Enzymatic Synthesis of Biodiesel via Alcoholysis of Palm Oil

The enzymatic alcoholysis of crude palm oil with methanol and ethanol was investigated using commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM). The effect of alcohol (methanol or ethanol), molar ratio of alcohol to crude palm oil, and temperature on biodiesel production was determined. The best ethyl ester yield was about 25 wt.% and was obtained with ethanol/oil molar ratio of 3.0, temperature of 50 °C, enzyme concentration of 3.0 wt.%, and stepwise addition of the alcohol after 4 h of reaction. Experiments with 1 and 3 wt.% of KOH and 3 wt.% of MgO were carried out to compare their catalytic behavior with the enzymatic transesterification results. The commercial immobilized lipase, Lipozyme TL IM, showed the best catalytic performance.     

Optimization of enzymatic production of biodiesel from castor oil in organic solvent medium

We studied the production of fatty acid ethyl esters from castor oil using n-hexane as solvent and two commercial lipases, Novozym 435 and Lipozyme IM, as catalysts. For this purpose, a Taguchi experimental design was adopted considering the following variables: temperature (35–65°C), water (0–10 wt/wt%), and enzyme (5–20 wt/wt%) concentrations and oil-to-ethanol molar ratio (1∶3 to 1∶10). An empirical model was then built so as to assess the main and cross-variable effects on the reaction conversion and also to maximize biodiesel production for each enzyme. For the system containing Novozym 435 as tatalyst the maximum conversion obtained was 81.4% at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶10. When the catalyst was Lipozyme IM, a conversion as high as 98% was obtained at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶3.     

Kinetics of enzyme-catalyzed alcoholysis of soybean oil in n-hexane

This work investigated the production of fatty acid ethyl esters (FAEEs) from soybean oil using n-hexane as solvent and two commercial lipases as catalysts, Novozym 435 and Lipozyme IM. A Taguchi experimental design was adopted considering the variables temperature (35–65°C), addition of water (0–10 wt/wt%), enzyme (5–20 wt/wt%) concentration, and oil-to-ethanol molar ratio (1:3–1:10). It is shown that complete conversion in FAEE is achieved for some experimental conditions. The effects of process variables on reaction conversion and kinetics of the enzymatic reactions are presented for all experimental conditions investigated in the factorial design.     

13C NMR quantification of mono and diacylglycerols obtained through the solvent‐free lipase‐catalyzed esterification of saturated fatty acids

In the present investigation, we studied the enzymatic synthesis of monoacylglycerols (MAG) and diacylglycerols (DAG) via the esterification of saturated fatty acids (stearic, palmitic and an industrial residue containing 87% palmitic acid) and glycerol in a solvent‐free system. Three immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) and different reaction conditions were evaluated. Under the optimal reaction conditions, esterifications catalyzed by Lipozyme RM IM resulted in a mixture of MAG and DAG at high conversion rates for all of the substrates. In addition, except for the reaction of industrial residue at atmospheric pressure, all of these products met the World Health Organization and European Union directives for acylglycerol mixtures for use in food applications. The products were quantified by ¹³C NMR, with the aid of an external reference signal which was generated from a sealed coaxial tube filled with acetonitrile‐d3. After calibrating the area of this signal using the classical external reference method, the same coaxial tube was used repeatedly to quantify the reaction products. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of Geraniol Esters in a Continuous-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modeling

With increasing demand for perfumes, flavors, beverages, and pharmaceuticals, the various associated industries are resorting to different approaches to enhance yields of desired compounds. The use of fixed-bed biocatalytic reactors in some of the processes for making fine chemicals will be of great value because the reaction times could be reduced substantially as well as high conversion and yields obtained. In the current study, a continuous-flow packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed for synthesis of various geraniol esters. Optimization of process parameters such as biocatalyst screening, effect of solvent, mole ratio, temperature and acyl donors was studied in a continuous-flow packed-bed reactor. Maximum conversion of ~ 87% of geranyl propionate was achieved in 15 min residence time at 70 °C using geraniol and propionic acid with a 1:1 mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested. Ternary complex mechanism with propionic acid inhibition was found to fit the data.     

Synthesis of isoamyl laurate and isoamyl stearate in supercritical carbon dioxide

The synthesis of isoamyl laurate and isoamyl stearate was studied in supercritical carbon dioxide with three lipases, Novozym 435, Lipolase 100T, and Candida rugosa. The maximum conversion of 37% and 53%, respectively for isoamyl laurate and isoamyl stearate was obtained when Novozym 435 was used. The effect of various parameters such as molar ratio of alcohol to acid, presence of water, time and temperature was investigated. An optimum temperature of 40–45°C was observed for all reactions. The kinetics of reactions was fast and equilibrium was achieved in 2–3 h. Although the presence of excess alcohol did not reduce conversion, excess water reduced conversion significantly.     

Development of Batch and Continuous Processes on Biodiesel Production in a Packed-Bed Reactor by a Mixture of Immobilized Candida rugosa and Rhizopus oryzae Lipases

In this study, transesterification and esterification were investigated in batch and continuous process using immobilized Candida rugosa and Rhizopus oryzae lipases. In the case of batch process, stepwise reaction method was investigated to prevent the lipase deactivation. Reaction conditions were as follows: temperature, 45 °C; agitation speed, 250 rpm; enzyme concentration, 20%; and water contents 10%. And then, conversion yield was 98.33% at 4 h. In the case of continuous process, circulation and long-term continuous system were investigated for development of efficient mass transfer system. Optimal reaction conditions were as follows: temperature, 45 °C; flow rate, 0.8 mL/min; and water contents, 10%. And then, conversion yield of biodiesel was 97.98% at 3 h. Especially, the maximum conversion yield using a mixture of immobilized lipases exceeded over 90% for 108 h in long-term continuous system under optimal reaction conditions (45 °C; flow rate, 0.8 mL/min; and water contents, 10%). These results should help in determining the best method for the biodiesel production and improving the design and operation of large scale by enzymatic systems.     

Optimization of lipase-catalyzed transesterification of lard for biodiesel production using response surface methodology

Biodiesel, an alternative diesel fuel made from renewable biological resources, has become more and more attractive recently. Combined use of two immobilized lipases with complementary position specificity instead of one lipase is a potential way to significantly reduce cost of lipase-catalyzed biodiesel production. In this study, the process of biodiesel production from lard catalyzed by the combined use of Novozym435 (non-specific) and Lipozyme TLIM (1,3-specific) was optimized by response surface methodology. The optimal reaction conditions were 0.04 of amount of lipase/oil (w/w), 0.49 of proportion of Novozym435/total lipases (w/w), 0.55 of quantity of tert-butanol/oil (v/v), 5.12 of quantity of methanol/oil (mol/mol), and 20 h of reaction time, by which 97.2% of methyl ester (ME) yield was attained, very close to the predicted value (97.6%). This optimal reaction condition could be true of other similar reactions with plant and animal oil resources; their ME yield could be higher than 95%. The lipases regenerated by washing with organic solvent after each reaction cycle could be continuously reused for 20 cycles without any loss of activity, exhibiting very high manipulation stability.     

Production of FAME and FAEE via Alcoholysis of Sunflower Oil by Eversa Lipases Immobilized on Hydrophobic Supports

The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.     

Monitoring lipase-catalyzed methanolysis of sunflower oil by reversed-phase high-performance liquid chromatography: elucidation of the mechanisms of lipases

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 210 nm was used to monitor the formation of the major compounds during the lipase-catalyzed transesterification reaction of sunflower oil with methanol. Individual triacylglycerols, diacylglycerols, monoacylglycerols as well as fatty acids and their corresponding methyl esters were separated using acetonitrile/acetone as a mobile phase and a combined linear gradient-isocratic-step gradient-isocratic elution procedure. Another relatively short method consisting of a linear gradient elution followed by an isocratic elution gave similar results, yet with lower resolution. HPLC/mass spectrometry with an ion trap analyzer and atmospheric pressure chemical ionization source was used for the identification of the individual compounds. Individual calibration curves obtained with UV detection at 210 nm were found to be of use for quantitative analyses of double-bond containing methyl esters and acylglycerols. The use of the RP-HPLC methods in the elucidation of the mechanisms of three immobilized lipases, namely Lipozyme TL IM, Lipozyme RM IM and Novozym 435, in biodiesel production was described.     

Complementary regioselective esterification of non-reducing oligosaccharides catalyzed by different hydrolases

The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is described. The lipases from Candida antarctica B (Novozym 435) and Thermomyces lanuginosus (Lipozyme TL IM) and the alkaline protease from Bacillus licheniformis (subtilisin Carlsberg) have been used with each carbohydrate to obtain different regioisomers. By using the sugars in their amorphous form, complete solubility is achieved in the reaction media (tert-butanol/pyridine mixtures for the lipases and pyridine for the protease) and high isolated yields of the corresponding monoesters are obtained. Good to excellent regioselectivity is observed for all the enzymes, showing a final complementary picture respect to the primary hydroxyls of the oligosaccharides studied.     

Enzymatic Biodiesel Synthesis in Semi-Pilot Continuous Process in Near-Critical Carbon Dioxide

A semi-pilot continuous process (SPCP) for enzymatic biodiesel synthesis utilizing near-critical carbon dioxide (NcCO₂) as the reaction medium was developed with the aim of reducing the reaction time and alleviating the catalyst inhibition by methanol. Biodiesel synthesis was evaluated in both lab-scale and semi-pilot scale reactors (batch and continuous reactors). In a SPCP, the highest conversion (～99.9 %) in four and a half hours was observed when three-step substrate (methanol) addition (molar ratio [oil/methanol]?=?1:1.3) was used and the reaction mixture containing enzyme (Lipozyme TL IM, 20 wt.% of oil) was continuously mixed (agitation speed?=?300 rpm) at 30 °C and 100 bar in a CO₂ environment. The biodiesel produced from canola oil conformed to the fuel standard (EU) even without additional downstream processing, other than glycerol separation and drying.     

A kinetic study on methylation of naphthalene over Fe/ZSM-5 zeolite catalysts

2,6-Dimethylnaphthalene is an important dimethylnaphthalene isomer which can be used in the production of polyethylene naphthalate. The novelty of this study is to reveal Langmuir–Hinshelwood and Eley–Rideal reaction rate equations for the methylation of naphthalene over Fe/ZSM-5 zeolite catalysts besides the proposed reaction rate equation. To investigate the kinetics and mechanisms of naphthalene methylation, the methylation experiments were carried out in a gas–solid catalytic fixed-bed reactor in the presence of Fe/ZSM-5 zeolite catalysts at two different temperatures (450 and 500 °C) and five different weight hourly space velocities (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 h^?1). A naphthalene:methanol:1,2,4-trimethyl benzene mixture having a 1:3:10 molar ratio was used as a feed stream. The methylation products were identified by using GC–MS. For the methylation kinetics of naphthalene, the reaction rates depending on the naphthalene and methanol concentration were determined. Furthermore, the effects of temperature and weight hourly space velocity on the conversion of naphthalene, the selectivity of 2,6-dimethylnaphthalene, and the ratio of 2-methylnaphthalene/1-methylnaphthalene were determined. The results of this study demonstrate that the Langmuir–Hinshelwood reaction mechanism for naphthalene methylation is more compatible at 450 °C and the Eley–Rideal reaction mechanism at 500 °C. Moreover, in addition to 2,6-dimethylnaphthalene, other dimethylnaphthalene and tri-methylnaphthalene isomers were formed in the methylation of naphthalene. The conversion of naphthalene reached approximately 70%. Moreover, the highest selectivity of 2,6-dimethylnaphthalene was almost 40%. The ratios of 2-methylnaphthalene/1-methylnaphalene demonstrate that the methylation of naphthalene to 2-methynaphthalene is much higher than to 1-methynaphthalene.     

Enzymatic conversion of waste cooking oils into alternative fuel—Biodiesel

Production of biodiesel from pure oils through chemical conversion may not be applicable to waste oils/fats. Therefore, enzymatic conversion using immobilized lipase based on Rhizopus orzyae is considered in this article. This article studies this technological process, focusing on optimization of several process parameters, including the molar ratio of methanol to waste oils, biocatalyst load, and adding method, reaction temperature, and water content. The results indicate that methanol/oils ratio of 4, immobilized lipase/oils of 30 wt% and 40°C are suitable for waste oils under 1 atm. The irreversible inactivation of the lipase is presumed, and a stepwise addition of methanol to reduce inactivation of immobilized lipases is proposed. Under the optimum conditions the yield of methyl esters is around 88–90%.     

Biodiesel Production from Various Oils Under Supercritical Fluid Conditions by Candida antartica Lipase B Using a Stepwise Reaction Method

In this study, we evaluate the effects of various reaction factors, including pressure, temperature, agitation speed, enzyme concentration, and water content to increase biodiesel production. In addition, biodiesel was produced from various oils to establish the optimal enzymatic process of biodiesel production. Optimal conditions were determined to be as follows: pressure 130 bar, temperature 45 °C, agitation speed 200 rpm, enzyme concentration 20%, and water contents 10%. Among the various oils used for production, olive oil showed the highest yield (65.18%) upon transesterification. However, when biodiesel was produced using a batch system, biodiesel conversion yield was not increased over 65%; therefore, a stepwise reaction was conducted to increase biodiesel production. When a reaction medium with an initial concentration of methanol of 60 mmol was used and adjusted to maintain this concentration of methanol every 1.5 h during biodiesel production, the conversion yield of biodiesel was 98.92% at 6 h. Finally, reusability was evaluated using immobilized lipase to determine if this method was applicable for industrial biodiesel production. When biodiesel was produced repeatedly, the conversion rate was maintained at over 85% after eight reuses.     

Polyesters from Macrolactones Using Commercial Lipase NS 88011 and Novozym 435 as Biocatalysts

The demand for environmentally friendly products allied with the depletion of natural resources has increased the search for sustainable materials in chemical and pharmaceutical industries. Polyesters are among the most widely used biodegradable polymers in biomedical applications. In this work, aliphatic polyesters (from globalide and ω-pentadecalactone) were synthesized using a new commercial biocatalyst, the low-cost immobilized NS 88011 lipase (lipase B from Candida antarctica immobilized on a hydrophobic support). Results were compared with those obtained under the same conditions using a traditional, but more expensive, commercial biocatalyst, Novozym 435 (lipase B from C. antarctica immobilized on Lewatit VP OC). When NS 88011 was used in the polymerization of globalide, longer reaction times (240 min)—when compared to Novozym 435—were required to obtain high yields (80–90 wt%). However, higher molecular weights were achieved. When poly(ω-pentadecalactone) was synthesized, high yields and molecular weights (130,000 g mol^?1) were obtained and the enzyme concentration showed strong influence on the polyester properties. This is the first report describing NS 88011 in polymer synthesis. The use of this cheaper enzymatic preparation can provide an alternative for polyester synthesis via enzymatic ring-opening polymerization.     

Lipozyme 435-Mediated Synthesis of Xylose Oleate in Methyl Ethyl Ketone

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates’ molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.     

New chiral building blocks from acetovanillone using lipase A and B from Candida antarctica

Acetovanillone has been used as the starting material for the synthesis of a series of secondary alcohols, which were resolved by lipase catalyzed esterification. 1-(4-Benzyloxy-3-methoxyphenyl)ethanol was efficiently resolved using immobilized lipase B from Candida antarctica (Novozym 435, CAL-B), whereas immobilized lipase A from C. antarctica (Novozym 735, CAL-A) was the lipase of choice for the resolution of the corresponding 2-bromo- and 2-chloro-derivatives. The enantioenriched alcohols are new building blocks for potential use in the synthesis of bioactive compounds.     

Synergism of the catalytic effect of nanosized gold-nickel catalysts in the reaction of selective acetylene hydrogenation to ethylene

Gold and nickel nanoclusters immobilized on aluminum oxide exhibited high activity in the reaction of acetylene hydrogenation to ethylene with molecular hydrogen at temperatures from 20 to 64°C. The reaction selectivity on Au-Ni nanocomposites with metal concentrations from 0.02 to 0.36 wt % was no lower than 99.99%, and the stability of catalysts was retained for at least 12 h. The simultaneous presence of gold and nickel in the systems resulted in the synergism of their catalytic effects: acetylene conversion on layer-by-layer immobilized metal clusters was higher than the total conversion on individual gold and nickel clusters. The dependence of the conversion of acetylene on bimetallic catalysts on the Au : Ni ratio exhibited an extremal character.     

Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents

The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl methyl ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of commercial lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that observed in other largely employed organic solvents [toluene and tert-butyl methyl ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E = 232) for the resolution of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, d-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resolution of racemic sulcatol, where high activity and enantioselectivity were obtained (E ≥ 1000). For the resolution of the racemic primary alcohol α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of two ionic liquids (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (v/v)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (v/v)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], respectively. However, in all cases ILs caused a decrease of enzyme activity.