Effect of Preparation Conditions on Structure and Activity of Sodium-Impregnated Oyster Shell Catalysts for Transesteriﬁcation

: The catalyst preparation technique plays a signiﬁcant role in its activity and durability. The present research investigated sodium hydroxide and sodium chloride as the precursor chemicals for impregnation on waste oyster shells that were tested as heterogeneous base catalysts for transesteriﬁcation of soybean oil. Effects of precursor concentration and calcination temperature on the surface structure and the activity of the catalysts were studied via the one-factor-at-a-time method. The optimal impregnation concentrations of sodium hydroxide and sodium chloride were determined to be 6 mol/L and 2.43 mol/L, respectively. The optimal calcination temperature was determined to be 800 ◦ C for both sodium hydroxide and sodium chloride-impregnated catalysts. Analyses of the catalysts via X-ray Diffraction and X-ray photoelectron spectroscopy indicated that different active species were formed on the surface depending on the calcination temperature. Results obtained from this study could be used to ﬁne-tune the procedure for the synthesis of transesteriﬁcation catalysts from aquatic animal shells. catalysts suggesting that no catalytically active sites were formed on the surface of the oyster shells at lower precursor concentrations. When the precursor concentrations were increased, significant differences were observed. For NaOH/shell experiments, FAME yields increased from 8% to 83% when precursor (NaOH) concentration increased from 2 mol/L to 6 mol/L. 0% (for 1000 °C calcination), respectively, while the amount of Ca increased from 5.8% (600 °C) to 10.3% (800 °C), and finally reached 15.8% (1000 °C). These observations were consistent with the XRD results, in which the major phases of the catalyst, as the calcination temperature increased, changed from carbonate to oxide for the Ca, and the intensity of NaCl phase was attenuated continuously and disappeared finally at 1000 °C.


Introduction
Solid base catalysts have been extensively studied for their catalytic performance in transesterification reactions [1]. CaO is one of such solid base catalysts capable of transesterification of various oils to fatty acid methyl esters (FAME). However, its activity was still found to be lower than that of NaOH [2]. Hence, to increase the catalytic activity of CaO, wet impregnation methods have been employed to modify the surface of CaO to generate more active sites. Doped could react with the oxygen present in CaO to produce defect sites on the surface, which served as the enhanced basic sites for transesterification reactions [3][4][5][6][7][8]. Watkins et al., impregnated CaO with a series of LiNO 3 solutions in the range of 1-20 wt. % [4]. Despite showing a continuous drop in specific surface area (with increasing Li content), all Li-impregnated CaO catalysts that were tested exhibited increased basic strengths when compared to CaO. The transesterification results indicated that the initial reaction rate increased with the increased Li content and reached a maximum when Li content reached 1.23 wt. %. Further, the characterization study indicated that the doped CaO helped the formation of surface OH − which enhanced the catalytic activity. However, it was noted that the high concentration of LiNO 3 may have covered the surface OH − group, and thus may have lowered the catalytic activity.
In addition to the alkali metal oxides and hydroxide groups generated from the impregnation-calcination method, calcium and halogen have also been identified by many research information on how the activity of oyster shell catalyst is influenced by preparation conditions. By elucidating how calcination temperature and impregnation conditions affect the catalyst activity will not only improve catalyst synthesis processes but also allow us to predict the performance of the catalyst. Hence, the goal of this research is to systematically investigate how the catalyst preparation conditions (precursor type, precursor concentration, calcination temperature) influence the structure and subsequent activity of the catalyst. Figure 1 summarized the effects of precursor type and concentration. For both precursors, the calcined oyster shell (COS) was used as a control for comparison purposes. When a concentration of 2 mol/L was used for impregnation, there were no significant differences between the FAME yields obtained from control (COS) and NaCl/shell and NaOH/shell catalysts suggesting that no catalytically active sites were formed on the surface of the oyster shells at lower precursor concentrations. When the precursor concentrations were increased, significant differences were observed. For NaOH/shell experiments, FAME yields increased from 8% to 83% when precursor (NaOH) concentration increased from 2 mol/L to 6 mol/L. information on how the activity of oyster shell catalyst is influenced by preparation conditions. By elucidating how calcination temperature and impregnation conditions affect the catalyst activity will not only improve catalyst synthesis processes but also allow us to predict the performance of the catalyst. Hence, the goal of this research is to systematically investigate how the catalyst preparation conditions (precursor type, precursor concentration, calcination temperature) influence the structure and subsequent activity of the catalyst. Figure 1 summarized the effects of precursor type and concentration. For both precursors, the calcined oyster shell (COS) was used as a control for comparison purposes. When a concentration of 2 mol/L was used for impregnation, there were no significant differences between the FAME yields obtained from control (COS) and NaCl/shell and NaOH/shell catalysts suggesting that no catalytically active sites were formed on the surface of the oyster shells at lower precursor concentrations. When the precursor concentrations were increased, significant differences were observed. For NaOH/shell experiments, FAME yields increased from 8% to 83% when precursor (NaOH) concentration increased from 2 mol/L to 6 mol/L. However, for precursor concentrations beyond, 6 mol/L, FAME yields appeared to be similar (FAME yield between 83% and 94%) (Figure 1a). Our results agree with those reported by de Luna et al. who investigated the activity of NaOH impregnated pumice stone as catalysts for transesterification of soybean oil [17]. The authors reported a significant increase in fractional conversion of soybean oil when the precursor concentration was increased due to the incorporation of sodium ions within pumice stone matrix. The results obtained from NaCl/shell exhibited a different trend. As shown in Figure 1b, the FAME yield increased (from 8% to 84%) when the precursor concentration was increased from 2 mol/L to 3.64 mol/L and started decreasing when the precursor concentration was further increased beyond 3.64 mol/L, suggesting that presence of excess precursor may inhibit catalytic activity as shown in Figure 1. However, no significant differences in FAME yields were found between precursor loadings of 2.43 and 3.64 mol/L. Hence, precursor concentrations of 6 mol/L (for NaOH) and 2.43 mol/L (for NaCl) were selected for subsequent studies to investigate the effect of calcination of catalyst activity. Figure 1. The FAME yield of the transesterification reaction catalyzed by various concentrations of NaOH (a) and NaCl (b) impregnated calcined oyster shell. Transesterification conditions: temperature = 62 °C; stirring rate = 800 rpm; MR = 12; CL = 10%.

Effects of the Precursor Type and Concentration
However, for precursor concentrations beyond, 6 mol/L, FAME yields appeared to be similar (FAME yield between 83% and 94%) (Figure 1a). Our results agree with those reported by de Luna et al. who investigated the activity of NaOH impregnated pumice stone as catalysts for transesterification of soybean oil [17]. The authors reported a significant increase in fractional conversion of soybean oil when the precursor concentration was increased due to the incorporation of sodium ions within pumice stone matrix. The results obtained from NaCl/shell exhibited a different trend. As shown in Figure 1b, the FAME yield increased (from 8% to 84%) when the precursor concentration was increased from 2 mol/L to 3.64 mol/L and started decreasing when the precursor concentration was further increased beyond 3.64 mol/L, suggesting that presence of excess precursor may inhibit catalytic activity as shown in Figure 1. However, no significant differences in FAME yields were found between precursor loadings of 2.43 and 3.64 mol/L. Hence, precursor concentrations of 6 mol/L (for NaOH) and 2.43 mol/L (for NaCl) were selected for subsequent studies to investigate the effect of calcination of catalyst activity.

Effect of Calcination Temperature
The effects of calcination temperature are summarized in Figure 2. For experiments with NaOH/shell, calcination temperature (400, 600, 800, and 1000 °C) did not affect the yields of FAME that ranged between 80 and 91% ( Figure 1a).   However, since the stability of catalyst depends on the calcination temperature, further experiments were conducted to evaluate the reusability of the catalysts calcined at all temperatures. The catalysts were reused consecutively for five times using fresh batches of methanol and oil to determine the yields of FAME. As shown in Figure 3, catalysts calcined at 400 • C started losing activity after the second run and provided a FAME yield of 26.2% after the fifth run. However, when calcination temperature was increased, the stability of the catalyst appeared to improve. Figure 2. The FAME yield of the transesterification reaction catalyzed by NaOH6/shell (a) and NaCl2.43/shell (b) calcined at various temperatures. Transesterification conditions: temperature = 62 °C; stirring rate = 800 rpm; MR = 12; CL = 10%.
However, since the stability of catalyst depends on the calcination temperature, further experiments were conducted to evaluate the reusability of the catalysts calcined at all temperatures. The catalysts were reused consecutively for five times using fresh batches of methanol and oil to determine the yields of FAME. As shown in Figure 3, catalysts calcined at 400 °C started losing activity after the second run and provided a FAME yield of 26.2% after the fifth run. However, when calcination temperature was increased, the stability of the catalyst appeared to improve. The catalysts calcined at 800 and 1000 °C exhibited similar FAME yields for all five runs whereas the catalyst calcined at 600 °C provided lower FAME yields after the third run, when compared to 800 and 1000 °C suggesting that calcination at higher temperatures allowed for adequate reaction between Na ions and oyster shell resulting in strong binding of active sites on the surface.
For NaCl/shell, almost no activity was observed at 600 °C (FAME yield of ~0.1%) ( Figure 2b). When the calcination temperature was increased to 800 °C the yield of FAME increased to 88.4% and started to decrease with an increase in calcination temperature beyond 800 °C.
Overall, it appears that NaOH/shell and NaCl/shell calcined at 800 °C provided best FAME yields and stability. Hence to elucidate the role of calcination and effects of precursor concentrations, the surface properties of catalysts synthesized under various conditions were studied in detail.

Catalyst Characterization
The specific surface areas and basicities of the catalysts synthesized are summarized in Table 1. The impregnation of NaOH or NaCl lowered the specific surface area of the oyster shell, probably because of deposition of precursor molecules within the pores of COS. Our results were similar to those noted by Alburquerque et al., who observed 90-98% reduction in surface area of SBA-15 support when 4-20% (w/w) CaO was impregnated [18]. Similarly, de Luna et al., impregnated NaOH The catalysts calcined at 800 and 1000 • C exhibited similar FAME yields for all five runs whereas the catalyst calcined at 600 • C provided lower FAME yields after the third run, when compared to 800 and 1000 • C suggesting that calcination at higher temperatures allowed for adequate reaction between Na ions and oyster shell resulting in strong binding of active sites on the surface.
For NaCl/shell, almost no activity was observed at 600 • C (FAME yield of~0.1%) ( Figure 2b). When the calcination temperature was increased to 800 • C the yield of FAME increased to 88.4% and started to decrease with an increase in calcination temperature beyond 800 • C.
Overall, it appears that NaOH/shell and NaCl/shell calcined at 800 • C provided best FAME yields and stability. Hence to elucidate the role of calcination and effects of precursor concentrations, the surface properties of catalysts synthesized under various conditions were studied in detail.

Catalyst Characterization
The specific surface areas and basicities of the catalysts synthesized are summarized in Table 1. The impregnation of NaOH or NaCl lowered the specific surface area of the oyster shell, probably because of deposition of precursor molecules within the pores of COS. Our results were similar to those noted by Alburquerque et al., who observed 90-98% reduction in surface area of SBA-15 support when 4-20% (w/w) CaO was impregnated [18]. Similarly, de Luna et al., impregnated NaOH on pumice stone to enhance its catalytic activity [17]. The authors reported a significant reduction in surface area and pore volume after impregnation due formation of active sites within the pores. In our research, the basicity, however, showed no substantial difference between all types of catalysts, indicating that the precursor type and concentration calcination temperature did not alter the catalysts' basicity. The XRD pattern of each catalyst is shown in Figure 4a. For NaOH-impregnated shells, major phases formed on the surface of NaOH 6 /shell-400 were CaCO 3 , Na 2 CO 3 and Ca(OH) 2 . Since CaCO 3 and Na 2 CO 3 are not catalytically very active for transesterification, it may be concluded that the catalytic activity of NaOH 6 /shell-400 may be due to Ca(OH) 2 . These results were similar to the results obtained by Kumar and Ali, who used KOH to modify the surface of CaO [19]. The as-synthesized nanocrystalline 3.5% K-CaO exhibited newly generated Ca(OH) 2 sites on the surface that contributed to the increased basic strength from 9.8< H_ <10.1 to 11.1< H_ <15.0. The activity was also improved as the FAME yield reached 98% in 75 min [19]. In our research, when the calcination temperature increased (from 400 • C to 800 • C), the type of surface species changed on the shells' surface. The major phases formed on the surface of NaOH 6 /shell-800 were CaO and Na 2 O 2 , both of which are catalytically active in transesterification. The formation of alkaline oxide was also observed in the KNO 3 impregnated CaO-ZnO catalyst synthesized by Istabdi et al. [20]. The as-synthesized catalysts acquired K 2 O on the surface, which might have resulted in the improved activity in the transesterification of soybean oil. A similar trend was observed in our research as well, in which, up to 90% FAME yields were obtained from the NaOH 6 /shell-800 ( Figure 1). It appeared that calcination at higher temperature allowed for decomposition of carbonate and hydroxide to respective oxides that are active and stable. In addition, as shown in the reusability experiments (Figure 3), NaOH 6 /shell-800 provided high FAME yield even after five successive runs whereas the FAME yields for NaOH 6 /shell-400 continually decreased to 26.2% after the fifth run, indicating a substantial loss of activity. on pumice stone to enhance its catalytic activity [17]. The authors reported a significant reduction in surface area and pore volume after impregnation due formation of active sites within the pores. In our research, the basicity, however, showed no substantial difference between all types of catalysts, indicating that the precursor type and concentration calcination temperature did not alter the catalysts' basicity. The XRD pattern of each catalyst is shown in Figure 4a. For NaOH-impregnated shells, major phases formed on the surface of NaOH6/shell-400 were CaCO3, Na2CO3 and Ca(OH)2. Since CaCO3 and Na2CO3 are not catalytically very active for transesterification, it may be concluded that the catalytic activity of NaOH6/shell-400 may be due to Ca(OH)2. These results were similar to the results obtained by Kumar and Ali, who used KOH to modify the surface of CaO [19]. The as-synthesized nanocrystalline 3.5% K-CaO exhibited newly generated Ca(OH)2 sites on the surface that contributed to the increased basic strength from 9.8< H_ <10.1 to 11.1< H_ <15.0. The activity was also improved as the FAME yield reached 98% in 75 min [19]. In our research, when the calcination temperature increased (from 400 °C to 800 °C), the type of surface species changed on the shells' surface. The major phases formed on the surface of NaOH6/shell-800 were CaO and Na2O2, both of which are catalytically active in transesterification. The formation of alkaline oxide was also observed in the KNO3 impregnated CaO-ZnO catalyst synthesized by Istabdi et al. [20]. The as-synthesized catalysts acquired K2O on the surface, which might have resulted in the improved activity in the transesterification of soybean oil. A similar trend was observed in our research as well, in which, up to 90% FAME yields were obtained from the NaOH6/shell-800 ( Figure 1). It appeared that calcination at higher temperature allowed for decomposition of carbonate and hydroxide to respective oxides that are active and stable. In addition, as shown in the reusability experiments (Figure 3), NaOH6/shell-800 provided high FAME yield even after five successive runs whereas the FAME yields for NaOH6/shell-400 continually decreased to 26.2% after the fifth run, indicating a substantial loss of activity.
(a)  The XRD patterns of NaCl2.43/shell-600, NaCl2.43/shell-800 and NaCl2.43/shell-1000 are also shown in Figure 4a. At 600 °C, the major phases identified were CaCO3 and NaCl and traces of CaO. As a result, NaCl2.43/shell-600 provided only 0.13% FAME yield after 2 h. When the calcination temperature increased to 800 °C, most CaCO3 had been decomposed into CaO, resulting in the disappearance of CaCO3 and increase in the intensity of CaO peaks as was also reported by Madhu et al. [21]. Because CaO is also an active catalyst, the FAME yield from NaCl2.43/shell-800 increased up to 88.4%. Further increase in the calcination temperature to 1000 °C, however, decreased the yield to 58.3%. As seen from the XRD pattern for NaCl2.43/shell-1000, only CaO was detected on the surface. The disappearance of NaCl in NaCl2.43/shell-1000 might have caused the FAME yield to decrease. These results implied that a synergistic effect between NaCl and CaO on the surface of NaCl2.43/shell-800 may have resulted in high FAME yields when compared to yields obtained from NaCl2.43/shell-1000.
The XPS analyses of NaOH-impregnated catalysts indicated different elemental distribution for NaOH6/shell-400 and NaOH6/shell-800 catalysts (Figure 4b and Table 2). The atomic percentage of Na in NaOH6/shell-400 was 42.6%, whereas the atomic percentage of Na dropped to 16.6% in NaOH6/shell-800 as the calcination temperature increased from 400 to 800 °C. However, the atomic percentage of Ca in NaOH6/shell-400 increased from 1.4% to 9.3% as the calcination temperature increased from 400 to 800 °C. Combined with the XRD results, the Na from the surface of NaOH6/shell-400 probably originated from Na2CO3. When the calcination temperature increased, the Na2CO3 and CaCO3 probably decomposed into Na2O2 and CaO as was revealed from XRD spectra.  Figure 5A presented the C 1s, Ca 2p, Na 1s, and O 1s regions for NaOH6/Shell-400 and NaOH6/Shell-800. In the C 1s region, both catalysts showed doublet peaks (285.0 eV and 289.2 eV for NaOH6/Shell-400 and 285.0 eV and 289.5 eV for NaOH6/Shell-800), suggesting that they contained C-H and carbonate groups on the surface, probably from the air contamination [18]. In the Ca 2p region, NaOH6/Shell-400 showed two peaks at 346.5 eV and 350.1 eV, suggesting that the Ca originated from CaCO3 [22]. NaOH6/Shell-800 showed two peaks at 346.8 eV and 350.3 eV, suggesting that the Ca originated from CaO [23]. Similarly, in the Na 1s region, both catalysts displayed doublet peaks. In The XRD patterns of NaCl 2.43 /shell-600, NaCl 2.43 /shell-800 and NaCl 2.43 /shell-1000 are also shown in Figure 4a. At 600 • C, the major phases identified were CaCO 3 and NaCl and traces of CaO. As a result, NaCl 2.43 /shell-600 provided only 0.13% FAME yield after 2 h. When the calcination temperature increased to 800 • C, most CaCO 3 had been decomposed into CaO, resulting in the disappearance of CaCO 3 and increase in the intensity of CaO peaks as was also reported by Madhu et al. [21]. Because CaO is also an active catalyst, the FAME yield from NaCl 2.43 /shell-800 increased up to 88.4%. Further increase in the calcination temperature to 1000 • C, however, decreased the yield to 58.3%. As seen from the XRD pattern for NaCl 2.43 /shell-1000, only CaO was detected on the surface. The disappearance of NaCl in NaCl 2.43 /shell-1000 might have caused the FAME yield to decrease. These results implied that a synergistic effect between NaCl and CaO on the surface of NaCl 2.43 /shell-800 may have resulted in high FAME yields when compared to yields obtained from NaCl 2.43 /shell-1000.
The XPS analyses of NaOH-impregnated catalysts indicated different elemental distribution for NaOH 6 /shell-400 and NaOH 6 /shell-800 catalysts (Figure 4b and Table 2). The atomic percentage of Na in NaOH 6 /shell-400 was 42.6%, whereas the atomic percentage of Na dropped to 16.6% in NaOH 6 /shell-800 as the calcination temperature increased from 400 to 800 • C. However, the atomic percentage of Ca in NaOH 6 /shell-400 increased from 1.4% to 9.3% as the calcination temperature increased from 400 to 800 • C. Combined with the XRD results, the Na from the surface of NaOH 6 /shell-400 probably originated from Na 2 CO 3 . When the calcination temperature increased, the Na 2 CO 3 and CaCO 3 probably decomposed into Na 2 O 2 and CaO as was revealed from XRD spectra.  Figure 5A presented the C 1s, Ca 2p, Na 1s, and O 1s regions for NaOH 6 /Shell-400 and NaOH 6 /Shell-800. In the C 1s region, both catalysts showed doublet peaks (285.0 eV and 289.2 eV for NaOH 6 /Shell-400 and 285.0 eV and 289.5 eV for NaOH6/Shell-800), suggesting that they contained C-H and carbonate groups on the surface, probably from the air contamination [18]. In the Ca 2p region, NaOH 6 /Shell-400 showed two peaks at 346.5 eV and 350.1 eV, suggesting that the Ca originated from CaCO 3 [22]. NaOH 6 /Shell-800 showed two peaks at 346.8 eV and 350.3 eV, suggesting that the Ca originated from CaO [23]. Similarly, in the Na 1s region, both catalysts displayed doublet peaks. In our analyses, the two peaks in both catalysts seemed to shift to lower BE by 1.0 eV compared with the references [24][25][26]. Combined with the details of the preparation method and the XRD results, the Na 1s peak at lower BE might have originated from Na 2 CO 3 , while the Na 1s peak at higher BE might be associated with NaOH and Na 2 O 2 for NaOH 6 /Shell-400 and NaOH 6 /Shell-800, respectively [24][25][26]. In the O 1s region, NaOH 6 /Shell-400 displayed doublet peaks at 530.3 eV and 531.2 eV, assigned to CaO and Ca(OH) 2 , respectively [27,28]. Similarly, NaOH 6 /Shell-800 exhibited doublet peaks at 530.3 eV and 531.5 eV, assigned to Na 2 O 2 and CaO [29], respectively, based on the XRD analysis. This doublet feature indicated that some of the surface oxygen had been shifted to lower BE for both catalysts, when compared to control (calcined oyster shell), acquiring a higher effective negative charge. As was also observed in our recent research [16], the shift in oxygen would have enhanced the electron donating ability, suggesting that the surface oxygen was more basic [30]. As a result, the increased electron donating ability may have allowed triglyceride and methanol to form a tetrahedral intermediate that may have resulted in FAME after structural rearrangement [2]. our analyses, the two peaks in both catalysts seemed to shift to lower BE by 1.0 eV compared with the references [24][25][26]. Combined with the details of the preparation method and the XRD results, the Na 1s peak at lower BE might have originated from Na2CO3, while the Na 1s peak at higher BE might be associated with NaOH and Na2O2 for NaOH6/Shell-400 and NaOH6/Shell-800, respectively [24][25][26]. In the O 1s region, NaOH6/Shell-400 displayed doublet peaks at 530.3 eV and 531.2 eV, assigned to CaO and Ca(OH)2, respectively [27,28]. Similarly, NaOH6/Shell-800 exhibited doublet peaks at 530.3 eV and 531.5 eV, assigned to Na2O2 and CaO [29], respectively, based on the XRD analysis. This doublet feature indicated that some of the surface oxygen had been shifted to lower BE for both catalysts, when compared to control (calcined oyster shell), acquiring a higher effective negative charge. As was also observed in our recent research [16], the shift in oxygen would have enhanced the electron donating ability, suggesting that the surface oxygen was more basic [30]. As a result, the increased electron donating ability may have allowed triglyceride and methanol to form a tetrahedral intermediate that may have resulted in FAME after structural rearrangement [2]. The XPS data obtained from NaCl-impregnated catalysts also revealed different elemental distributions for NaCl2.43/shell-600, NaCl2.43/shell-800, and NaCl2.43/shell-1000 (Table 3). As the calcination temperature increased, the amount of Na and Cl decreased continuously from 32.4% (Na) and 15.0% (Cl) (for 600 °C calcination) to 10.0% and 2.1% (for 800 °C calcination), and finally dropped to 1.2% and 0% (for 1000 °C calcination), respectively, while the amount of Ca increased from 5.8% (600 °C) to 10.3% (800 °C), and finally reached 15.8% (1000 °C). These observations were consistent with the XRD results, in which the major phases of the catalyst, as the calcination temperature increased, changed from carbonate to oxide for the Ca, and the intensity of NaCl phase was attenuated continuously and disappeared finally at 1000 °C. The XPS data obtained from NaCl-impregnated catalysts also revealed different elemental distributions for NaCl 2.43 /shell-600, NaCl 2.43 /shell-800, and NaCl 2.43 /shell-1000 (Table 3). As the calcination temperature increased, the amount of Na and Cl decreased continuously from 32.4% (Na) and 15.0% (Cl) (for 600 • C calcination) to 10.0% and 2.1% (for 800 • C calcination), and finally dropped to 1.2% and 0% (for 1000 • C calcination), respectively, while the amount of Ca increased from 5.8% (600 • C) to 10.3% (800 • C), and finally reached 15.8% (1000 • C). These observations were consistent with the XRD results, in which the major phases of the catalyst, as the calcination temperature increased, changed from carbonate to oxide for the Ca, and the intensity of NaCl phase was attenuated continuously and disappeared finally at 1000 • C.  Figure 5B presented the C 1s, Ca 2p, Na 1s, O 1s and Cl 2p regions for NaCl 2.43 /Shell-600, NaCl 2.43 /Shell-800 and NaCl 2.43 /Shell-1000.
In the C 1s region, NaCl 2.43 /Shell-600 and NaCl 2.43 /Shell-800 exhibited triplet peaks while NaCl 2.43 /Shell-1000 showed doublet peaks. In addition to peaks at~289.4 eV and~285.0 eV (assigned to C-H and carbonate groups, respectively) [18], NaCl 2.43 /Shell-600 and NaCl 2.43 /Shell-800 also displayed a third C 1s peak at 292.4 eV, which may be attributed to the existence of C-Cl [31]. NaCl 2.43 /Shell-1000 did not contain Cl, and thus no such peak was detected. In the Ca 2p region, all three catalysts showed doublet peaks. The peaks at 346.6 eV and 350.1 eV in NaCl 2.43 /Shell-600 were assigned to CaCO 3 [22]. The peaks at 347.1 eV and 350.6 eV in NaCl 2.43 /Shell-800 and at 347.2 eV and 350.7 eV in NaCl 2.43 /Shell-1000 were assigned to CaO [29]. In the Na region, NaCl 2.43 /Shell-600 and NaCl 2.43 /Shell-800 displayed doublet peaks at~1073.6 eV and~1071.5 eV, assigned to NaCl [32] and Na 2 CO 3 [25], respectively, while NaCl 2.43 /Shell-1000 only showed single peak at 1071.6 eV, assigned to Na 2 CO 3 [25]. The Na 2 CO 3 in NaCl 2.43 /Shell-600 may be attributed to adsorbed Na and undecomposed carbonate in the shell matrix, while the Na 2 CO 3 in NaCl 2.43 /Shell-800 and NaCl 2.43 /Shell-1000 might be attributed to the air contaminant on the surface since no carbonate group was detected based on the XRD analysis. Because NaCl 2.43 /Shell-1000 did not contain Cl, it did not exhibit a peak representing NaCl. In the O 1s region, all three catalysts exhibited a single peak. The peak at 531.0 eV of NaCl 2.43 /Shell-600 was assigned to CaCO 3 [23] and the peak at 531.5 eV of NaCl 2.43 /Shell-800 and the peak at 531.6 eV of NaCl 2.43 /Shell-1000 were assigned to CaO [29]. In the Cl 2p region, both NaCl 2.43 /Shell-600 and NaCl 2.43 /Shell-800 displayed doublet peaks at 200.3 eV and 202.0 eV, for 2p3/2 and 2p1/2 components of Cl, respectively, with a separation of 1.7 eV that was also observed by Wu et al. [33]. In addition, NaCl 2.43 /Shell-800 also had a third peak at lower BE, 198.4 eV. Comparing the FAME yields of NaCl 2.43 /Shell-800 with NaCl 2.43 /Shell-1000 indicated that NaCl 2.43 /Shell-800 resulted in higher FAME yield than NaCl 2.43 /Shell-1000. Considering that the BE of O 1s for NaCl 2.43 /Shell-800 was comparable with NaCl 2.43 /Shell-1000, the enhanced activity of NaCl 2.43 /Shell-800 may have been due to the formation of surface Cl with lower BE (198.4 eV). As shown in Figure 6, the negatively shifted Cl probably behaved similarly as the negatively shifted O, thereby increasing the effective negative charge that led to an increase in the electron donating ability and enhanced the FAME production rate by facilitating the formation of the tetrahedral intermediate between adsorbed triglyceride and methanol [2,30]. This synergistic effect between NaCl and CaO appears to have occurred at 800 • C because the catalysts calcined at 600 • C and 100 • C did not exhibit high activity despite equipped with identical NaCl loading.

Catalyst Preparation
Discarded oyster shells were procured locally. The dirt on the shells were cleaned in running water and allowed to dry overnight. Subsequently, the shells were calcined (2h @ 500 °C) and crushed (1-2 mm).
Two types of Na-based precursor solutions were employed in this research: NaOH and NaCl. Typically, 20 g of oyster shells were impregnated with 60 mL of precursor solutions (90 °C for 5 h) of predetermined concentration on a process-controlled hot plate (Isotemp, Fisher Scientific, Pittsburgh, PA). Subsequently, the impregnated shells were filtered and calcined at a predetermined temperature for 3 h (with a flow of nitrogen).

Catalyst Characterization
The specific surface area of the catalyst synthesized was determined using Quantachrome Monosorb Surface Area Analyzer (Quantachrome Instruments, Boynton Beach, FL, USA) based on the Brunauer-Emmett-Teller (BET) nitrogen adsorption method [16]. Prior to the measurement, all catalysts were degassed at 120 °C for 8 h.
The basicity of the catalyst was determined as described by Mo et al. [34]. 0.1 g sample was soaked into 20 mL of the 0.1 N HCl solution for 20 min flowed by titration using 0.15 N NaOH with Phenolphthalein.
The samples were also analyzed via X-ray photoelectron spectroscopy (XPS) using a SPECS FlexMod system (SPECS GmbH, Berlin, Germany) equipped with a hemispherical analyzer PHOIBIS 150 and a Mg Kalpha (1254 eV) X-ray source @ 10 −10 mbar) using adventitious Carbon (C 1s line at 285.0 eV binding energy) as reference.

Catalyst Preparation
Discarded oyster shells were procured locally. The dirt on the shells were cleaned in running water and allowed to dry overnight. Subsequently, the shells were calcined (2h @ 500 • C) and crushed (1-2 mm).
Two types of Na-based precursor solutions were employed in this research: NaOH and NaCl. Typically, 20 g of oyster shells were impregnated with 60 mL of precursor solutions (90 • C for 5 h) of predetermined concentration on a process-controlled hot plate (Isotemp, Fisher Scientific, Pittsburgh, PA). Subsequently, the impregnated shells were filtered and calcined at a predetermined temperature for 3 h (with a flow of nitrogen).

Catalyst Characterization
The specific surface area of the catalyst synthesized was determined using Quantachrome Monosorb Surface Area Analyzer (Quantachrome Instruments, Boynton Beach, FL, USA) based on the Brunauer-Emmett-Teller (BET) nitrogen adsorption method [16]. Prior to the measurement, all catalysts were degassed at 120 • C for 8 h.
The basicity of the catalyst was determined as described by Mo et al. [34]. 0.1 g sample was soaked into 20 mL of the 0.1 N HCl solution for 20 min flowed by titration using 0.15 N NaOH with Phenolphthalein.
The samples were also analyzed via X-ray photoelectron spectroscopy (XPS) using a SPECS FlexMod system (SPECS GmbH, Berlin, Germany) equipped with a hemispherical analyzer PHOIBIS 150 and a Mg Kalpha (1254 eV) X-ray source @ 10 −10 mbar) using adventitious Carbon (C 1s line at 285.0 eV binding energy) as reference.

Transesterification
Batch experiments were performed (in duplicates) 250 mL three-neck glass reactors. 20 mL of soybean oil was reacted (62 • C and 800 rpm) with predetermined amount of methanol and catalyst on a process-controlled hot plate (Isotemp, Fisher Scientific, Pittsburgh, PA, USA). In this research, the molar ratio of methanol to oil (MR) and catalyst loading (CL) were set to 12 and 10%, respectively. After a 2-h reaction, 3 mL of the reaction mixture was transferred to 15 mL plastic tubes for centrifugation (5000 rpm for 5 min) to remove the catalyst. Subsequently, 800 µL of the supernatant liquid was drawn and washed three times with DI water from which a 300 µL sample was collected for further gas chromatographic analysis.

Gas Chromatography Analysis
A gas chromatograph with a mass spectrometer (Agilent 7890/5975C) was used to separate, detect, and quantify FAME using Methyl Laurate (99% purity, CAS 111-82-0) as an internal standard as described in Jin et al., (2017b). FAME yields were calculated using the equation below, as proposed by Liu et al and Chung et al [2,35].
where ∑ A is the total area of peak areas of all methyl esters, A IS is the peak area of Methyl Laurate, C IS is the concentration of Methyl Laurate in the solution after dilution (g/mL), V is the volume of the final diluted solution (mL), m FAMEs is the theoretical maximum mass of FAMEs (g), m oil is the mass of pure soybean oil of volume V (g), ρ oil is the density of soybean oil (g/mL), r is the dilution ratio of FAMEs sample, C oil is the corresponding concentration of soybean oil (g/mL) in the solution after dilution, if dilution ratio is r.

Experimental Design
All experiments were performed in duplicates. For the synthesis of NaCl/shell catalyst, NaCl solutions of 2, 2.43, 3.64, 4, and 6 mol/L were employed. Similarly, for the synthesis of NaOH/shell catalyst, NaOH solutions of 2, 4, 6, 8, 10, and 12.5 were used. All catalysts synthesized were tested in batch reactors and the FAME yields were analyzed using SAS (SAS Institute Inc., Cary, NC, USA).
The optimal conditions of each precursor type were chosen based on the FAME yield. The corresponding catalyst was then re-prepared but calcined at various temperatures. For NaCl-impregnated catalysts, calcination temperatures of 600 • C, 800 • C, 1000 • C and for NaOH-impregnated catalysts, calcination temperatures of 400 • C, 600 • C, 800 • C, and 1000 • C were used.
Additional experiments were performed to test the reusability of the various NaOH-impregnated calcined oyster shells. The spent catalyst was used for five consecutive runs. After each run, the FAME yield was determined and the recovered catalyst was applied to subsequent run without any treatment. The data were analyzed via a Tukey's HSD test.

Conclusions
This research investigated how preparation conditions affected the structure and activity of transesterification catalysts derived from waste oyster shells. Oyster shells that were impregnated with various concentrations of NaOH and NaCl and calcined at different temperatures were tested as catalysts in batch experiments. Based on results obtained from batch experiments and catalyst characterization, it appears that precursor type and calcination temperature determined the formation of newly generated surface species that influenced the activity of the catalyst. In general, higher calcination temperature converted more carbonates and hydroxides into oxides that enhanced catalyst activity due to a higher electron donating ability.
Author Contributions: H.J. designed and conducted the experiments, performed the data analysis, and wrote the manuscript; P.K. conceived the idea, supervised the research and edited the manuscript; J.J.C. and S.W.P. contributed to the catalyst characterization and reaction mechanisms of the transesterification reaction. J.A.O. contributed to the experimental design and data analysis.