Ion Exchange Resin and Entrapped Candida rugosa Lipase for Biodiesel Synthesis in the Recirculating Packed-Bed Reactor: A Performance Comparison of Heterogeneous Catalysts

Abstract

Ion exchange resins and immobilized lipase as heterogeneous catalysts are used to synthesize biodiesel for alternative fossil fuels. The use of ion exchange resins in the solid and activated phase can ease the separation process. Furthermore, resins can be reactivated and used repeatedly, reducing the need for catalysts. On the other hand, an immobilized enzyme is biodegradable and can catalyze the transesterification process to produce biodiesel with a lower alcohol-to-oil ratio, minimizing side reactions and impurities. Therefore, the catalysts used in this study are ion exchange resins, such as Lewatit MP-64, Amberlite IRA410Cl, and Diaion PK208LH, as well as immobilized Candida rugosa lipase. By using vegetable oil as a feedstock and methanol for the transesterification, biodiesel production was carried out in a packed bed reactor. The present study aims to investigate the optimum process parameters, including the concentration of resin and enzyme, resin activation time, resin types, flowrate, and stability of resin and enzyme on the biodiesel yield. The results showed that the optimum conditions for biodiesel production with ion exchange resin were 4 g of resin, activated for 3 h, and synthesized for 3 h; Lewatit obtained a biodiesel yield of 94.06%, Amberlite obtained 90.00%, and Diaion obtained 73.88%. Additionally, the stability test of the reactivated Lewatit resin showed that it still has the capability of producing biodiesel with a yield of more than 80% after three regeneration cycles. In contrast, Candida rugosa lipase as was immobilized by entrapment in sodium alginate before being used in the biodiesel production for 12 h. The results showed that lower flowrate in enzymatic biodiesel synthesis produced a higher amount of biodiesel, of up to 71.1%. Nonetheless, immobilized lipases can be used up to three times without a significant loss in biodiesel yield.

1. Introduction

Along with the need for renewable energy, the rising global energy demand has recognized biofuels as a future element of the energy mix. A substitute fuel must be technically viable, commercially competitive, environmentally acceptable, and widely accessible [1]. Biodiesel, a fatty acid ethyl ester produced from triglyceride by transesterification reaction, is one of the most well-known renewable energy sources. As a renewable source of biomass with a high triglyceride content, vegetable oil is in high demand for the production of biodiesel. Triglycerides from any sources can be converted into biodiesel. However, the challenge lies in the selection of a feedstock that is abundant and widely available while still managing to generate a sufficient biodiesel yield.

Biodiesel synthesis uses supercritical conditions, and provides a rapid process compared to the other catalytic processes, as well as removing the need for a catalyst. However, it is inefficient in its use of alcohol as well as due to its need for high temperature and pressure [2]. Meanwhile, transesterification is one of the ideal alternative methods used to produce biodiesel and generates physical properties of fatty acid esters closely resembling those of diesel fuel [3]. Other prominent transesterification techniques involve the use of a heterogeneous catalyst that reduces alcohol consumption and reduces operational temperature. It has been noted that a heterogeneous base catalyst has numerous advantages over a homogenous base catalyst, such as allowing simple catalyst recovery, higher biodiesel yield, high-purity glycerin byproduct, and cheaper catalyst and maintenance costs [2].

Triglycerides are the primary source of carbon chain for biodiesel synthesis. In terms of oil production, palm oil is superior to all other triglyceride sources and oil-producing plants. Annually, 4.5 tons of palm oil are generated per hectare of oil palm plants, whereas other plants may only be able to produce up to 30% of oil [4]. Therefore, the use of palm oil as a biodiesel raw material is supported by its low cost and abundant source compared to other oils [5].

Triglyceride can react with water and NaOH through saponification to generate soap, decreasing the biodiesel yield. Ion exchange resin is a solution for mitigating this issue. An extensive study has investigated the potential and limitations of biodiesel synthesis by employing ion-exchange resin. Biodiesel synthesis via anion-exchange resin has been reported to be successful for triglyceride transesterification, although the cation-exchange resin is preferable for acid oil [6,7,8,9]. Recent research highlights the use of various ion-exchange resins in biodiesel synthesis, with the capacity, functional group, and physical structure of each resin influencing the reaction yield [10].

Enzyme-catalyzed replacements for chemical catalysts in biodiesel synthesis are gaining popularity because they eliminate the disadvantages of chemical catalysts. Enzymes do not form soaps and can catalyze free fatty acids and triglycerides [11]. To lower the production costs associated with enzyme catalyzation, we employ entrapment immobilization, which is easily carried out by forming calcium alginate gel from sodium alginate and calcium chloride. Entrapment by sodium alginate is an established and well-known approach for immobilizing lipase [12,13,14,15,16]. Methanol, a low-chain alcohol, is likewise capable of inactivating lipase [17]. As described by Watanabe [18], the two-step transesterification, which consists of the addition of methanol at the beginning of the reaction and a second injection of methanol at a specific time, is needed to avoid a high methanol concentration that can inactivate the enzyme. Since only a moderate amount of methanol was added, it is miscible through simple mixing and does not inactivate the enzymes. Palm oil is abundant and easy to obtain in Indonesia compared to any other type of oil. It contains high oleic and palmitic acid in the form of triglyceride [19]. Candida rugosa lipase (CRL) has been known to synthesize biodiesel from palm oil and its transesterification capability has been studied. Besides, CRL also has a large market availability, and thus may reduce production costs [20].

The packed-bed reactor is the appropriate configuration for catalyzing ion-exchange resin or immobilized lipase. This type of reactor is the most practical and successful method for biodiesel synthesis with heterogeneous catalysts [18,21,22,23] because it can extend the contact duration between the liquid substrate and the solid catalyst. Another advantage of a heterogeneous catalyst is that it can be regenerated and used in several production cycles. Previously, a co-solvent (e.g., hexane) was required to dissolve oil with alcohol in a packed bed reactor, which is typically operated in a continuous process [22,24,25,26,27]. The use of a co-solvent would increase the diffusivity of reactants, thus maximizing the mass transfer in the catalyst. However, the production cost would also increase by incorporating a solvent. This problem arises further in enzymatic-packed bed reactors since a high amount of methanol can inhibit lipase [28]. Thus, the reactor is configured to circulate the feedstock with fatty acid methyl esters (FAME). FAME formation in the reaction increases alcohol miscibility, which eliminates the need for high methanol amount and co-solvent requirement. In the present study, we examined the ability of ion exchange resins and immobilized lipase from Candida rugosa by using a specific substrate to convert palm oil into biodiesel in a circulating fixed-bed reactor. This research determines the type of heterogeneous catalysts that are preferable for improving biodiesel conversion and recovery in a continuous system.

2. Materials and Methods

2.1. Materials

Cooking palm oil was used as a triglyceride substrate and obtained from PT. Salim Ivomas Pratama, Tbk. (Jakarta, Indonesia) with a free fatty acid content below 0.3% (according to SNI 7709-2019). PK208LH resins were obtained from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Amberlite IRA-410 CL Lewatit MP-64 chloride, lyophilized Candida rugosa lipase (CRL) Type VII, bovine serum albumin (BSA), and all other reagents (methanol, calcium chloride, and sodium hydroxide) were purchased from Merck & Co (Rahway, NJ, USA). Lastly, n-hexane was obtained from PT. Smart Lab Indonesia, Banten, Indonesia.

2.2. Resin Activation

Resin activation was accomplished by weighing the Amberlite and Lewatit resins and 6 M NaOH solution with a weight ratio of 1:4 (g). The resins were placed in a series of three-neck flask batch reactors (with a condenser of running cold water) at 60 °C. After activation, the resin and base were separated using vacuum filtering. The resins were dried in an oven at 110 °C for 24 h. Then, the dried resins were placed in a desiccator for further use in biodiesel synthesis. An illustration of resin activation can be seen in Figure 1.

2.3. Enzyme Immobilization

To produce immobilized lipase, sodium alginate was first dissolved in deionized water through homogenous mixing. After mixing lipase powder in deionized water by using a vortex at 1000 rpm for 1 min, it was added to a sodium alginate solution to produce 1.5% sodium alginate containing 50–200 mg of lipase. The mixture was extruded into a 2% CaCl₂ solution using a syringe with a 26 G needle. Then, it is cured at room temperature for 1 h. The beads were then suspended in a fresh 2% CaCl₂ solution and stored for 24 h at 4 °C. To avoid high viscosity, a phosphate buffer solution was substituted with deionized water (pH 7). The immobilized lipase was filtered using vacuum filtration and rinsed with deionized water. The beads can be stored in deionized water at 4 °C for one month when not in use [29].

2.4. Enzyme Loading

Loading efficiency was determined by using the amount of CRL used in bead preparation as the initial amount of enzyme. The amount of solute enzyme in the washing solution was determined using the Bradford method and measured at the 595 nm absorbance.

$$\mathit{Loading} \mathit{efficiency}\left(\mathit{\%}\right)=\left(\frac{C_i{V}_i-C_f{V}_f}{C_i{V}_i}\right)\times 100$$

(1)

Loading efficiency shows the percentage of enzyme mass retained in calcium alginate beads. C_i, V_i, C_f, and V_f represent the initial and final concentration and volume of the enzyme, respectively.

2.5. Continuous Transesterification in a Packed Bed Reactor

The dimensions of the packed bed reactor were 1.1 cm in diameter, 15 cm in length, and 14.255 mL in volume. To synthesize biodiesel, the packed bed reactor was first heated with a hot water jacket at 60 °C. The substrate was created by combining cooking oil and methanol in a 1:6 ratio and by stirring it at 100 rpm with a magnetic stirrer. A peristaltic pump with a rotational speed of 5 rpm was prepared to transport the substrate into the reactor. The reactor bed was filled with resin, and the process was carried out continuously for 3 h in each cycle. The same packed bed concept was employed for the two-step methanol addition during the biodiesel production by using immobilized lipase with a methanol-to-oil mol ratio of 1:3. Additional methanol was added to the substrate at a mol ratio of 2:3 after 4 h. The flowrate of the peristaltic pump was measured and set to 0.4–2.5 mL/min. Immobilized lipase was recycled by vacuum filtration and washing with deionized water. The set of a recirculated packed bed reactor can be seen in Figure 2.

2.6. Resin Regeneration

In order to regenerate resin, it must first be dried for 30 min at 110 °C in the oven. The resin was vacuum-filtered and washed with 40 mL of n-hexane. The resin was then redried in the oven for 3 h at 110 °C. The stability of dried resin in biodiesel production can be evaluated by comparing biodiesel yields produced by using the regenerated resin in further biodiesel production cycles.

2.7. Biodiesel Washing

Following the biodiesel synthesis, vacuum filtration was used to separate the resin from the biodiesel. The crude biodiesel product was then rinsed with water to remove the glycerol. Afterwards, biodiesel was heated to eliminate any water content. The crude biodiesel is separated from the glycerol in a separatory funnel for 30 min to 2 h. The bottom layer containing glycerol was then removed. Then, water was added to the funnel at a ratio of 1:1 with biodiesel and the separation process was completed around 30 min to 2 h later. The bottom layer, consisting of water coupled to the glycerol content, was eliminated. This procedure was performed twice with a 2:1 water-to-biodiesel ratio. The biodiesel was then transferred to a glass beaker and heated for 30 min at 110 °C by using a hot plate stirrer. Lastly, biodiesel samples were collected for further analysis.

2.8. Analysis of FAME Content

The FAME content of the biodiesel was determined under the same operating conditions using regenerated and dried resins. The composition of biodiesel was analyzed using Gas Chromatography-Flame Ionization Detector (GC-FID) (Clarus 680, PerkinElmer^®, Waltham, MA, USA), and the calculation of methyl ester component (in mass percentage) was carried out using Equation (1).

$$E=\frac{\sum (A-A_{EI})}{A_{EI}}\times \frac{W_{EI}}{W}\times 100$$

(2)

where:

• $E$ = ester content (% w/w)
• $\sum A$ = total peak area of methyl ester C6:0–C24:1
• $A_{EI}$ = peak area of internal standard C19
• $W_{EI}$ = weight of internal standard (mg)
• $W$ = weight of sample (mg).

3. Results

3.1. The Use of Ion Exchange Resins for Biodiesel Synthesis in Recirculating Packed Bed Reactors

3.1.1. The Effect of Resin Types on the Biodiesel Yield

The effect of different resin types on biodiesel yield can be seen in Figure 3. Based on the figure, activated Lewatit MP-64 resin produced 94.06% biodiesel yield under optimal operating conditions. Meanwhile, Amberlite IRA410Cl and Diaion PK208LH produced 90.00% yield and 73.88% under the same conditions, respectively. The results are consistent with a previously reported study [8], where the alcohol adsorption strength was found to be higher than that of ester. This explains Lewatit MP-64 and Amberlite IRA410Cl, being the anion exchange resins, showed higher catalytic activity compared to cation exchange resins such as Diaion PK208LH.

A study of biodiesel synthesis using anion exchange resin D261 in a packed bed reactor obtained 95.2% yield, and another study that utilized PK306 anion exchange resin managed to reach 98.8% biodiesel yield [8,22]. In another study conducted by Kitakawa et al. [30], the synthesis of biodiesel using Diaion PK208LH cation exchange resin reached 81.4% conversion in batch operation and 91.1% in continuous operation during 12 h of reaction. A reaction time of 3 h is insufficient to reach a high yield using PK208LH resin. Thus, increasing the reaction time could increase the biodiesel production. However, it is unfavorable since changing to other types of resin could improve the yield.

3.1.2. Effect of Dry Weight of Activated Ion Exchange Resin in Biodiesel Yield

Figure 4 depicts the increase in biodiesel yield based on the amount of resin that is utilized. The yield of biodiesel that was formed using 3 g dry weight of Lewatit MP-64 resin was 75.74% and increased by 6.89% with the addition of 0.5 g of resins. A further increase of 6.43% was also observed by adding an additional 0.5 g of resins, making the optimal amount of activated resins in a packed bed reactor to be 4 g. Thus, when the three dry weight variations were synthesized in the same synthesis time, i.e., 2 h, it was assumed that the highest dry weight variation, i.e., 4 g or 0.8% w/v_oil, experienced a faster conversion rate than the lesser variation.

A study on the effect of catalyst concentrations on biodiesel production showed that using higher concentrations of the acid catalyst resulted in higher conversions of biodiesel in a shorter time [31]. The study showed that biodiesel conversion increased from 70 to 90% when the catalyst concentration increased from 0.5 to 5%. A similar study was conducted by Hartono et al. [32], who utilized used cooking oil and 16.7% w/v_oil of Lewatit MP-64 resin and successfully obtained a biodiesel yield of 85.94% in 3 h. A higher amount of activated resins provides better catalytic activity due to the increased active sites, improving the yield with the same amount of reaction time.

3.1.3. Effect of Synthesis Time in Biodiesel Yield

Figure 5 represents the duration of biodiesel synthesis by Lewatit MP-64 in the reactor. The experimental results showed that the reaction time affects the yield value of the produced biodiesel. The highest value of 84.84% was obtained at a reaction time of 3 h. However, there is an insignificant difference compared to a shorter amount of synthesis time.

The synthesis time has implications on the contact between the active site of the catalyst and the substrate, including methanol and cooking oil. The biodiesel reaction is reversible. Thus, when equilibrium is reached, the synthesis process needs to be stopped to make more efficient usage of the energy [33]. The biodiesel production achieved 84.25% after 1 h of synthesis time. Further increasing the reaction time to 3 h only improve the yield by 0.59%. The increase in the percentage of biodiesel yield is not significant, presumably because the reaction has almost or has reached equilibrium. Thus, further increasing the reaction time will not affect the biodiesel yield.

In a study by Ting et al. [31], the reaction rate was increased when feedstock and methanol reacted in the presence of 0.5–1% sulfuric acid during a reaction time of 15–45 min. In another study by Pan et al. [34], an increase from 82.2% to 97.0% in biodiesel yield was obtained when the reaction time increased from 6 to 10 h. However, the biodiesel yield did not increase significantly once the esterification reached the equilibrium time at 10 h. The increase in the biodiesel conversion is attributed to the high concentration of reactants and the low concentration of products [35].

3.1.4. Reusability Test for Activated Ion Exchange Resin

The reusability of the Lewatit MP-64 resin can be seen from the yield of the obtained biodiesel. The stability of resin in catalyzing the reaction can be deduced if the biodiesel yield remains stable without a significant decrease over each cycle. The test was done using 4 g of Lewatit MP-64, and the yield of biodiesel during the four cycles is shown in Figure 6.

Based on the results of the stability tests (Figure 6), the yield of biodiesel decreases as the catalyst being reused. In the first cycle of using the Lewatit MP-64 resin, the yield of biodiesel produced was 94.06%. After the resin was regenerated and used for the next cycle, the yield decreased by 5.17%. Then for the second regeneration, the yield obtained decreased further by 4.03% from the previous cycle. In the last cycle, which is the third regeneration, the yield obtained decreased by 1.53% from the previous cycle. Although it continues to decline, the biodiesel yield in the three regeneration cycles is still above 80%.

Ren et al. [20] obtained the same yield of esters after regeneration. However, the study noticed that the decrease in biodiesel yield was unavoidable without regeneration due to the buildup of glycerol on resin. A study by Feng et al. [33] also obtained unchanged biodiesel conversion at the first 10 runs using NKC-9 type resin. Hartono observed that the performance of the catalyst dropped significantly after the initial use [32], reducing the yield from 85.94% to 41.88% after the fourth cycle [32]. The difference between this research and the research of Hartono lies in the synthesis process, activation time, dry weight of the resin used, stirring speed, substrate, and FAME test method. Therefore, the results of this study produce different biodiesel yields compared to the study of Hartono.

Patino et al. [36] observed the reusability of resin catalyst and obtained constant biodiesel yield (values from 31.8% to 33.2%) for six production cycles. However, a noticeable decrease in the catalyst activity started to show in the seventh and eighth runs, with a decrease of 16.6% and 29.7%, respectively, from the initial value of the biodiesel yield [36]. This performance loss is likely caused by the adsorption of water onto the catalyst surface. Thus, the presence of water coverage on the catalyst hinders the access of apolar fatty acids to its active site, as reported in the study by Kusakabe et al. [37].

3.2. The Use of Immobilized Lipase for Biodiesel Synthesis in Recirculating Packed Bed Reactors

3.2.1. Lipase Loading for Biodiesel Synthesis

The enzyme loading determines the amount of enzyme successfully immobilized in the gel matrix. The enzyme loading investigated solute protein concentration in the curing solution (calcium chloride solution). A lower amount of enzyme was successfully immobilized with entrapment. However, with the increase of enzyme amount, the enzyme loading was decreased. As shown in Figure 7, the lower amount of enzyme was mostly immobilized. However, the high concentration of enzyme could not achieve high enzyme loading. The amount of sodium alginate was constant for all variations. For enzyme amounts of 0.1% and 0.2%, distilled water was used as the sole solvent to dissolve both the enzyme and sodium alginate. However, when 0.3% enzyme was used, the distilled water was replaced with sodium phosphate buffer as it reduced the viscosity of the solution.

The cause of lower enzyme loading on 0.3% of lipase may be related to the solvent used to dissolve sodium alginate and lipase. The possible explanation for this may be related to the formation of calcium phosphate when phosphate ion from lipase or alginate or phosphate buffer solution reacts with the CaCl₂ solution. As for the loading efficiency of the entrapped enzyme, a low amount of enzyme shows 96.82% and 89.13% of enzyme loading for 0.1% and 0.2%, respectively, while 0.3% enzyme only reaches 60.25% enzyme loading. This indicates that a higher amount of enzyme is not efficiently entrapped as compared to a low enzyme concentration. Besides, using buffer solution as solvent may further reduce the loading efficiency. Vetrano et al. [15] already suspected this phenomenon when they compared their result with previous research [18,28].

The total dry weight amount of immobilized enzymes at 0.2% and 0.3% enzyme variations is almost the same, resulting in similar biodiesel yields between the two variations. Thus, it is better to use 0.2% (w/v_oil) lyophilized enzyme powder when producing immobilized enzyme using calcium alginate. In order to determine its biocatalysis ability to convert oil to methyl esters, all immobilized lipase was used to synthesize biodiesel.

It is known that Lipase enzymes are prone to inactivation because of methanol [28]. A two-step methanol addition has been used to avoid this drawback. Figure 8 illustrates the effect of enzyme concentration (w/v_oil). Biodiesel reached the highest yield when using 0.2% lipase (the ratio of lipase to oil). Generally, a higher lipase-to-oil ratio produces a better reaction rate because a more active site is available. Our experiment demonstrated that an enzyme amount of 0.2% can produce better yield of biodiesel compared to 0.1% and 0.3%.

Commercially available immobilized lipase (Novozyme 435) is able to synthesize 28.95 g of vegetable oil using 4% immobilized lipase, producing 96.1% biodiesel with two-step methanol addition in 27 h [38]. Compared with Novozyme 435, at 12 h, a higher biodiesel content is observed, particularly when using immobilized 0.2%(w/v_oil) of the enzyme. Transesterification and esterification by Novozyme 435 also appears to be affected by the amount of substrate used and the ratio of methanol-to-oil, in which the optimum ratio of methanol increases the reaction rate [39]. A variation on the substrate has been shown to affect the biodiesel yield. Nguyen et al. [40] used insect fat and methyl acetate as the substrate with Novozyme 435 as the biocatalyst, and was able to produce a biodiesel yield of 96.97% in 12 h. The same author also produced up to 96.18% by using the same biocatalyst, insect fat, and methanol as the acyl acceptor [41].

3.2.2. Effect of the Substrate Flow Rate on Biodiesel Synthesis

Previous research has shown the influence of flowrate in enzymatic biodiesel synthesis using a packed bed. Flowrate affects the residence time, in which a longer residence time facilitates more contact time between the enzyme with triglyceride and methanol. However, the rate of conversion will be reduced when the enzyme is saturated with the product. Thus, finding the optimal residence time is crucial for maximum conversion.

Figure 9 shows the biodiesel production with variation in the flowrate. Transesterification for 12 h indicates that the flowrate has insignificant effect on the yield. It was also observed that the yield change during the first 4 h is insignificant after the second addition of methanol. This may be caused by either absorption or adsorption of glycerol on the calcium alginate matrix, which may hinder methanol diffusion into enzymes active sites inside the calcium alginate. Based on Figure 9, variation in the flowrate formed a similar trend in the biodiesel production—regardless of the enzyme concentration. The biodiesel yield increased from 16% to 38% with an increase in the synthesis time from 8 h to 12 h. The lowest change was observed when 0.1% enzyme concentration is used, while the highest change was seen when using 0.2% enzyme concentration. The highest average change of biodiesel yield is 36.81% (regardless of the flowrate), which was obtained during biodiesel synthesis using 0.2% enzyme concentration, as shown in Figure 9B. It was also noted that a low flowrate (1 and 0.4 mL/min) produces a higher yield of biodiesel, thus the reusability test was done using 0.4 mL/min flowrate.

Lee Jong Ho et al. [42] uses immobilized Rhizopus oryzae and Candida rugosa lipases to catalyze soybean oil to biodiesel in circulated packed bed reactor [42]. The optimum reaction with a 97.98% conversion yield was achieved using 0.8 mL/min flowrate at 3 h. Chen, HC et al. [43] managed to produce a molar conversion of at least 82.81%, with an optimal flowrate of 0.1 mL/min, at 52.1 °C and using substrate molar ratio of 1:4 and tert-butanol as solvent. Another configuration using downflow packed bed reactor has been reported to produce a higher yield by increasing the substrate flowrate to 25 L/h and further increasing the flowrate caused reduction in the yield [44]. Enzymatic activity is also shown to be improved when using lower flowrates [45]. Reduction in the biodiesel production with increased flowrate might be caused by low residence time when higher flowrate are used, thus, reducing contact time between the bed with the substrate. Under high flow rates, substrate attachment to enzymes may also be disrupted

3.2.3. Stability Test for the Immobilized Enzyme

Figure 10 demonstrates the reusability test of the immobilized enzyme (0.2% w/v_oil). Based on the results, the enzymatic biodiesel synthesis performed under optimal conditions with three cycles can maintain more than 90% of enzyme activity. A similar result was obtained by Vetrano et al. (2022), where the catalytic activity of enzymes began to decrease in the fourth cycle [15]. Immobilization of lipase enzymes in the calcium alginate matrix can protect lipase well up to at least three cycles.

Batch synthesis of biodiesel using a calcium alginate entrapped enzyme has been previously reported by Kareem et al. [46], reaching 96.9% in 48 h while being able to retain most of its catalytic activity in 10 cycles. Other types of enzymes, such as invertase, are also able to be entrapped using calcium alginate, however, with an optimal reusability of nine cycles [47]. The advancement in immobilization of CRL by Rial et al. [48] using p-nitrobenyl cellulose xanthate (NBXCel) was also capable of producing >90% yield using 4.31% (ratio of lipase mass with NBXCel mass). The immobilization can retain >50% conversion even after 15 cycles. Aspergillus niger lipase cross-linked into Lewatit MP-64 can produce 69.07% yield within 50 h synthesis time using methyl acetate as the acyl acceptor, and 48.77% biodiesel yield could be observed on the fourth cycle on the reuse test [49]. Enzymatic biodiesel synthesis utilizing insect fat with Novozyme 435 catalyst has shown that the biocatalyst can withstand at least 20 cycles with a biodiesel yield mostly above 95% [40]. The same catalyst type was also used to synthesize biodiesel using insect fat with methanol. The result shows that the catalyst was also stable for 20 cycles and most of the yield was above 90% [41].

4. Discussions

Biodiesel production using enzyme offers advantages such as lower temperature and pressure, offering a more efficient process from an economic standpoint. In this study, FAME were formed using ion exchange resin and enzymatic catalysts. The resin used for biodiesel production were Lewatit MP-64, Amberlite IRA410Cifful, and Diaion PK208LH. Biodiesel was also successfully produced using Candida rugosa lipase encapsulated in calcium alginate gel.

The reactivity of catalysts is dependent on reaction conditions and variables, which include feedstock, the molar ratio of alcohol to oil, temperature, and reactor configuration. Therefore, selecting these variables at an optimum condition are crucial [50].

The selectivity of enzymatic reactions is high, and the enzymes can be immobilized to further preserve and reuse their function. The drawback is that the enzymes are relatively expensive. Calcium alginate immobilization offers a cheap and easy process to preserve and reuse enzymes. The use of commercially common lipase, Candida rugosa lipase, also helps to reduce the operational cost. However, our result shows that the resulting reaction rate is much lower than when using a resin catalyst. Resin catalysts have the advantage of separating the remaining reactants and products. They are also resistant to high temperatures and have higher reaction rates.

4.1. Enzyme vs. Resin Amount

Biodiesel yield is affected by the quantity of the used catalyst. In the case of ion exchange resins, a higher biodiesel yield is obtained when a higher concentration of catalyst is used. This is caused by an increase in the activation energy when more catalysts are used, which will increase the number of activated molecules that causes the reaction rate to improve and subsequently increase the conversion of the product [31]. The enzymatic process also shows an increase in the biodiesel yield by increasing the concentration of the enzyme. However, the entrapped enzyme was considerably reduced when increasing the enzyme concentration. Biodiesel synthesis using 0.2% enzyme concentration generated a higher yield at the end of the reaction, showing that enzyme concentration is a dominant factor in synthesizing biodiesel (Figure 9B). Calcium alginate matrix could not retain a high concentration of lipase, so the immobilization process may pose several problems and better entrapment is needed. The amount of activated resin used for biodiesel synthesis was in the range of 6–8% w/v_oil while usage of the enzyme (ignoring the weight of calcium alginate) was in the range of 0.1–0.3% w/v_oil. The imbalance of the percentage was due to the limited enzyme-retaining capability of calcium alginate and the size of the beads, which were considerably larger than the resin and thus unable to fit more into the reactor. This also shows that resin could retain more active sites while also having a wider surface area compared to calcium alginate-containing enzymes.

4.2. Synthesis Time

Conversion in transesterification may be increased by increasing the concentration of the catalyst and increase the reaction time [31]. Once the esterification has reached equilibrium, further prolonging the reaction time will not significantly increase the conversion. The resin catalyst produced a high yield with 1 h reaction time. Increasing the reaction time to 3 h produced an insignificant difference in the biodiesel yield. The 12-h reaction gives the highest yield of biodiesel when using immobilized Candida rugosa lipase. When the reaction of enzymatic catalysis was terminated after 12 h, low reaction rates were observed and may be caused by inaccessible enzymes due to the gel matrix and low methanol ratio in the substrate. The reaction time for enzymatic biodiesel synthesis is generally conducted for 24 h [23,44,48]. Conversely, resin catalyst can achieve a high yield of biodiesel within 3 h of reaction time [7,8,51]. Biodiesel synthesis using 4 g of activated Lewatit MP-64 resin produces the highest biodiesel yield compared to any other resin type and amount. The resins provide the H+ ions needed for the transesterification of triglyceride. Meanwhile, the triglyceride and methanol were adsorbed into the active site of the resin. The adsorption, conversion, and diffusion process of substrate and product was much faster on resin compared to the enzyme. The activated resin contains active sites within macro and microporous. On the other side, a gel catalyst such as calcium alginate was reported to have limited mass transfer of substrate to enzymes, which reduced the reaction rate [52].

4.3. Reusability Test

The reusability of catalysts is crucial to reduce and maintain biodiesel prices. As ion exchange catalyst has been previously investigated, aside from exhibiting high catalytic ability in FAME conversion, ion exchange resins can be regenerated and reused over 10 times without a noticeable performance loss [51,52]. Our result also proves that during at least four cycles, ion exchange resins can be employed as a potential heterogeneous catalysts as they provide an efficient pathway for biodiesel production. However, the drying process must be repeated for every cycle because the presence of water could reduce the biodiesel yield by forming unwanted byproducts.

Enzyme catalyst have different degree of reusability and activity depending on the immobilization strategy being applied. Recent research has demonstrated that they can be regenerated at least nine times without noticeable reduction of yield [46]. Conversely, Vetrano et al. (2022) reported variation in the degree of immobilized enzyme reusability, that is generally below 10 cycle to maintain 80% of enzyme residual activity [15,47]. Research utilizing methyl acetate as an acyl acceptor to synthesize biodiesel enzymatically was able to maintain 48.77% of yield in fourth cycle and experienced a 20.3% reduction in yield compared to the first cycle. Aspergillus niger Lipase was immobilized by cross-linking into Lewatit MP-64 and was used to synthesize biodiesel in 50 h flask transesterification [49]. Calcium alginate entrapment showed better immobilization capability and higher biodiesel yield than cross-linked immobilization on macroporous resin. There was a possibility that cross-link immobilization changed enzyme conformation, thus inactivating the enzymes. The entrapment process using biocompatible material may prevent inactivation due to the reaction of the material with enzyme active sites. However, the entrapment process may block the access of the substrate into the enzyme, leading to a reduction of the effective enzyme needed for transesterification. There was an alternative process involving liquid enzyme in an attempt to reduce the production cost. Nguyen et al. [53] attempted to synthesize biodiesel using liquid lipase with the addition of superabsorbent polymer. The study succeeded in producing a maximum conversion of 96.73%. However, the author noted that the liquid lipase was only able to be reused once. Therefore, activated resins (specifically anion exchange resin) and immobilized lipase can be used as an alternative heterogenous catalysts for the transesterification process in biodiesel synthesis. Resins can produce higher yields of biodiesel in a shorter time. Conversely, while enzymes require a longer time, it is hard to make a fair comparison because of the different reaction mechanisms and costs.

4.4. Catalyst Cost Comparison

The cost of biodiesel is significantly influenced by the synthesis time, material, and catalyst preparation cost. Compared to the resin catalyst, the biocatalyst (immobilized enzyme) preparation was comparatively simple. The immobilization of enzyme is easily performed in room temperature with common materials such as sodium alginate, deionized water, and calcium chloride. The biocatalyst might also be reused by washing the immobilized enzyme with deionized water. Because the temperature of resin activation is 60 °C and the drying process requires 110 °C in the oven, resin catalyst synthesis requires more energy than enzyme immobilization. The cost of producing activated resin was undeniably higher. Enzyme synthesis requires a purification step, which adds to high enzyme pricing [54], and consequently higher production costs. Compared to enzymes, the resin catalyst material cost was cheaper [55].

The rate of biodiesel generation would be affected by the synthesis time. By employing activated resins as a catalyst, biodiesel production could be improved to 80–94% in 1–3 h. Immobilized enzyme yields 64–71% biodiesel in 12 h, which is 4 times the time required for an activated resin-catalyzed process. Activated resins could boost the production capacity and quality. Production capacity and quality the critical aspects of biodiesel for industrial utilization, as slow-quality biodiesel (with a greater triglyceride content) would damage diesel-based machinery over time [56].

5. Conclusions

The maximum biodiesel yield was produced using Lewatit MP-64, which used 4 g of resin dry weight, 3 h of synthesis time, and 3 h of resin activation time, yielding 94.06% biodiesel. The Lewatit MP-64 resin was found to be reusable, and the output was reduced by 10.73% on the fourth cycle. The yield obtained via enzymatic catalysis is 71.1%, employing 0.2% (w/v_oil) enzyme and a reaction flowrate of 0.4–1 mL/min. Calcium alginate entrapment managed to retain most of the enzyme activity, and biodiesel yield was reduced by 4.6% in the third cycle.

Resins have greater reaction rates than enzyme catalysts, generating a larger biodiesel yield and may lead to more efficient manufacturing. Considering their reusability, both might be used as alternative catalysts in the production of biodiesel, reducing the need for homogeneous catalyst and ecologically harmful byproducts. Future studies and reports on the viability of either enzymatic or activated resin usage for biodiesel synthesis must be completed to investigate production costs, waste output, and production volume to produce biodiesel efficiently on an industrial scale.