained data was performed using SPSS version 26 (Informer Technologies, Inc., Los Angeles, CA, USA). Tukey was used to evaluate the signifcant diferences between the obtained based on a signifcant interval of 95% (p � < 0.05).

Efect of Lipase
Loading. Te prepared nanobiocatalyst (E-NH 2 -Pr-SBA-15) with a loading ratio of 1% (Figure 3(a)) expressed the maximum lipase activity, recording 45 ± 2 U/ mL (p < 0.0001 at α � 0.05 level, 95% confdence interval). However, any further increment in lipase loading ( Figure 3(a)) did not show any elevation in lipase activity (0.153 ≤ p ≤ 0.895 at α � 0.05 level, 95% confdence interval). Te 1% loading was selected for further experiments to avoid the possible occurrence of retardation in the substrate and product difusion. According to Li et al. [26], a relatively high enzyme loading ratio would cause intermolecular steric hindrance. As calculated by the method reported by Sun et al. [45], the specifc enzyme activity reached approximately 44,550 U/g. Tat was higher than that reported for immobilized Candida Antarctica lipase onto amine SBA-15, which recorded 12,000 U/g [32].

Te Stability of the Prepared E-NH 2 -Pr-SBA-15 under Shaking Conditions.
Tere was a continuous activity loss with time, under vigorous shaking at 200 rpm ( Figure 3(b)). However, the prepared nanobiocatalyst (E-NH 2 -Pr-SBA-15) expressed reasonable stability, as it retained approximately 77.14 ± 1.4% of its initial activity after 120 h (p < 0.0001 at α = 0.05 level, 95% confdence interval). Tat was much better than those reported by Gao et al. [22], where the immobilized Candida sp. 99-125 lipase onto mesoporous silica via adsorption and cross-linking retained only up to 9.6% and 60% of its initial activity after 120 h under shaking at 200 rpm, respectively. It is also worth mentioning that approximately 67.70 ± 1.4% and 56.92 ± 1.4% of the immobilized PSP8 lipase initial activity was retained after 7 and 10 d, respectively (p < 0.0001 at α = 0.05 level, 95% confdence interval). Consequently, it would indicate that the prepared NH 2 -Pr-SBA-15 efectively prevented the leaching of of the immobilized lipase. Tus, this recommends the application of the prepared E-NH 2 -Pr-SBA-15 in the industrial transesterifcation process, as it usually takes place under vigorous shaking conditions to overcome the mass transfer limitations.

Catalyst Characterization.
Te XRD patterns of the prepared catalysts are illustrated in Figure 4(a) and prove their crystallinity. Besides, the prominent peak at (100) and the lower intensity peaks that appeared at (110) and (200) are typical for the ordered mesoporous SBA-15 with hexagonal arrays and p6mm symmetry [46]. Moreover, the NH 2 -Pr-SBA-15 nearly retained the same pattern as the unfunctionalized SBA-15, proving that grafting by APTES did not afect its structural integrity. Disappeared peaks at (110 and 200) with the decreased intensity at (100) in the E-NH 2 -Pr-SBA-15 XRD pattern (Figure 4(a)) might be attributed to the presence of organic moieties. Consequently, it would confrm the successful immobilization of PSP8 lipase onto the prepared NH 2 -Pr-SBA-15. A similar observation was reported by Veisi et al. [47] and attributed to the possible disorderliness occurring by the direct functionalization of SBA-15, which might cause partial shielding of the tiny pores and/or the presence of organic moieties, which might afect the scattering angle within the unit cell. Te FTIR spectra of SBA-15, NH 2 -Pr-SBA-15, and E-NH 2 -Pr-SBA-15 are illustrated in Figure 4(b). Te SBA-15 showed FTIR peaks at 1075 and 800 cm -1 of the-Si-O asymmetric and symmetric stretching vibration, respectively, a peak around 458 cm -1 of the bending vibration of Si-O-Si group and a peak around 945 cm -1 of Si-OH bending vibration [46]. Besides, the broad peak appeared around 3452 cm -1 , which might be attributed to the terminated hydroxyl groups, in addition to the band of-OH deformation vibration around 1630 cm -1 [48]. Te functionalized NH 2 -Pr-SBA-15 nanoparticles displayed additional peaks at 2928 and 2856 cm -1 that could be assigned to C-H's asymmetric and symmetric stretching vibration in the 3-aminopropyltriethoxysilane, and the weak band at 3290 cm −1 might be ascribed to-NH 2 group [49]. Te disappearance of the Si-OH bending vibration band around 945 cm -1 might indicate the involvement of Si-OH in the functionalization reaction [46]. Moreover, the FTIR peak around 1229 cm -1 is due to Si-C bond. Te weak FTIR peaks around 1540 and 1400 cm -1 are attributed to the asymmetric and symmetric vibrations of-NH 2 , confrming the successful preparation of NH 2 -Pr-SBA-15 nanoparticles. Comparing the spectrum of NH 2 -Pr-SBA-15 to that of E-NH 2 -Pr-SBA-15 nanobiocomposite, the increase in the intensities of the peaks around 2930 and 600 cm -1 might be attributed to the C-H and N-H stretching vibrations of the lipase enzyme [46]. Besides, the appearance of peaks within 1780 to 1860 cm -1 would be attributed to the C�O of peptide and carboxylic moieties in the lipase enzyme [50]. Te increase in intensities of peaks around 1440 and 1530 cm -1 besides the appearance of the peak around 1640 cm -1 indicates-CH and-NH 2 moieties of the lipase enzyme [21]. Furthermore, the sharp decrease in intensity of peak at 3450 cm −1 gave another indication of electrostatic interaction between the lipase enzyme and -OH of the silica [46].
Te FTIR analysis would prove the involvement of the surface functional groups of SBA-15 and the functionalized NH 2 -Pr-SBA-15 into the lipase immobilization via covalent bonding, not only via the conventional hydrophobic and/or electrostatic interactions. According to Salvi and Yadav [51]; the observed high stability and activity of the immobilized PSP8 lipase onto the NH 2 -Pr-SBA-15 might be attributed to the immobilization via covalent bonding that provides efcient strengthened immobilized constancy between the lipase molecules and the immobilizing supporting material and consequently reduces the enzyme leakage.
It can be depicted from the N 2 -physisorption analysis illustrated in Figure 4(c) Tat all isotherms of the prepared SBA-15, NH 2 -Pr-SBA-15, and E-NH 2 -Pr-SBA-15 exhibited type IV with H1-hysteresis loops and indicated according to Betiha et al. [52] the presence of mesopores. Te materials showed a steep increase in N 2 -adsorption (capillary condensation step) at P/P 0 of 0.57-0.85, suggesting high uniform mesoporous materials according to Gao et al. [22]. Upon functionalization, the capillary condensation shifted to lower P/P 0 , confrming shrinkage in pore diameter due to the presence of pendant organic moieties (-Si-CH 2 CH 2 CH 2 -NH 2 ), that further decreased upon the enzyme immobilization. Te desorption hysteresis loop likely became broader after enzyme immobilization, which might be attributed, according to Pinto et al. [36]; to the presence of protein inside the pores, causing a delay in the nitrogen desorption. After introducing-Si-CH 2 CH 2 CH 2 -NH 2 and enzyme, the specifc surface area decreased from 802 m 2 /g for virgin SBA-15 to 612 and 579 m 2 /g, respectively. Tat trend was consistent with decreasing pore volume and pore size, recording 1.02 cc/g and 3.55 nm for SBA-15, 0.911 cc/g, and 2.9 nm for NH2-Pr-SBA-15 0.89 cc/g and 2.81 nm for E-NH 2 -Pr-SBA-15, respectively.
Tat recorded decrease in specifc surface area, porevolume, and size confrmed, according to Salvi and Yadav [51]; the successful functionalization and enzyme immobilization. A similar observation was reported by Kou et al. [46] for SBA-15 functionalization by N(-2-aminoethyl)-3aminopropyl and 3-aminopropyl groups followed by lactase enzyme immobilization. Moreover, according to Badiei et al. [53] that would also indicate the occurrence of modifcation within the inner surface of the silica wall. Besides the homogenous distribution of the immobilized enzyme onto the SBA-15 surface and within its pores [36]. Not only that, but according to Kou et al. [46] those results would also indicate entrapment of the enzyme molecules within the silica channels besides its surface adsorption onto the mesoporous  Figure 5(e)) confrmed the XRD-analysis as mentioned above and showed more observable arrangements of conservative mesoporous channels. Tis, according to Kou et al. [46]; might indicate the intact SBA-15 Si-O-Si network and proves that SBA-15 pores did not collapse or break down during the functionalization reaction. However, these pores appeared embedded with organic moieties in the   International Journal of Chemical Engineering E-NH 2 -Pr-SBA-15 image, and the pore wall was less specifc than virgin the SBA-15, but the ordered structure was retained ( Figure 5(f )).

Mathematical Representation of the Transesterifcation
Process. Based on the obtained experimental results listed in Table 1, the transesterifcation process has been represented by two quadratic model equations; one was predicted for the % conversion (Y 1 eq.(4)), and the other was elucidated for the biodiesel yield (Y 2 , eq. (5)). Ten, the rationality of the two predicted model equations was validated by the F-test and the analysis of variance ANOVA, as represented in Table 2.   (Table 2) and Pareto charts (Figure 7), i.e., the signifcant efect of each of the studied parameters. In comparison, all of the independent variables within the studied range expressed a very high statistically signifcant efect on the transesterifcation process (p < 0.0001) except for the higher mixing rate (E * E), which expressed a negative statistically signifcant efect (0.001 ≤ p ≤ 0.0022). Moreover, the curvatures (Figure 7)  also confrmed that within a low range of M : O, its increase reduced the transesterifcation efciency, while vice versa occurred within a high range of M : O. Te perturbation plots also proved the high sensitivity of the transesterifcation reaction towards the E-NH 2 -Pr-SBA-15 nanobiocatalyst concentration, reaction temperature, and time. Moreover, the perturbation plots (Figure 7) presumptively indicated that, across the studied range, the transesterifcation efciency was better at high and relatively low M : O molar ratio, high catalyst concentration, relatively high mixing rate, low-temperature, and time near to the center point.
Te 2D contour plots and 3D RSM plots (Figure 8) elucidate the interactive efect of the studied parameters on the transesterifcation process. Te elliptical shape (Figure 8(a)) of the possible statistical signifcant negative interactive efect of M : O and nanobiocatalyst concentration (p � 0.0177) indicated that the transesterifcation efciency  Te very high statistically signifcant negative interactive efect of M : O and reaction time (p < 0.0001) was pronounced in the 2D and 3D plots (Figure 8(b)). In contrast,  Te inverted elliptical shape (Figure 8(c)) represents the highly statistically signifcant negative interactive efect of reaction time and temperature on the transesterifcation efciency (p � 0.0003). Te transesterifcation efciency decreased at higher and longer reaction temperature and time (>50°C and three h, respectively) and was also low at lower and shorter reaction temperature and time (<50°C and three h, respectively).
Moreover, the statistically signifcant negative interactive efect of reaction time and mixing rate (p � 0.0039) on the transesterifcation efciency (Figure 8(d)) indicated the decrease in % conversion at longer reaction time (>3 h). Nevertheless, it increased with the increase of mixing rate regardless of the reaction time, up to ≈450 rpm, and then slightly increased and/or remained nearly sustained at a higher mixing rate (>450 rpm).
Te 2D and 3D plots of the interactive efect of M : O and reaction temperature and those of nanobiocatalyst concentration and reaction time pronounced their very high statistically signifcant adverse efects (p < 0.0001) onto the transesterifcation efciency (Figure 8(e), 8(f )). It showed that at low M : O (<4.5 : 1), the biodiesel yield was recognizably increased with the increase in reaction temperature, but vice versa occurred at high M : O (>4.5 : 1). Moreover,     International Journal of Chemical Engineering the noticeable increase in the biodiesel yield with the increase in the nanobiocatalyst concentration decreased regardless of the catalyst concentration with a longer reaction time (>3 h). Nevertheless, the exceptionally high statistically significant positive interactive efect of M : O and mixing rate on biodiesel yield was evident in the 3D plot (Figure 8(g), p < 0.0001). Regardless of the M : O molar ratio, the biodiesel yield increased with the increase of the mixing rate until reaching ≈450 rpm and then slightly increased and/or remained nearly sustained at a higher mixing rate (>450 rpm).
Te enhancement of the transesterifcation efciency with the relatively higher mixing rate ≈450 rpm would indicate the possible elimination of mass transfer limitations and the manifestation of good mixing of the reactants. However, the decreased or sustained transesterifcation effciency at a relatively higher mixing rate >450 rpm might be due to the possible occurrence of turbulence and increased mass transfer opposition. However, lipases are sensitive to high methanol concentrations and reaction temperature [18,19,24]. However, the prepared E-NH2-Pr-SBA-15 showed reasonably good tolerance to relatively elevated methanol concentration and process temperature, which adds to the advantages of immobilized PSP8 lipase E-NH2-Pr-SBA-15. Tat contradicted the observation reported by Shimada et al. [54]; where the immobilized Candida Antarctica lipase was inhibited at a high M : O (>1.5) molar ratio. It also contradicted the data reported by Arumugam and Ponnusami [32] for the immobilized Candida Antarctica lipase onto amino-functionalized SBA-15, where the biodiesel yield decreased at reaction temperature higher than 35 o C. Te increment of transesterifcation efciency with relatively high reaction temperature might be, according to Ismail et al. [18]; due to the decrease in the viscosity of reaction mixture and the abolition of the mass transfer limitations. However, at higher elevated temperatures, denaturation of enzymes [32] and vaporization of methanol [18] might occur, consequently decreasing the transesterifcation efciency. According to Nassar et al. [2] the elevated temperatures afect the methanol polarity, decreasing the available methoxide moieties required for commencing the transesterifcation reaction towards the forward direction producing the FAME.
Te recorded sufcient transesterifcation efciency over a wide range of the applied nanobiocatalyst concentrations also added to the advantages of the prepared E-NH 2 -Pr-SBA-15 as it would indicate the nonagglomeration of the immobilized PSP8 lipase beads, the overwhelmed difusion limitation, and the easiness of substrates reaching the active sites of the enzyme molecules, even at higher nanobiocatalyst concentrations. However, it was also noticed that the transesterifcation of WCO into biodiesel using the immobilized PSP8 lipase onto the NH 2 -Pr-SBA-15 was mutually low at short and long reaction times. According to Ismail et al. [18]; that might be due to the initial time needed by the reactants to overcome the mass transfer limitation and get in contact with the active sites of the enzyme molecules. Nevertheless, according to Yang et al. [55] and Mohammadi et al. [37]; the production and accumulation of the oil insoluble hydrophilic glycerol with the transesterifcation progressive reaction time (Scheme 1), would straightforwardly adsorb onto the immobilized lipase surface active sites, negatively afect the enzyme activity and operating constancy and consequently the transesterifcation reaction. Moreover, according to Nassar et al. [2]; the accumulated glycerol  would dissolve in the unreacted methanol, shifting the transesterifcation process backward to the reverse direction, lowering the biodiesel yield.

Statistical Optimization of the Transesterifcation Process.
Te Design-Expert software, version 6.0.7, was applied to optimize the operating conditions to achieve the maximum transesterifcation efciency. Te desired goal for each operational condition (M : O, E-NH 2 -Pr-SBA-15 concentration, reaction time, reaction temperature, and mixing rate) was chosen "within" the studied range, and the responses (% conversion and biodiesel yield wt.%) were defned as maximum. Additional experiments were then performed applying the predicted optimum conditions listed in (  . Te reusability of the prepared nanobiocatalyst under the predicted optimum conditions was examined. Te transesterifcation efciency over eight successive cycles is illustrated in Figure 9, which proved that E-NH 2 -Pr-SBA-15 had been used for two successive cycles without a decrease in its activity (p > 0.05 at α � 0.05 level, 95% confdence interval). Moreover, there was no noteworthy decrease in E-NH 2 -Pr-SBA-15 activity after three successive cycles (p � 0.001 at α � 0.05 level, 95% confdence interval). Te prepared E-NH 2 -Pr-SBA-15 kept approximately 96%, 75%, and 68% of its initial activity after four, seven, and eight successive cycles (p < 0.0001 at α � 0.05 level, 95% confdence interval). Tat ascertained its operational fexibility, easy separation, stability, and long lifetime, which reinforce the recommended commercial application of E-NH 2 -Pr-SBA-15 for the valorization of WCO into biodiesel, whatever on continuous or batch industrial scale.

Reusability and Feasibility of the Prepared E-NH 2 -Pr-
Te Bacillus stratosphericus PSP8 lipase immobilized amine-functionalized SBA-15 expressed a comparable transesterifcation activity relative to the previously published literature (Table 4) at a one-step methanol feeding process without the need of any co-solvents. Te comparatively large biodiesel yield obtained using the Bacillus stratosphericus PSP8 lipase immobilized amine-functionalized SBA-15 within a quite short-time and mild temperature transesterifcation process. Besides, a relatively low alcohol and nanobiocatalyst consumption would, according to Nassar et al. [2]; indicate the dimensioned mass transfer limitations which usually occur in the heterogeneously catalyzed biodiesel production processes. Tat adds to the advantages of the applied Bacillus stratosphericus PSP8 lipase immobilized amine-functionalized SBA-15. However, the recorded loss of activity (≈9%-32%) within the 5 th to the 8 th cycles may be ascribed to; the possible conformational changes of PSP8 lipase, the blocking of the active lipase sites, or the gradual loss of the bounded lipase during the reaction procedures [23], the lipase leaching [43], and/or enzyme denaturation [21].

Physicochemical Characterization of the Produced
Biodiesel. Te produced biodiesel met the international biodiesel standard specifcations JUS [56,57] (Table 5). Tus, it can be ranked as a realistic fuel to be applied as a substitute and/or complementary to petro-diesel. Te drop in the values of biodiesel TAN, density, and viscosity relative to those of the WCO by 89.12, 4.08, and 92.4%, respectively, confrmed the excellent transesterifcation efciency. Te iodine value, a measure of unsaturation degree, dramatically infuences the fuel oxidation tendency and stability [2]. Te I 2 value of the produced biodiesel was recorded at 110 mg I 2 / 100 g oil and agreed well with the EN14214 standards (<120 mg I 2 /100 g oil). Te acid value indicates the content of free fatty acids (FFAs) in the sample and infuences fuel aging [18]. Te acid value of the produced biodiesel recorded 0.31 mg KOH/g oil and agreed well with the international biodiesel standards (<0.5 mg KOH/g oil). Tus, no operational problems, such as corrosion and pump plugging, would occur. Te relatively good cold fow characteristics, with a cloud point of 3 o C and a pour point of 9 o C (Table 5), might be ascribed to the high unsaturated FAME content of the produced biodiesel (≈64.5%) and recommend its application in cold weather. Not only that, but good fow properties also characterized the produced biodiesel; density of 0.8893 g/ cm 3 and viscosity of 3.7 cSt and rational values of specifc gravity and API, recording 0.8862 and 26.32, respectively. Although the produced biodiesel has a lower CV∼39.381 MJ/ kg than that of the Egyptian petro-diesel standards, it is higher than that of the biodiesel standards.
Moreover, the produced biodiesel was characterized by four signifcant advantages (Table 5); it has no sulfur, so it meets the goal of the petroleum industry for ultra-low to zero-sulfur-diesel; and its burning would not emit sulfur oxides, triggering engine corrosion, air pollution, and acid rain. Te produced biodiesel had a relatively high fash point of 155°C. So, it is much less fammable than conventional petro-diesel, and hence, it is much safer in usage, storing, and transference. In addition, the viscosity of the produced biodiesel was 3.7 cSt, which is competitive with that of conventional petro-diesel. Hence, no hardware or engine amendments would be needed to apply the produced biodiesel to the market. Te distillation temperatures (DTs) characterize the volatility of the fuel and have a signifcant efect on the burning efciency of the diesel engine [18]. Te fairly observed high DTs for the produced biodiesel (Table 5) would reduce the ignition delay of the fuel and minimize the possibility of knocking in the diesel engine.

Conclusion
Te valorization of WCO into eco-friendly biodiesel using sustainable immobilized lipase enzymes has positive impacts on the dilemma of food versus fuel, waste management, climate change, and the depletion of renewable energy resources. Most of the published immobilized lipase/SBA-15 catalyzed transesterifcation processes used nonedible oil or standard fatty acids to produce biodiesel. Tus, as far as our knowledge, this is the frst time to apply Bacillus stratosphericus PSP8 lipase immobilized onto amine-functionalized SBA-15 (E-NH 2 -Pr-SBA-15) to valorize WCO into highly qualifed biodiesel in the absence of any co-solvent. Furthermore, the statistical optimization of the transesterifcation process using RSM based on FCCCD proved to be very suitable for maximizing the production yield of highly qualifed eco-friendly biodiesel. Te produced E-NH 2 -Pr-SBA-15 proved to have many advantages; it had a large biodiesel yield throughout a relatively mild transesterifcation operational process, in addition to low alcohol and enzyme consumption. Furthermore, the high purity of the obtained biodiesel without the need for a washing step, usually applied in a homogenous transesterifcation reaction, would decrease the water consumption in the biodiesel industry. E-NH 2 -Pr-SBA-15 is also characterized by a long lifetime, sufcient operational stability, and reusability, which add to its feasibility and recommend its application in continuous industrial-scale processes.

Data Availability
Data are available on request.

Disclosure
Abdallah R. Ismail and Hamdy Kashtoh are co-frst authors.

Conflicts of Interest
Te authors declare they have no conficts of interest.