Lipase immobilization and production of fatty acid methyl esters from canola oil using immobilized lipase

Lipase enzyme from Aspergillus oryzae (EC 3.1.1.3) was immobilized onto a micro porous polymeric matrix which contains aldehyde functional groups and methyl esters of long chain fatty acids (biodiesel) were synthesized by transesterification of crude canola oil using immobilized lipase. Micro porous polymeric matrix was synthesized from styrene-divinylbenzene (STY-DVB) copolymers by using high internal phase emulsion technique and two different lipases, Lipozyme TL-100L^® and Novozym 388^®, were used for immobilization by both physical adsorption and covalent attachment. Biodiesel production was carried out with semi-continuous operation. Methanol was added into the reactor by three successive additions of 1:4 M equivalent of methanol to avoid enzyme inhibition. The transesterification reaction conditions were as follows: oil/alcohol molar ratio 1:4; temperature 40 °C and total reaction time 6 h. Lipozyme TL-100L^® lipase provided the highest yield of fatty acid methyl esters as 92%. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation occurred after used repeatedly for 10 consecutive batches with each of 24 h. Since the process is yet effective and enzyme does not leak out from the polymer, the method can be proposed for industrial applications.     

Enzymatic transesterification of soybean oil with ethanol using lipases immobilized on highly crystalline PVA microspheres

Polyvinyl alcohol (PVA) microspheres with different degree of crystallinity were used as solid supports for Rhizomucor miehei lipase immobilization, and the enzyme-PVA complexes were used as biocatalysts for the transesterification of soybean oil to fatty acid ethyl esters (FAEE). The amounts of immobilized enzyme on the polymeric supports were similar for both the amorphous microspheres (PVA4) and the high crystalline microspheres (PVA25). However, the enzymatic activity of the immobilized enzymes was depended on the crystallinity degree of the PVA microspheres: enzymes immobilized on the PVA4 microspheres have shown low enzymatic activity (6.13 U mg⁻¹), in comparison with enzymes immobilized on the high crystalline PVA25 microspheres (149.15 U mg⁻¹). A synergistic effect was observed for the enzyme-PVA25 complex during the transesterification reaction of soybean oil to FAEE: transesterification reactions with free enzyme with the equivalent amount of enzyme that were immobilized onto the PVA25 microspheres (5.4 U) have yielded only 20% of FAEE, reactions with the pure highly crystalline microsphere PVA25 have not yielded FAEE, however reactions with the enzyme-PVA25 complexes have yielded 66.3% of FAEE. This synergistic effect of an immobilized enzyme on a polymeric support has not been observed before for transesterification reaction of triacylglycerides into FAEE. Based on ATR-FTIR, ²³Na- and ¹³C-NMR-MAS spectroscopic data and the interaction of the polymeric network intermolecular hydrogen bonds with the lipases residual amino acids a possible explanation for this synergistic effect is provided.     

Activity of homogeneous and heterogeneous catalysts,spectroscopic and chromatographic characterization of biodiesel: A review

Biofuel has got tremendous attraction for the last decade as an alternative source of energy. Bioethanol and biodiesel are two main products of first generation biofuel. Biodiesel is chemically fatty acid methyl esters prepared from various edible and non-edible oils. It has been used as a substitute to mineral diesel during the last decade. This review is about generation, transesterification, factors affecting transesterification, catalysts (homogeneous and heterogeneous) and physico-chemical characterization of biodiesel by chromatographic and spectroscopic techniques. The alkaline homogeneous catalysts (NaOH or KOH) have been used on commercial scale for production of biodiesel because these are cheap and reaction occurs in less time. The heterogeneous catalysts such as metal oxides, e.g., CaO, MgO, SrO, ZnO, La₂O₃, Mg–Al hydrolalcite have been used for transesterification of vegetable oil due to their easy separation and reuse but these catalysts take more time for completion of reaction. The yield of biodiesel may be affected by alcohol/oil ratio, concentration of catalyst, time required for reaction, temperature free fatty acid moisture. The prepared biodiesel has been characterized by chromatographic techniques like gas chromatography, gas chromatography–mass spectroscopy, high performance liquid chromatography and spectroscopic techniques like nuclear magnetic resonance and infrared spectroscopy.     

Optimization of transesterification conditions for the production of fatty acid methyl ester (FAME) from Chinese tallow kernel oil with surfactant-coated lipase

Surfactant-coated lipase was used as a catalyst in preparing fatty acid methyl ester (FAME) from Chinese tallow kernel oil from Sapium sebiferum (L.) Roxb. syn. Triadica sebifera (L.) small. FAME transesterification was analyzed using response surface methodology to find out the effect of the process variables on the esterification rate and to establish prediction models. Reaction temperature and time were found to be the main factors affecting the esterification rate with the presence of surfactant-coated lipase. Developed prediction models satisfactorily described the esterification rate as a function of reaction temperature, time, dosage of surfactant-coated lipase, ratio of methanol to oil, and water content. The FAME mainly contained fatty acid esters of C16:0, C18:0, C18:1, C18:2, and C18:3, determined by a gas chromatograph. The optimal esterification rate was 93.86%. The optimal conditions for the above esterification ratio were found to be a reaction time of 9.2 h, a reaction temperature of 49 °C, dosage of surfactant-coated lipase of 18.5%, a ratio of methanol to oil of 3:1, and water content of 15.6%. Thus, by using the central composite design, it is possible to determine accurate values of the transesterification parameters where maximum production of FAME occurs using the surfactant-coated lipase as a transesterification catalyst.     

The influence of free fatty acid intermediate on biodiesel production from soybean oil by whole cell biocatalyst

The whole cell of lipase-producing Rhizopus oryzae was employed as biocatalyst for transesterification of soybean oil containing oleic acid. The free fatty acid (FFA) intermediate, playing an important role in the kinetics of transesterification of soybean oil, was thoroughly investigated and characterized. The conversion was more than 97% at the initial FFA content of 5.5%. A high content of FFA could protect the lipase from denaturation. The 34.6 percent of FFA with the optimal 26-mg mL⁻¹ methanol resulted in a specific reaction rate of 420 mg h⁻¹g-dry cell⁻¹. In addition, the methanol/FFA ratio at 0.83-1.7 provides a good indication of the fatty acid methyl esters conversions for different initial FFA contents. In the transesterification process, more FFA intermediate present would become beneficial to conversion of retrograde feedstock to biodiesel. The immediately generated and original FFA content become the major rate-determining factor in the FFA-mixed transesterification process.     

Lipase immobilized carbon nanotubes for conversion of Jatropha oil to fatty acid methyl esters

Lipase has been immobilized on multi-walled carbon nanotubes (MWCNTs) through the reaction of the carboxylated nanotubes with the enzyme in the presence of N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride. Successful immobilization has been verified by infrared spectroscopy and scanning electron microscopy. The synthesized biomaterial has been used for catalyzing the conversion of Jatropha oil to the fatty acid methyl esters (FAME). Progress of the conversion reaction has been monitored with NIR spectroscopy. Gas chromatographic studies have indicated that the use of lipase–MWCNT bioconjuagate leads to almost quantitative conversion of Jatropha oil to the FAME. Around 1 h of the reaction, in the presence of around 15% of catalyst (mass fraction of catalyst on the total mixture), has yielded the desired results. The above used catalyst support has been regenerated 10 times without any adverse effect on the enzyme activity.     

Solid base catalysts for production of fatty acid methyl esters

A solid base catalyst Na₂SiO₃ was prepared by microwave heating. The catalyst was used to catalyze the transesterification reactions for the production of fatty acid methyl esters from cottonseed oil. The optimum conditions of the catalyst preparation and transesterification reactions were investigated by orthogonal experiments. The catalyst with the highest catalytic activity was obtained using microwave power of 640 W, microwave irradiation time of 6 min, catalyst particle size of 60 mesh. The catalyst was characterized with X-ray diffraction (XRD), scanning electron micrographs (SEM), and the results showed the catalyst Na₂SiO₃ has good microstructure. Under the transesterification conditions of methanol/oil molar ratio of 6:1, catalyst dosage of 5%, reaction temperature of 65 °C, reaction time of 100 min and stirring speed of 400 rpm, the yield of methyl esters was 97.6%. The lifetime of the solid base catalysts by different process methods (microwave heating and conventional electric heating) was no significant differences, but microwave heating may be more economical than conventional electric heating.     

A rapid ultrasound-assisted production of biodiesel from a mixture of Karanj and soybean oil

Karanj oil having high free fatty acid was neutralized with a dilute alkali solution and then mixed with soybean oil in different ratios in order to reduce the free fatty acid content significantly. The mixture of the oils was then transesterified with methanol to produce fatty acid methyl ester. The transesterification was carried out using ultrasonication energy of 20 kHz in pulse mode. It was found that up to 60% Karanj oil in the blended mixture could produce good quality biodiesel that met the ASTM standards. However, the lesser content of Karanj oil in the mixture, the lesser the reaction parameters viz. alcohol to oil molar ratio, catalyst concentration, and reaction time. About 99% yield of methyl esters was obtained when the Karanj oil content in the mixture was 20% with a reaction time of 30 min, catalyst concentration 1 wt%, and a temperature of 55°C.     

Biodiesel production from waste cooking oil over sulfonated catalysts

Biodiesel production from waste cooking oil with methanol was carried out in the presence of poly(vinyl alcohol) with sulfonic acid groups (PVA-SO₃H) and polystyrene with sulfonic acid groups (PS-SO₃H), at 60°C. The PVA-SO₃H catalyst showed higher catalytic activity than the PS-SO₃H one. In order to optimize the reaction conditions, different parameters were studied. An increase of waste cooking oil conversion into fatty acid methyl esters with the amount of PVA-SO₃H was observed. When the transesterification and esterification of WCO was carried out with ethanol over PVA-SO₃H, at 60°C, a decrease of biodiesel production was also observed. The WCO conversion into fatty acid ethyl ester increased when the temperature was increased from 60 to 80°C. When different amounts of free fatty acids were added to the reaction mixture, a slight increase on the conversion was observed. The PVA-SO₃H catalyst was reused and recycled with negligible loss in the activity.     

Transesterification of non-edible seed oil for biodiesel production: characterization and analysis of biodiesel

Evaluation of Radish (Raphanus sativus) seed oil (RSO) as a non-edible feedstock for biodiesel production was the main target of the present study. Extraction by solvent disclosed that radish seeds contains 33.50 wt.% of oil. Therefore, biodiesel production from it could be beneficial. Optimized base-catalyzed transesterification of RSO with methanol, ethanol and mixed methanol/ethanol was performed, to produce fatty acid methyl esters, fatty acid ethyl esters and mixed fatty acid methyl ethyl esters, respectively. The optimal yields of the methyl esters, ethyl esters and mixed methyl ethyl esters, were 95.55wt.%, 90.66 wt.% and 93.33 wt.%, respectively when the optimal reaction conditions were attained. Fuel properties of the parent oil were positively changed as consequence of transesterification reaction such that they fulfilled the standard limits as prescribed by ASTM D6751. Moreover, fuel properties of (biodiesels + petro diesel) blends conformed ASTM D7467-17 standards indicating their suitability as a fuel for diesel engines. Biodiesels form RSO were analyzed by thin layer chromatography and FTIR spectroscopy, and both techniques conformed its conversion into its corresponding alkyl esters.     

Biodiesel production from waste sunflower oil by using Amberlyst 46 as a catalyst

In this study, simultaneous transesterification and esterification of high acid value sunflower oil to fatty acid methyl esters was studied using Amberlyst 46 as a heterogeneous catalyst. The influence of reaction conditions such as molar ratio of methanol/oil, reaction time, and reaction temperature was investigated. The highest fatty acids methyl esters yield of 75.8% was obtained in presence of 6 wt% oleic acid content under reaction conditions of 20 wt% Amberlyst 46 catalyst amount, 6/1 methanol/oil molar ratio, reaction temperature of 130°C, and reaction time of 10 h.     

Biocatalytic production of biodiesel from cottonseed oil: Standardization of process parameters and comparison of fuel characteristics

The enzymatic production of biodiesel by transesterification of cottonseed oil was studied using low cost crude pancreatic lipase as catalyst in a batch system. The effects of the critical process parameters including water percentage, methanol:oil ratio, enzyme concentration, buffer pH and reaction temperature were determined. Maximum conversion of 75–80% was achieved after 4 h at 37 °C, pH 7.0 and with 1:15 M ratio of oil to methanol, 0.5% (wt of oil) enzyme and water concentration of 5% (wt of oil). Various organic solvents were tested among which a partially polar solvent (t-butanol) was found to be suitable for the reaction. The major fuel characteristics like specific gravity, kinematic viscosity, flash point and calorific value of the 20:80 blends (B20) of the fatty acid methyl esters with petroleum diesel conformed very closely to those of American Society for Testing Materials (ASTM) standards.     

Acid-catalyzed production of biodiesel from waste frying oil

The reaction kinetics of acid-catalyzed transesterification of waste frying oil in excess methanol to form fatty acid methyl esters (FAME), for possible use as biodiesel, was studied. Rate of mixing, feed composition (molar ratio oil:methanol:acid) and temperature were independent variables. There was no significant difference in the yield of FAME when the rate of mixing was in the turbulent range 100 to 600 rpm. The oil:methanol:acid molar ratios and the temperature were the most significant factors affecting the yield of FAME. At 70 °C with oil:methanol:acid molar ratios of 1:245:3.8, and at 80 °C with oil:methanol:acid molar ratios in the range 1:74:1.9–1:245:3.8, the transesterification was essentially a pseudo-first-order reaction as a result of the large excess of methanol which drove the reaction to completion (99±1% at 4 h). In the presence of the large excess of methanol, free fatty acids present in the waste oil were very rapidly converted to methyl esters in the first few minutes under the above conditions. Little or no monoglycerides were detected during the course of the reaction, and diglycerides present in the initial waste oil were rapidly converted to FAME.     

Biodiesel from corn germ oil catalytic and non-catalytic supercritical methanol transesterification

Transesterifications of grain of corn oil samples in KOH catalytic and in supercritical methanol were studied without using any catalyst. Biodiesel, an alternative biodegradable diesel fuel, is derived from triglycerides by transesterification with methanol and ethanol. The transesterification reaction is affected by the molar ratio of glycerides to alcohol, catalysts, reaction temperature, reaction time and free fatty acids and water content of oils or fats. It was observed that increasing the reaction temperature, especially to supercritical temperatures, had a favorable influence on methyl ester (biodiesel) conversion. The molar ratio of methanol to corn germ oil is also one of the most important variables affecting the yield of methyl esters. Higher molar ratios result in greater ester production in a shorter time. In the transesterification, free fatty acids and water always produce negative effects, since the presence of free fatty acids and water causes soap formation, consumes catalysts, and reduces catalyst effectiveness, all of which result in a low conversion.     

Development of novel Ag/bauxite nanocomposite as a heterogeneous catalyst for biodiesel production

Ag/bauxite nanocomposites have been prepared using in situ reduction of aqueous AgNO₃ solution in a bauxite matrix and investigated for the transesterification of sunflower oil with methanol in order to study their potential as heterogeneous catalysts. The prepared nanocopmosites were characterized by XRD, SEM, EDX, FT-IR, and TG- DTA. The Central Composite Design of the Response Surface Methodology was used to optimize the effect of reaction temperature, reaction time, catalyst loading and methanol to oil molar ratio on the yield of fatty acid methyl esters. The highest yield was obtained at 67 °C reaction temperature, 3 h reaction time, 0.3 wt.% catalyst loading and 9:1 methanol to oil molar ratio. Under the optimal conditions, the methyl ester content was 94% and the catalyst successfully reused for at least 7 cycles without significant deactivation.     

Muskmelon (Cucumis melo) seed oil: A potential non-food oil source for biodiesel production

Methanolysis of muskmelon seed oil was optimized employing RSM (response surface methodology). Four process variables were evaluated at two levels: methanol/oil molar ratio (3:1–12:1), catalyst concentration in relation to oil mass (0.25–1.25 wt % KOH), reaction temperature (25–65 °C) and methanolysis reaction time (20–90 min). Multiple regression analysis was employed to get the quadratic polynomial equation for predicting transesterification using RSM. The result indicated that catalyst concentration and reaction temperature were the important factors that significantly affect the yield of MMOMEs (muskmelon oil methyl esters)/biodiesel. The RSM methodology was used to obtain methyl esters yield (89.5%) were found at following reaction conditions; 5.8:1 methanol-to-oil ratio, 0.79% catalyst concentration, 55 °C reaction temperature and 72.5-min reaction time. There was a linear correlation between observed and predicted values. The biodiesel was analyzed using GC/MS (gas chromatography/mass spectrometry) which indicated four FAMEs (fatty acid methyl esters) (linoleic-, oleic-, palmitic- and stearic acids) as its major components. The FT-IR (fourier transform infraRed) spectrum of MMOMEs was also acquired to ensure the confirmation of methyl esters formation. Fuel properties of MMOMEs were determined and found to satisfy the ASTM D 6751 and EU 14214 specifications.     

Pumpkin (Cucurbita pepo L.) seed oil as an alternative feedstock for the production of biodiesel in Greece

In recent years, the acceptance of fatty acid methyl esters (biodiesel) as a substitute to petroleum diesel has rapidly grown in Greece. The raw materials for biodiesel production in this country mainly include traditional seed oils (cotton seed oil, sunflower oil, soybean oil and rapeseed oil) and used frying oils. In the search for new low-cost alternative feedstocks for biodiesel production, this study emphasizes the evaluation of pumpkin seed oil. The experimental results showed that the oil content of pumpkin seeds was remarkably high (45%). The fatty acid profile of the oil showed that is composed primarily of linoleic, oleic, palmitic and stearic acids. The oil was chemically converted via an alkaline transesterification reaction with methanol to methyl esters, with a yield nearly 97.5 wt%. All of the measured properties of the produced biodiesel met the current quality requirements according to EN 14214. Although this study showed that pumpkin oil could be a promising feedstock for biodiesel production within the EU, it is rather difficult for this production to be achieved on a large scale.     

Synthesis of fatty acid methyl esters via the transesterification of waste cooking oil by methanol with a barium-modified montmorillonite K10 catalyst

The transesterification of waste cooking oil (WCO) with methanol to produce fatty acid methyl esters (FAMEs) in the presence of barium-modified montmorillonite K10 (BMK10) catalyst was investigated in a batch reactor. The influence of the reaction parameters on the yield of FAME was investigated. The highest value of 83.38% was obtained with 3.5 wt% catalyst loading at 150 °C with a methanol: oil molar ratio of 12:1 during a reaction time of 5 h. BMK10 is a promising low-cost catalyst for the transesterification of WCO to produce FAME.     

Biodiesel production using gel‐type cation exchange resin at different ionic forms

In this study, a strong acidic‐type cation exchange resin was used in the transesterification of corn oil to fatty acid methyl esters (FAME). The gel‐type cation exchange resin (Purolite‐PD206) was used in H⁺ and Na⁺ forms to utilize ion‐exchange resin as effective heterogeneous catalyst in the production of biodiesel. Effect of ionic forms of ion exchange resin on free fatty acid (FFA) conversion and composition was investigated by using different amounts of ion exchange resin (12, 16, and 20 wt%), various mole ratios of methanol to oil (1:6, 1:12, and 1:18 mol/mol), reaction temperatures (63, 65, and 67°C), and reaction time (24, 36, and 48 h) during transesterification reaction. The highest FFA conversions of 73.5% and 79.45% were obtained at conditions of 20 wt% of catalyst, 65°C of reaction temperature, 18:1 as methanol to oil ratio, and 48 h of reaction time for H⁺ and Na⁺ forms of ion exchange resin, respectively. These results were obtained from regression equations established by using analysis of variance (ANOVA) model according to the experimental results of selected parameters. Gas chromatography analysis revealed that FAME is mainly composed of C16:0 (palmitic), C18:1 (oleic), and C18:2 (linoleic) acids of methyl ester.     

Transesterification of Prunus armeniaca oil using calcium methoxide as a catalyst: Identification of methyl esters and proposed mechanistic pathways

The utilization of nonedible feedstocks for biodiesel production is getting top priority in the recent years, as they do not interfere with the global food economy. In the present study, calcium methoxide (Ca(OCH₃)₂) catalyst was synthesized for the production of biodiesel from Prunus armeniaca oil via transesterification reaction. Under optimized reaction conditions, the biodiesel yield of 89.93% was achieved and catalyst could be reused for four times with slight loss in activity. The fatty acid methyl esters in the produced biodiesel were detected by gas chromatography/mass spectrometry (GC/MS), and six fragments were identified based on mass-to-charge ratio (m/z) at different retention time. The fuel properties of the biodiesel obtained were in conformity with the ASTM standards.