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Synthesis of Lipase-Immobilized CeO2 Nanorods as Heterogeneous Nano-Biocatalyst for Optimized Biodiesel Production from Eruca sativa Seed Oil

Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
Institute of Industrial Biotechnology, Government College University, Lahore 54000, Pakistan
Institute of Advanced Technology, Universiti Putra Malaysia, Serdang 43400 UPM, Malaysia
Department of Civil and Environmental Engineering, Universiti Teknologi PETRONAS, Seri Iskandar 32610, Malaysia
Centre for Urban Resource Sustainability, Institute of Self-Sustainable Building, Universiti Teknologi PETRONAS, Seri Iskandar 32610, Malaysia
Chemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
Laboratoire de Recherche LR18ES08, Chemistry Department, Science College, Tunis El Manar University, Tunis 2092, Tunisia
Catalysis Science and Technology Research Centre, Faculty of Science, Universiti Putra Malaysia, Serdang 43400, Malaysia
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang 43400, Malaysia
Authors to whom correspondence should be addressed.
Catalysts 2020, 10(2), 231;
Submission received: 6 November 2019 / Revised: 2 December 2019 / Accepted: 2 December 2019 / Published: 15 February 2020
(This article belongs to the Special Issue Lipases and Phospholipases in Biocatalysis)


Biodiesel has emerged as one of the most attractive alternative energy sources to meet the growing needs of energy. Many approaches have been adopted for biodiesel synthesis. In the present work, biodiesel was produced from non-edible Eruca sativa oil using nano-biocatalyst-catalysed transesterification. Nano-biocatalyst (CeO2@PDA@A. terreus Lipase) was developed via the immobilization of lipase on polydopamine coated ceria nanorods, and CeO2 nanorods were developed via a hydrothermal process. The mean diameter of nanorods were measured to be 50–60 nm, while their mean length was 150–200 nm. Lipase activity before and after immobilization was measured to be 18.32 and 16.90 U/mg/min, respectively. The immobilized lipase depicted high stability at high temperature and pH. CeO2@PDA@A. terreus Lipase-catalysed transesterification resulted in 89.3% yield of the product. Process optimization through response surface methodology was also executed, and it was depicted that the optimum/maximum E. sativa oil-based biodiesel yield was procured at conditions of 10% CeO2@PDA@A. terreus Lipase, 6:1 methanol/oil ratio, 0.6% water content, 35 °C reaction temperature, and 30 h reaction time. The fuel compatibility of synthesized biodiesel was confirmed via the estimation of fuel properties that were in agreement with the ASTM D standard. The nanorods and dopamine-modified nanorods were characterized by FTIR spectroscopy, SEM, and energy dispersive X-ray (EDX), while conversion of E. sativa oil to biodiesel was confirmed by GC/MS and FTIR spectroscopy. Conclusively, it was revealed that CeO2@PDA@A. terreus Lipase has potential to be employed as an emphatic nano-biocatalyst.

1. Introduction

Depletion of petroleum reservoirs has stimulated the scientific community worldwide to search for alternate energy resources. Biodiesel has notable potential as an imperative green fuel [1]. The additional impacts of biodiesel production are to increase agricultural production and productiveness, to increase the income of rural communities, to lessen pollution, and to create new jobs [2]. Biodiesel is a biodegradable, relatively nontoxic, and green fuel with reduced CO and unburned hydrocarbons (UHC) emission. Moreover, biodiesel has benefits such as appropriate lubricity, and no sulphur [3]. Biodiesel is widely manufactured using the transesterification process, which is catalyzed by numerous catalysts. Transesterification lowers the viscosity of feedstock oils [4] which reacts with alcohol, resulting in biodiesel. Many acidic and basic catalysts have been employed for the said purpose, but enzymatic catalysis is being promoted. The chemical methods of transesterification are considered to be problematic due to the production of poor-quality glycerol, the production of alkaline wastes, washing requirements, side reactions, and issues related to biodiesel recovery and purification. Therefore, enzymes are preferred over chemical catalysts in the transesterification to prepare biodiesel [5].
Various lipases are frequently being used in the transesterification process. It has been observed that the reproducibility and catalytic activity of immobilized enzymes are better than the non-immobilized enzymatic system [6]. Immobilization also makes the enzyme reusable and stable. Different microbial lipases have been immobilized on various surfaces, such as gels, ceramics, and alginates beads [7]. Recently, nanoparticles have also gained much attraction as effective supports for enzyme immobilization. Nanoparticles of a very small size provide a large surface area for enzyme immobilization, and characteristic Brownian moment of nanoparticles provide higher enzymatic activity [8]. Lipases are mostly immobilized on magnetic nanoparticles; other supports used for this purpose are nanoparticles of silver, magnesium, and carbon nanotubes. Successful immobilization of lipase on nano-materials/supports in biodiesel synthesis enhances the efficiency of the transesterification process and makes lipase reusable, which finally reduces the cost of the process without compromising the yield and quality [9]. The production of biodiesel from nano-immobilized lipase involves (1) the preparation of enzyme; (2) the synthesis of nanoparticles (NPs); (3) the attachment of enzyme with nanomaterials, directly or through suitable a linkage or mediator; and (4) the transesterification of feedstock oil using synthesized nano-biocatalyst [10]. Bearing in mind the significance of nano-immobilized lipase, this work was planned to develop CeO2-based nano-biocatalyst for the synthesis of fatty acid methyl esters/biodiesel from E. sativa oil.

2. Results and Discussion

2.1. Scanning Electron Microscopy (SEM)-Based Characterization

From the SEM micrographs (Figure 1a,b), it can be seen that the morphology of prepared CeO2 is rod-like. These nanorods are uniform in shape and size, and they are not agglomerated. At different magnification levels, the SEM images revealed that the nanorods have mean diameter of 50–60 nm, whereas their mean length was 150–200 nm.

2.2. Energy Dispersive X-Ray Spectroscopy (EDX)

The constituent elements of the nanorods were determined using energy dispersive X-ray (EDX) spectroscopy. The plot between energy of X-rays vs. X-rays counts is presented in (Figure 2). Each peak in the plot corresponds to a specific element. EDX plot confirms the presence of cerium and oxygen in the product. Absence of extra peaks may be attributed to the purity of synthesized nanorods, while peak for carbon is due to the carbon plates of instrument. Atomic% and weight% of elements obtained by EDX are shown in Table 1. Ratio of atomic% between cerium and oxygen affirms the formation of CeO2 nanorods.

2.3. X-Ray Diffraction (XRD)

XRD pattern (Figure 3) was used to characterize the CeO2 nano-powder, and instrument was operated within range of 20–80°. The peaks appeared in the XRD pattern with 2θ values at 27.95°, 32.71°, 46.93°, 56.21°, 59.27°, and 69.03°, ascribed to the d-values (with cubic phase) 0.319 (111), 0.275 (200), 0.194 (220), 0.164 (311), 0.156 (222), and 0.136 (400), respectively. The XRD pattern of CeO2 nanorods was in fine arrangement with the standard JCPDS card no# (JCPDS-34-0394). The spectrum indicates that there are no additional impurity peaks present.

2.4. Fourier Transform Infrared Spectroscopic (FTIR) Analysis of Naked and Polydopamine Coated CeO2 Nanorods

Both the polydopamine-coated and uncoated CeO2 nanorods were scanned for FTIR spectroscopy in the range of 600–4000 cm−1 (Figure 4). FTIR spectrum (blue colored) reveals the functional groups and the chemical bonds that are present in the synthesized, uncoated CeO2 nanorods, whereas FTIR (black colored) spectrum confirms the functionalization of CeO2 nanorods with polydopamine layer. The broad band at 3402.47 cm−1 corresponds to (the stretching vibration of O-H bond) OH-groups. The peak around 1569.67 cm−1 is ascribed to the bending vibration of N-H group. The absorption peak around 1496.92 cm−1 indicates the -CH2 vibrations. The intense band at 691.17 cm−1 corresponds to the Ce-O stretching vibrations [11]; another additional peak at 1312.27 cm−1 corresponds to the C-O bonds in dopamine molecule that verifies the dopamine addition on CeO2 nanorods [12].

2.5. Lipase Activity Assay

The activity titer of free lipase (which was produced from Aspergillus terreus AH-F2) was found to be 18.32 U/mg/min, while the activity titer of CeO2@PDA@A. terreus Lipase was found to be 16.90 U/mg/min. It was found that, by using polydopamine, high immobilization efficiency was achieved by using CeO2 nanorods because polydopamine and CeO2 nanorods formed the complex, which was found to be efficient regarding immobilization of lipase. The wider surface was available during the reaction, which gave higher conversion rate in a short period of time by using the lipase-immobilized nanorods for biodiesel production [13]. To the best of our knowledge, the synthesized nano-biocatalyst with CeO2 nanorods as immobilizing support for Lipase (Aspergillus terreus AH-F2) may be novel nano-biocatalyst for biodiesel synthesis.

2.6. Effect of pH and Temperature on Activity of Nano-Biocatalyst

The impact of pH on the activity of free lipase and synthesized nano-biocatalyst is presented in (Figure 5a). The effect of pH was studied within pH range of 5 to 10, and it was found that maximum lipase activity, i.e., 18.32 U/mg/min, was observed at pH 7.0 in case of free lipase, while at pH 8, CeO2@PDA@A. terreus Lipase showed maximum activity (i.e., 16.90 U/mg/min). The relationship plot between activities of Lipase (Free and immobilized) and pH depicted that CeO2@PDA@A. terreus Lipase could tolerate high pH values. The comparison of free and immobilized lipase revealed that immobilizing A. terreus Lipase on CeO2@PDA increased the flexibility of lipase to a wide pH range, compared to free lipase. Our results were comparable to the studies of Baharfar and Mahajer [13].
The impact of temperature (ranging from 25 to 50 °C) on the activity of free lipase (A. terreus Lipase) and CeO2@PDA@A. terreus Lipase is presented in Figure 5b. It was depicted that highest free lipase activity was revealed at 30 °C, while the CeO2@PDA@A. terreus Lipase showed highest/maximum activity at 40 °C, so it showed that CeO2@PDA@A. terreus Lipase was tolerant to high temperature conditions and was stable at a wider temperature range. The increased tolerability of lipase may have been due to the formation of covalent bonds during immobilization. Comparable observations have been reported by Baharfar & Mahajer [13] and Dumri & Hung [14].

2.7. Physico-Chemical Properties of E. sativa Seed Oil

Physico-chemical properties of E. sativa oil are described below in (Table 2). Density is measure of mass per unit value of a sample (E. sativa oil) [15]. Peroxide value is the measure of amount of peroxides in the sample, i.e., oil or fats [16]. The ratio of density of sample to the water density is described by specific gravity [17]. Acid value is the amount of potassium hydroxide (mg) required for the deactivation of the fatty acids present in the 1.0 g of sample (oil) [18]. Iodine value is the measurement of unsaturation in the given oil; if the iodine value is high, then degree of unsaturation is high. The milligram of KOH required to saponify each gram of oil under specific set of conditions is referred as saponification value [19].

2.8. Biodiesel Yield (%) and Optimum Reaction Conditions

Optimum reaction conditions for CeO2@PDA@A. terreus Lipase-catalyzed transesterification of E. sativa are given below in (Table 3), with maximum biodiesel yield of 89.3%. Optimum reaction conditions revealed were reaction time (30 h), temperature (35 °C), alcohol/oil ratio (6:1), water content (0.6%), and CeO2@PDA@A. terreus Lipase concentration (10%). The same enzyme (A. terreus Lipase) has also been immobilized on magnetite nano-support in another study performed by our research group, and comparable reaction conditions were revealed for optimal biodiesel production (92%) with slight variation in reaction temperature (37 °C). When methanol is added in oil, the viscosity of oil decreases and reaction rate increases, which is why methanol is significant for the reaction rate, but if an excessive amount of methanol is added in oil then emulsification of the glycerol may take place, deactivating the lipase and minimizing the biodiesel yield [20].
The other factor is catalyst concentration that influences the reaction rate. In the current study, 10% CeO2@PDA@A. terreus Lipase concentration was observed as optimum concentration for the synthesis of E. sativa seed oil-based biodiesel. The maximum yield of biodiesel (89.3%) may be increased by taking higher concentration of nano-biocatalyst or by using different lipase source for immobilization on functionalized CeO2. Another factor is reaction temperature, when the reaction is carried out at a more optimum temperature than reaction rate and biodiesel yield is maximum. However, high temperature denatured the lipase and reduced the biodiesel yield. Water content is also significant for the reaction rate, as it affects the activity and stability of lipase and protects it from deactivation via short chain alcohols [21].
Previously different researchers have employed response surface methodology (RSM) for biodiesel production using various feedstock oils and catalysts [22,23]. Aghababaie et al. has described bio-catalytic biodiesel synthesis from the crude E. sativa oil and obtained the highest FAME yield at 3:1 methanol to oil ratio, 5 mg lipase, 40% water content, and 21 °C temperature. The results that have been reported in literature are comparable to the present work [24,25,26,27], but still some variations are present that might be due to the varying fatty acid profiles of feedstock, different activities of the catalysts, and ranges of reaction parameters selected for optimization process.

2.9. RSM Model Fitting

Central composite design was applied to optimize biodiesel production procedure. Among the different models (viz; linear, quadratic, cubic polynomial, and two-factor interaction 2FI), quadratic model was revealed to be the most significant model for the responses with p-value < 0.0001 for CeO2@PDA@A. terreus Lipase catalyzed transesterification of sample oil. Fitness of quadratic model was also confirmed by the lack of fitness test, which was insignificant for the model with p-value 0.0728 in addition to higher R2 and adjusted R2 values (Table 4).
Fitness of the model was also affirmed by the normality and predicted vs. actual values graph (Figure 6). The linear distribution of data along the straight line in normality plot of the model depicts the fitness of quadratic models, while the small difference b/w predicted, and actual values of biodiesel yield further advocates the significance of quadratic models.

2.10. ANOVA for Biodiesel Yield Response

Table 5 represents the ANOVA for the selected quadratic model describing the impact of various reaction parameters (as linear terms, 1st order interaction and quadratic terms) on response (% biodiesel yield) catalyzed by CeO2@PDA@A. terreus Lipase.
A (methanol/oil ratio), B (CeO2@PDA@A. terreus Lipase concentration), and D (reaction time) were significant linear terms, while C and E were non-significant (>0.05) with p-value 0.1344 and 0.6091, respectively. The AB, AC, AE, and BC were significant interaction terms, having p-values of 0.0069, 0.0283, 0.0243, and 0.0013, respectively (<0.05). As for the quadratic terms, quadratic terms B2, C2, and D2 were the most significant quadratic terms, with p-values of 0.0001, 0.0001, and 0.0240, respectively, while A2 and E2 were non-significant quadratic terms that had p-values > 0.05.
Comparable results have been obtained in previous research. Mehmood et al. (2018) described the ANOVA for the significant quadratic model for biodiesel production from E. sativa oil catalysed by H2SO4 and reported that A (amount of catalyst) and D (methanol/oil ratio) impact significantly on the response (biodiesel yield), while B (temperature) and C (time) were non-significant terms. Regarding the 1st order interaction terms, BC and CD were significant, whereas AB, AC, AD, and BD were non-significant. Among the quadratic terms, D2 and B2 were significant [28].
Mumtaz et al. also reported the ANOVA for bio-catalytic synthesis of palm oil-based biodiesel. According to their findings, liner terms that showed a significant effect on response (% biodiesel) were A (amount of bio-catalyst), B (reaction time), and D (methanol/oil ratio). Among first-order interacting terms, AD showed significant influence on biodiesel yield. Among quadratic terms, B2 and D2 were depicted to be significant [29]. In another previous study, Chang et al. reported the analysis of variance for experimental data obtained by Lipase catalyzed biodiesel production from Novozym 435. The significant linear terms were X2 (reaction temperature), X3 (enzyme concentration), X4 (substrate molar ratio), and X5 (water content), while the non-significant term was X1 (reaction time) [30].
Razack and Duraiarasan also reported ANOVA for the biodiesel production from waste cooking oil catalyzed by encapsulated mixed enzyme in which the only significant linear factor was C (reaction temperature); while A (enzyme), B (molar ratio) and D(time) had p-value > 0.05. Significant quadratic factors were revealed to be A2, B2, and C2. AC was the only significant first order interaction term [31].
The results reported previously are comparable with the results obtained in present work with some variation. The significance of methanol-to-oil ratio and enzyme concentration is obvious, as they directly influence the reaction rate. The enzymatic transesterification is a slow process so the significance of reaction time for enzymatic transesterification cannot be denied, as reported by other researchers as well [32]. Water is also considered as an imperative factor; however, water concentration (as linear term) has not been proved significant in the present work.

2.11. 3D Surface Graphs for % Biodiesel Yield

Response surface plots of interaction terms depicting significant influence on response (biodiesel yield) are presented in (Figure 7). 3D graph describing the cumulative impact of CeO2@PDA@A. terreus Lipase and CH3OH/oil ratio indicated that the % biodiesel increases with an increase in nano-biocatalyst level and methanol/oil ratio, and optimal biodiesel yield is obtained when nano-biocatalyst level and methanol/oil ratio were 10% and 6:1, respectively. Deviation from these values, however, results in lower biodiesel yield. 3D graph between temperature and methanol/oil ratio showing their impact on response is shown in (Figure 7b), which shows that the highest response was observed at 6:1 methanol/oil ratio and 35 °C reaction temperature. However, high temperature can denature the active sites of the enzyme. Response surface plot for methanol-to-oil ratio and water content is presented in Figure 7c, which predicts that the combination of these two variables also affect the response. Figure 7d shows the combined effect of temperature and nano-biocatalyst concentration on the response, which indicates that the yield rises with rise in reaction temperature and nano-biocatalyst amount until it reaches an optimum point, i.e., 35 °C and 10%, respectively, while beyond this point a decline in % biodiesel yield is observed.

2.12. FTIR Spectroscopic Analysis of Eruca Sativa Oil and Biodiesel

The transesterification of E. sativa oil and biodiesel was monitored by FTIR spectroscopy (Figure 8). The IR absorption band at 1438.0935 cm−1 corresponds to the -CH3 asymmetric bending that was present in E. sativa oil-based biodiesel but absent in E. sativa oil FTIR spectra. Similarly, absorption band at 1196.7202 cm−1 ascribed to O-CH2 stretching which was absent in E. sativa oil but present in E. sativa biodiesel spectra. The bands at 1161.1 cm−1, 1459.3 cm−1, and 1099.6 cm−1, corresponding to C-O stretching, C=C stretching, and OCH2C asymmetric stretching, respectively, were absent in E. sativa oil biodiesel but present in E. sativa oil FTIR spectra. The bands at 1700–1800 cm−1 and 2800–3000 cm−1 (vibrational frequency band of C=O stretching and CH2 symmetric stretching, respectively) were present in both E. sativa oil and E. sativa oil biodiesel spectra. Tariq et al. reported the conversion of E. sativa oil into biodiesel by disappearance of peaks in E. sativa oil at 1465 and 1095 cm−1 and formation of new peaks at 1435 and 1195 cm−1 in the biodiesel [33]. Similar results have been reported in other research [34].

2.13. Major Fatty Acid Methyl Esters of Synthesized Biodiesel

GC-MS analysis for the profiling of major fatty acids methyl esters in product (biodiesel) is presented in (Table 6). Palmitic acid (1.448% composition), oleic acid (28.181% composition), stearic acid (0.186%), gondoic acid (4.712%), and erucic acid (65.111%) were the major fatty acid methyl esters identified in sample.
Mumtaz et al. reported the fatty acids profile of E. sativa oil that consists of palmitic acid (2.8%), linoleic acid (10.3%), stearic acid (0.90%), oleic acid (16.3%), linolenic acid (12.56%), and erucic acid (47.7%) [35]. Chakrabarti and Ahmad also investigated and reported palmitic acid (10.2%), stearic acid (1.6%), oleic acid (22.8%), linoleic acid (6.4%), linolenic acid (11.9%), and erucic acid (40.8%) as major fatty acids in E. sativa oil [36].

2.14. Recovery and Reusability of Nano-Biocatalyst

Centrifugation was used to recover the nano-biocatalyst, which was then examined for lipase activity. It was revealed that after first use, there was no change in the CeO2@PDA@A. terreus Lipase activity, i.e., 16.90 U/mg/min (Table 7). So, recovered CeO2@PDA@A. terreus Lipase was reused multiple times for the production of biodiesel and after each reuse, the activity of lipase was assayed, which showed that a considerable decrease in the activity of lipase was observed after five uses, and after seven uses the activity was decreased to 3.9 U/mg/min. The immobilization of lipase is important for the economy of the biodiesel production process, as was observed/revealed by the results. The nano-biocatalyst can be used up to five times without significant decrease in activity, while free enzymes are readily deactivated at high temperatures, with little variations in pH and in the presence of short-chain alcohols, especially methanol [20]. So nano-immobilized lipases can appreciably help in cost reduction of biodiesel production process. After five uses, the biodiesel synthesis rate was reduced, which might have been due to the contact of nano-biocatalyst to organic compounds present in the reaction mixture during biodiesel production or recurring contact with heat. Similar studies have also been reported by Dumri and Hung [14].

2.15. Fuel Characteristics

Biodiesel fuel properties/characteristics were evaluated based on standard methods. The estimated fuel properties values of E. sativa-based biodiesel are presented in (Table 8).

3. Materials and Methods

Research/analytical grade chemicals were used in the whole experimental work. CeNO3.6H2O, NaOH, ethanol, lipase, methanol, acetone, polydopamine, phosphate buffer, tris-HCl buffer, and phenolphthalein were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Eruca sativa seeds were obtained from Directorate of Land Reclamation Agriculture Department, Lahore, Pakistan.

3.1. Preparation of CeO2 Nanorods

CeO2 nanorods were prepared by using the hydrothermal method, during which 7.2 g of NaOH was dissolved in 20.0 mL distilled H2O along with stirring in a beaker. In another beaker, 2.17 g of CeNO3·6H2O was dissolved in 10 mL distilled H2O. Then, the aqueous solution of CeNO3·6H2O was added dropwise in the above solution of NaOH with continuous stirring. After 10 min, white precipitates were obtained. Then, the mixture was filled in 60 mL Teflon lined autoclave and kept in oven for 24 h at 120 °C. Light-yellow-colored product was acquired that was washed with distilled H2O and ethanol. For calcination, the dried product was also kept in furnace for 2 h at 500 °C [37].

3.2. Coating of Dopamine on CeO2 Nanorods

For dopamine coating on CeO2 nanorods, 0.3 g of prepared CeO2 nanorods was dispersed in 20 mL of distilled H2O in a flask, then 20 mL of 20 mM tris buffer having pH 8.5 was added in the flask. 0.1 g of polydopamine was added in the above mixture and stirred for 1 h. The obtained suspension was separated by centrifugation and washed with the tris-HCl buffer to remove the unwanted polydopamine. Addition of polydopamine on nanorods was due to the polymerization of dopamine in the basic conditions [38].

3.3. Characterization of CeO2 Nanorods

CeO2 nanorods were characterized by means of the SEM, XRD, FT-IR, and EDX spectroscopy. XRD pattern for CeO2 nanorods was obtained by using the X’pertpro (PANalyatical) with radiations of Cu k alpha of wavelength 1.54 Å in the scan range of 20–80°, 2θ with scan step size of 0.02. XRD gave the information about the particle size, crystal phase, and dimensions of CeO2 nanorods. The particle size and surface morphology of CeO2 nanorods were confirmed by using the scanning electron microscope (JSM5910, JEOL, Tokyo, Japan) with 30 kV energy, (300,000×) maximum magnification, and 2.3 nm resolving power. The formation of CeO2 nanorods and their modification with polydopamine were performed by using the Cary 630 Agilent FT-IR spectroscopy. EDX gives information about the purity, composition, and elemental analysis of the CeO2 nanorods. EDX spectroscopic analysis was performed using EDX (JSM5910) (INCA200/Oxford instruments, High Wycombe, UK).

3.4. Immobilization of Lipase on Modified Nanorods

0.4 g of lipase was added in the 40 mL of phosphate buffer. The mixture of polydopamine-coated nanorods was slowly added in the lipase mixture with continuous stirring for 3 h at 4 °C. The resulting nano-biocatalyst (CeO2@PDA@A. terreus Lipase) was washed with the phosphate buffer several times to remove the un-reacted lipase and dried in desiccator at low temperature [39]. Figure 9 shows the lipase immobilization process. The immobilization efficiency was revealed by investigating the protein content in solution (before and after immobilization) using Bradford’s method.

3.5. Lipase Activity Assay in Free and Immobilized Form

Titrimetric method was performed for free and immobilized lipase activity assay [40]. The assay mixtures consisted of specific amounts of nano-biocatalysts, along with 10 mL of homogeneous mixture of olive oil in gum acacia, 5 mL of phosphate buffer (pH 7), and 2.0 mL of CaCl2 (0.6%); then, the mixture was incubated at 37 °C for 1 h. Afterward, the reaction was stopped using 20 mL of ethanol:acetone (1:1) followed by the titration with 0.1 N NaOH solution. The lipase activity assay was executed by the titration of fatty acids produced from olive oil after reaction with the lipase.
One unit of lipase activity was defined as “the amount of enzyme which released one micro mole (µmol) of fatty acid per min. under specified assay conditions”.
Lipase units were determined as follows:
Lipase Activity = (∆V × N × 1000)/M × 60
where ΔV = V2 − V1; V1 = vol. of NaOH used against control flask; V2 = vol. of NaOH used against experimental flask; N = normality of NaOH; M(sample) = mass of enzyme extract; and 60 = time of incubation (min) for bacterial lipase.

3.6. Effect of pH and Temperature on Activity of Free and Immobilized Enzyme

The effects of temperature and pH were studied on the activity of free lipase (A. terreus Lipase) and immobilized lipase (CeO2@PDA@A. terreus Lipase). The impact of temperature in the range of 25 to 50 °C and the pH in the range of 5 to 10 (using phosphate buffer) were investigated. Reactions were performed in triplicates.

3.7. Collection of Feedstock, Extraction of Oil, and Its Quality Assessment

The Eruca sativa seeds were subjected to solvent extraction based on Soxhlet technique for the extraction of oil using hexane as solvent. The extracted E. sativa seed oil was analyzed for its initial quality check by evaluating its iodine value, peroxide value, viscosity, acid value, specific gravity, and saponification value by means of AOCS standard protocols [35].

3.8. Central Composite Response Surface Methodology (CCRSM) Experimental Design

CCRSM was used for optimized biodiesel production from CeO2@PDA@A. terreus Lipase catalyzed transesterification of E. sativa oil. Five independent variables—A (methanol/oil ratio), B (biocatalyst amount), C (reaction temperature), D (reaction period), and E (water content)—were optimized within the ranges 3:1–9:1, 1–10%, 20 °C–50 °C, 12–48 h, and 0.2–1%, respectively. Fifty reactions were performed as per CCRD experimental design. In a typical biodiesel production reaction, a conical flask was used containing the reaction mixture oil, methanol, bio-catalyst, and distilled H2O. The reaction conditions were set according to the RSM experimental design. When the reactions were completed, glycerol was separated from biodiesel. Biodiesel was washed with warm water and using the rotary evaporator the residual methanol was recovered under the reduced-pressure conditions.
The model used for response surface methodology is given below:
Y y i e l d = b 0 + i = 1 k b i   X i + i = 1 k b i   X i 2 + i = 1 k   j = 1 i > j b i j   X i X j + e

3.9. Recovery and Recycling of CeO2@PDA@A. Terreus Lipase

After completion of transesterification reactions, the nano-biocatalyst (CeO2@PDA@A. terreus Lipase) was recovered by centrifugation of glycerol and methyl esters. The recovered nano-biocatalyst was washed with water, dried in air, and reused for transesterification to produce biodiesel [41].

3.10. Characterization of Biodiesel

The resulted biodiesel was characterized by FTIR spectroscopic and gas chromatographic techniques. Fuel properties were also determined using standard ASTM methods [42]. Fourier transform infrared spectroscopic analysis was performed by using the Cary 630 Agilent FTIR spectrometer for the E. sativa oil and biodiesel in the range of 400–4000 cm−1.
Gas chromatography-mass spectrometry (GC-MS) analysis was carried out for the estimation of fatty acids methyl ester in the synthesized biodiesel. For this purpose, GCMS QP 2010 instrument dB 5 column of diameter 0.15 mm was used. Sample size and split ratio were 1 µL and 1:100, respectively. Helium was the carrier gas, and 1.2 mL/min flow rate was selected for sample elution. Oven temperature was set between 150 to 250 °C, at rate of 4 °C per minute. MS mass scanning range was 30–550 m/z. NIST MS library of GCMS was used to identify the alkyl esters. Biodiesel produced was further analyzed according to ASTM D methods to estimate the fuel properties to check their suitability as fuel.
Gas chromatography was performed using polar BPX_70 capillary column 30 m × 0.25 mm and FID detector, to determine biodiesel yield/ester content. Helium at 1.5 mL/min flow rate was used as carrier gas. The column oven temperature was kept at 100 °C and then increased to 260 °C, at rate of 10 °C/min. 1 g sample was taken in hexane having methyl heptadecanoate as internal standard, and 1 µL of this mixture was injected in column. FAME (%) yield was determined by using the following formula:
FAME   ( % )   =     AME A A × C × V M × 100
where ∑AME denotes the sum of peak areas of all FAMAs. A is the peak area of methyl heptadecanoate, C is the concentration of the internal standard, V is the volume of the internal standard, and M is mass of biodiesel.

4. Conclusions

In the current study, CeO2@PDA@A. terreus Lipase was utilized for conversion of E. sativa oil into methyl esters and the optimization of process through response surface methodology was carried out. 89.3% FAME yield was obtained by carrying out reactions under optimum conditions of 10%CeO2@PDA@A. terreus Lipase, 6:1 methanol/oil ratio, and 0.6% water at 35 °C for 30 h reaction time. The fuel properties of synthesized product (biodiesel) were compatible with ASTM standards. CeO2@PDA@A. terreus Lipase was ascertained to exhibit efficient catalytic characteristics. Hence, CeO2@PDA@A. terreus Lipase may further be explored for the transesterification of other feedstock to attain a maximum yield of environmentally friendly fuel.

Author Contributions

A.F., M.W.M., and U.R. conceived of this article; the methodology was designed by A.F., M.W.M., and U.R.; the writing—original draft was prepared by A.F.; S.A., T.T., and M.W.M., who also helped with the editing and supervision. H.M. helped in lipase immobilization studies and I.A.N., U.R., and M.R.U.M. reviewed the manuscript for final version improvement. M.I.S. also helped to revise the manuscript. All authors have read and agreed to the published version of the manuscript.


The authors acknowledge their gratitude to King Saud University (Riyadh, Saudi Arabia) for the support of this research through Researchers Supporting Project number (RSP-2019/80).

Conflicts of Interest

The author declares no conflict of interest.


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Figure 1. SEM micrographs of CeO2 nanorods (a) low resolution, 1 µm (b) high resolution, 500 nm.
Figure 1. SEM micrographs of CeO2 nanorods (a) low resolution, 1 µm (b) high resolution, 500 nm.
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Figure 2. EDX analysis of CeO2 nanorods.
Figure 2. EDX analysis of CeO2 nanorods.
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Figure 3. XRD pattern of CeO2 nanorods.
Figure 3. XRD pattern of CeO2 nanorods.
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Figure 4. FTIR of CeO2 and polydopamine-coated CeO2 nanorods.
Figure 4. FTIR of CeO2 and polydopamine-coated CeO2 nanorods.
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Figure 5. (a) The impact of pH on the activity of free lipase and synthesized nano-biocatalyst at 37 °C and (b) the impact of temperature on the activity of free lipase and synthesized nano-biocatalyst at pH 7.
Figure 5. (a) The impact of pH on the activity of free lipase and synthesized nano-biocatalyst at 37 °C and (b) the impact of temperature on the activity of free lipase and synthesized nano-biocatalyst at pH 7.
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Figure 6. Normality plot of residuals for model and correlation graph between predicted and actual values.
Figure 6. Normality plot of residuals for model and correlation graph between predicted and actual values.
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Figure 7. 3D graphical plots describing the impact of significant first-order interactions between (a) enzyme concentration × methanol to oil ratio (b) reaction temperature × methanol to oil ratio (c) water content × methanol to oil ratio (d) reaction temperature × enzyme concentration on biodiesel yield nano-biocatalyst reactions.
Figure 7. 3D graphical plots describing the impact of significant first-order interactions between (a) enzyme concentration × methanol to oil ratio (b) reaction temperature × methanol to oil ratio (c) water content × methanol to oil ratio (d) reaction temperature × enzyme concentration on biodiesel yield nano-biocatalyst reactions.
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Figure 8. FTIR spectrum of E. sativa oil and biodiesel.
Figure 8. FTIR spectrum of E. sativa oil and biodiesel.
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Figure 9. Schematic presentation of lipase immobilization process.
Figure 9. Schematic presentation of lipase immobilization process.
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Table 1. Constituent elements of CeO2 nanorods.
Table 1. Constituent elements of CeO2 nanorods.
Table 2. Physico-chemical characteristics/properties of E. sativa oil.
Table 2. Physico-chemical characteristics/properties of E. sativa oil.
Sr#Physicochemical PropertyUnitsValue
1Density(g/cm3)0.81 ± 0.29
2Peroxide value(meq)3.97 ± 0.163
3Specific gravityg/cm30.75 ± 0.0041
4Acid value(mg KOH/g)1.197 ± 0.0058
5Iodine valuemg/g102.17 ± 0.671
6Saponification valuemg KOH/g5.36 ± 1.70
Table 3. Optimum reaction conditions for biodiesel production.
Table 3. Optimum reaction conditions for biodiesel production.
CatalystReaction Time (hours)Reaction Temp (°C)Methanol: Oil RatioWater (%)Catalyst Conc. (%)Yield (%)
CeO2@PDA@A. terreus Lipase30356:10.61089.3
Table 4. Response surface methodology (RSM) model fitting for optimization of E. sativa oil-based biodiesel.
Table 4. Response surface methodology (RSM) model fitting for optimization of E. sativa oil-based biodiesel.
FeedstockCatalystSelected ModelModel Significance
R2 ValueAdj. R2 ValueLack of Fit
Eruca sativa seed oilCeO2@PDA@A. terreus LipaseQuadratic<0.00010.98020.99030.0728
Table 5. ANOVA for biodiesel yield response.
Table 5. ANOVA for biodiesel yield response.
SourceSum of SquaresDfMean SquareF Valuep-Value
Methanol to oil ratio (A)182.171182.1771.83<0.0001
Enzyme conc. (B)10,283.40110,283.404054.96<0.0001
Reaction temp. (C)6.0116.012.370.1344
Reaction time (B)157.171157.1761.97<0.0001
Water content (E)0.6810.680.270.6091
A × B21.45121.458.460.0069
A × C13.52113.525.330.0283
A × D1.5311.530.600.4434
A × E14.31114.315.640.0243
B × C32.00132.0012.620.0013
B × D9.4619.463.730.0632
B × E0.06110.0610.0240.8776
C × D0.02010.0207.886 × 1030.9298
C × E0.02010.0207.886 × 1030.9298
D × E3.2513.251.280.2668
Lack of Fit66.39223.022.950.0728
Pure Error7.1671.02
Cor Total12,848.3849
Table 6. Major FAMEs of biodiesel.
Table 6. Major FAMEs of biodiesel.
Peak #Retention Time (mints)Fatty AcidPercentage (%)
114.5991Palmitic acid (16:0)1.448 ± 0.012
218.896Oleic acid (18:1)28.181 ± 0.432
317.8101Stearic acid (18:0)0.186 ± 0.002
420.30Gondoic acid (20:1)4.712 ± 0.132
525.9340Erucic acid (22:0)65.111 ± 1.44
Table 7. Reusability of lipase-immobilized on CeO2 nanorods.
Table 7. Reusability of lipase-immobilized on CeO2 nanorods.
CyclesLipase Activity U/mg/min
116.9 ± 0.4
216.7 ± 0.5
316 ± 0.4
412.3 ± 0.7
511.0 ± 0.52
67.1 ± 0.08
74.4 ± 0.1
83.9 ± 0.3
Table 8. Fuel properties of biodiesel.
Table 8. Fuel properties of biodiesel.
PropertiesUnitValueASTM D Std
Flash point°C192.4 ± 1.9>130
Pour point°C−3.22 ± 0.51−15 to 16
Cloud point°C−10 ± 0.018−3 to −12
Fire point °C208.5 ± 1.4>130
Kinematic viscositymm2/s5.2 ± 0.31.9–6.0 mm2/sec

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Fatima, A.; Mumtaz, M.W.; Mukhtar, H.; Akram, S.; Touqeer, T.; Rashid, U.; Ul Mustafa, M.R.; Nehdi, I.A.; Saiman, M.I. Synthesis of Lipase-Immobilized CeO2 Nanorods as Heterogeneous Nano-Biocatalyst for Optimized Biodiesel Production from Eruca sativa Seed Oil. Catalysts 2020, 10, 231.

AMA Style

Fatima A, Mumtaz MW, Mukhtar H, Akram S, Touqeer T, Rashid U, Ul Mustafa MR, Nehdi IA, Saiman MI. Synthesis of Lipase-Immobilized CeO2 Nanorods as Heterogeneous Nano-Biocatalyst for Optimized Biodiesel Production from Eruca sativa Seed Oil. Catalysts. 2020; 10(2):231.

Chicago/Turabian Style

Fatima, Anam, Muhammad Waseem Mumtaz, Hamid Mukhtar, Sadia Akram, Tooba Touqeer, Umer Rashid, Muhammad Raza Ul Mustafa, Imededdine Arbi Nehdi, and Mohd Izham Saiman. 2020. "Synthesis of Lipase-Immobilized CeO2 Nanorods as Heterogeneous Nano-Biocatalyst for Optimized Biodiesel Production from Eruca sativa Seed Oil" Catalysts 10, no. 2: 231.

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