3.1. Characteristic analysis of magnetic nanoparticles and immobilized RML
In this study, an immobilization strategy for magnetic RML CLEAs was developed and studied. As shown in Fig. 1a, APTES-Fe3O4 can be easily obtained and then used for the immobilization of RML.
The magnetic characterization was shown in Fig. 2a. The magnetization hysteresis curves of magnetic RML CLEAs indicates the superparamagnetic character, and such a decrease compared to the Fe3O4 attributes to enzyme-loading. Moreover, the XRD patterns show that the incorporation of APTES and enzyme had no effect on the crystal structure of Fe3O4, and the main reflections completely match with pure Fe3O4 (Fig. 2b). TGA curves provide information on the thermal stability of Fe3O4, APTES-Fe3O4 and magnetic RML CLEAs (Fig. 2c). The TGA curve of Fe3O4 nanoparticles shows an initial weight loss (6%) corresponding to the removal of water molecules and unreacted ligand on the surface within the range of 200–300°C. The APTES-Fe3O4 exhibits two weight losses from room temperature to 200°C and 220 to 450°C, because of the removal of free water and the decomposition of the APTES, respectively (Li Q et al. 2020). For the magnetic RML CLEAs composite, a first weight loss similar to APTES-Fe3O4 nanoparticles was observed, related to free water molecules. When the temperature reached 200°C, a rapid loss started to occur as a result of the decomposition of the enzyme, structural water, and other guest molecules until 700°C (Lin CP et al. 2019).
FT-IR spectra of Fe3O4, APTES-Fe3O4 and magnetic RML CLEAs are shown in Fig. 2d. Presence of surface functional groups, binding of enzyme onto magnetic nanoparticles were vindicated by FT-IR spectroscopy. The peak near 540–560 cm− 1 is consistent with the stretching vibration of Fe-O in Fe3O4, indicating the presence of Fe3O4 in the microspheres, which proves the successful preparation of magnetic RML CLEAs nanoparticles. In addition, compared to the black curve, new peaks at 2969 cm− 1, 2923 cm-1 and 2883 cm− 1 appear in the red curve, which correspond to the C-H vibrations on the aliphatic chain in APTES, confirming the modification of APTES on Fe3O4 microspheres. The peaks at 3351 cm− 1 and 3307 cm− 1 in the blue curve correspond to the stretching vibration of the N-H bond in the enzyme molecules, while peaks at 1646 cm− 1 and 1600 cm− 1 were detected corresponding to the C = O bond vibration in -CONH2, confirming the successful immobilization of the enzyme on the APTES-modified magnetic nanoparticles (Miao CL et al. 2018). The acquired peaks matched those in previous reports well (Xie WL and Ma N 2009).
Figure 3 shows the surface morphology of the magnetic nanoparticles in scanning electron microscopy (SEM) images. As shown in Fig. 3A, the average diameters of the bare Fe3O4 particles range from 10 to 20 nm. An increase of 30 nm in the size of Fe3O4 can be found after the modification of APTES. Notably, compared to bare Fe3O4, the APTES-Fe3O4 particles showed a looser surface structure. The agglomeration of the magnetic nanoparticles can be weakened by modifying APTES on the surface of bare Fe3O4 nanoparticles, which was beneficial to improve the efficiency of immobilized enzyme. It is noteworthy that the formed APTES-Fe3O4 nanoparticles not only displayed magnetic behaviors but also had a broad active surface available for lipase immobilization, which is essential and important in term of protein immobilization (Miao CL et al. 2018).
To further confirm that lipase was immobilized on the support, fluorescein isothiocyanate (Rhodamine B)-labelled lipase was prepared and used for detecting the process of immobilization. The confocal laser scanning microscopy (CLSM) images of the Rhodamine B-labelled RML@Fe3O4 (Fig. 3d, single-channel mode of CLSM) showed red particles, and the confocal microscope image revealed that the labeled enzymes (red) were distributed uniformly on the support(Fig. 3f).
3.2. Catalyst performance of the magnetic RML CLEAs
In order to study the properties of the immobilized enzyme, the oil/IL quality ratio, temperature, reaction time, catalyst amount and the methanol/oil molar ratio were explored.
To determine the optimal solvent, the biocatalytic synthesis of biodiesel was carried out through methanolysis of jatropha oil in these fifteen different ILs as reaction media at 50°C, using a 1:5 molar ratio of oil and methanol as a substrate (Table 1). Transesterification reaction cannot be performed in the ILs used in this work in the absence of lipase. The catalytic efficiency of the immobilized lipase (magnetic RML CLEAs) on the methanolysis reaction was then checked in media containing methyl imidazolium ionic liquids (MIM) of different cation chain lengths (C2, C4, C8, C12 and C16) and in combination with four different anions ([BF4], [PF6], [N(CN)2] and [NTf2]).
Table 1
Effect of different ionic liquids on biodiesel production a.
Cation | Biodiesel yield (%) b |
Anion |
[NTf2] | [BF4] | [PF6] | [N(CN)2] |
[EMIM] | 23.5 ± 3.5 | 2.5 ± 2.1 | 24.5 ± 4.1 | 3.1 ± 2.4 |
[BMIM] | 24.8 ± 2.8 | 8.6 ± 3.1 | 31.3 ± 2.9 | 2.2 ± 1.8 |
[OMIM] | 23.7 ± 3.7 | 17.6 ± 2.6 | 22.3 ± 5.4 | 1.7 ± 1.1 |
[C12 MIM] | 15.3 ± 2.7 | 14.0 ± 3.7 | | |
[C16 MIM] | 12.9 ± 2.9 | | | |
Solvent free | 3.0 ± 1.5 | | | |
a The reaction conditions: time 48h, oil/IL weight ratio 1: 2.0, immobilized lipase 4 wt% and methanol/oil molar ratio 5:1. |
b Determined by GC analysis. |
When the anion was changed, the FAME conversion of ILs based on the [PF6] and [NTf2] anions was higher than that of ILs based on the [BF4] and [N(CN)2] anions. This may be due to the ILs with [BF4] and [N(CN)2] anions are more nucleophilic than ILs with [PF6] and [NTf2] anions. Therefore, they can promote the change of the secondary structure of the protein, resulting in a loss of activity (Zhao J et al. 2015).
The activity and stability of enzymes can be substantially affected by the alkyl chain length of cation in ILs (Itoh T 2017). The results showed that with the increase in alkyl chain length of the corresponding imidazolium cation, the synthetic activity increased first and then decreased. The initial increase of synthetic activity results from the higher solubility of the oil substrate in the more hydrophobic ILs, given that the hydrophobicity of the IL increased with the alkyl chain length on the cation (Diego TD et al. 2011). Nevertheless, as for the same anion, the viscosity of the IL increases with the increasing length of the alkyl chain substituted on the imidazolium ring. Regarding the [Cn MIM] cations, the viscosity increases in this order with the anion type: [PF6]> [BF4] > [NTf2] (Diego TD et al. 2011). The mass transfer resistance increases due to the increase of viscosity, which negatively affects the operating process. The highest production of FAME was obtained in [BMIM][PF6] and its production was over ten times higher than in a solvent-free system. Such a low yield in the solvent-free system may be due to the low solubility of methanol in triglycerides or to the inactivation of lipase by methanol (Lozano P et al. 2013; Rafiei S et al. 2018).
The influence of the oil-to-ionic liquid (oil/IL) weight ratio on the immobilized-RML-catalyzed biodiesel synthesis was studied after 48 h of reaction at 50°C. The oil: ionic liquid weight ratio shown in Fig. 4a, and in all cases, the initial system for the transesterification reaction was a homogenous medium. For the case of [BMIM][PF6], the yield of FAME increased when the oil: IL ratio decreased, due to the higher concentration of methanol in the reaction mixture the greater lipase inactivation (Rafiei S et al. 2018).
These results may also be due to the special protection of ILs on lipases in non-aqueous environments, which prevents lipase from deactivating by certain organic solvents (Sheldon RA 2016).
As the temperature is a crucial factor to be considered in the enzymatic synthesis of biodiesel, the influence of reaction temperature on the yield of FAME was studied by carrying out the reaction in [BMIM][PF6] at different temperatures. As can be seen from Fig. 4B, the immobilized RML was more active than free RML in the range of 20–60°C. The optimum temperature interval of RML CLEAs was found to be 30–60°C and when the temperature was higher than 37°C its activity decreased slightly. It was indicated that immobilization could effectively protect lipases from thermal denaturation (Otari SV et al. 2020). Based on the results, 37°C was selected for further studies.
The effect of reaction time on the preparation of biodiesel was studied from 12 h to 72 h. According to Fig. 4c, the longer reaction time, the higher yield, and the yield tended to be a constant after reaction time was longer than 48 h. Especially, the yield of FAME was 51% after 48 h while the yield reached only 58% after 72 h. Therefore, the comparatively optimal reaction time is 48h.
In this study, methanol was used as a substrate for transesterification reaction with jatropha oil. Excess methanol can drive the chemical equilibrium to biodiesel formation, but it was harmful to the enzymes (Xie WL and Wan F 2019). The reaction rate and the degree of transesterification can be increased by adding a proper amount of methanol into the reaction mixture. The influence of the methanol/oil molar ratio on the production of biodiesel is shown in Fig. 4d, the biodiesel yield increased with the methanol to oil ratio from 3:1 to 7:1, the highest biodiesel yield (60%) could be acquired at the ratio of 4:1. However, the yield of biodiesel reached the maximum and further increased in the alcohol concentration resulted in a dramatic decrease in conversion due to the inactivation of the enzyme. Consequently, the appropriate methanol/oil molar ratio is 4:1.
The lipase dosage has a direct impact on the reaction rate (Sun S et al. 2015). The effect of catalyst dosage on the conversion of jatropha oil into biodiesel was studied by varying its dosage in the range of 0–12 wt% (as referred to the oil mass). Figure 5a indicates that the FAME yield increased significantly as the lipase dosage increased. The high yield was obtained at 10 wt%. Further increase in catalyst concentration did not enhance the conversion yield. In fact, increasing the dosage of catalyst by more than 10 wt% caused a decrease in biodiesel yield. This result indicated that the excess magnetic nanoparticles tended to form enzyme aggregates, which reduced the accessibility of lipase to substrates (Palomo JM et al. 2003)
In many European countries, the most commonly used vegetable oils in the production of biocatalytic biodiesel are obtained from soybean seeds and sunflower seeds. Countries can use different types of raw materials based on the abundance or availability of the region, such as palm oil and coconut oil in tropical countries (Christopher LP et al. 2014; Rahimi V and Shafiei M 2019).
The influence of oil feedstock on the initial yield of biodiesel was analyzed in [BMIM][PF6]. In the present work, four different vegetable oils were used (soybean oil, olive oil, canola oil, and castor oil) in addition to jatropha oil (Fig. 5b). Notably, compared to free RML, the magnetic RML CLEAs exhibited better activity towards five different oils in catalyzing biodiesel production. Especially,the catalytic effect of free enzyme for five oils are poor, and the mostly yield are less than 10 %. There was no significant difference for the different oils when free RML was used, but slightly higher for jatropha oil.
3.3. The reusability and storage of the magnetic RML CLEAs
No matter the reusability of the IL and the enzyme, it is always an important advantage for the commercial viability of a process (Nadar SS et al. 2020). We further demonstrated the recyclable use of the enzyme in this solvent system (Fig. 6a). Surprisingly, the spent IL and immobilized enzyme exhibited favorable recyclability in the FAME production, and the catalytic activity of immobilized RML remained 60 % even after 5 cycles. This indicated that the lipase-IL had great reusability, which was satisfying for reducing the cost-effectiveness in the enzymatic synthesis of biodiesel. Furthermore, the nano-biocatalyst exhibited excellent reusability in biodiesel production might be due to: (i) The protection of hydrophobic ILs. (ii) The suitability of the proposed methodology to extract biodiesel and glycerol by an easy and sustainable approach that permits full recovery and reuse of enzyme–IL systems (Elgharbawy AA et al. 2018). In addition, the slight decrease of catalytic activity after several times of recycling may be caused by some loss of enzyme during the cycle or residual products in the IL accumulate and inactivate the enzyme.
Storage stability is one of the most important aspects in the development of reliable biocatalyst for commercial-scale application (Cui JD et al. 2017). Hence, a time-course study on the storage stability of immobilized RML was carried out by leaving the composite at -20°C for a period of time. As shown in Fig. 6b, the activity of the immobilized RML was almost no decrease after storage of the magnetic RML CLEAs for 98 days. This result showed that nano-biocatalyst performed well in long-term storage, which can be explained by the covalent binding of the enzyme on the carrier, thus increasing the strength and stability of lipase by effectively decreasing its denaturation (Talekar S et al. 2012).