Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa

Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x₁), temperature (x₂) and methanol to canola oil molar ratio (x₃) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x₁ = 6.3 mL/min, x₂ = 38 °C and x₃ = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432 h), where the methyl ester yield was maintained above 97%.     

Enzyme catalyzed transesterification of waste cooking oil with dimethyl carbonate

The detrimental effects of waste cooking oil on sewer system attracted attention toward its proper management and reusing this waste oil for making biodiesel provides commercial and environmental advantage. In the present study, biodiesel has been successfully produced from waste cooking oil and dimethyl carbonate by transesterification, instead of the conventional alcohol. In this optimization study, the effect of various reaction conditions such as solvent, time and temperature, molar ratio of DMC to oil, enzyme loading and reusability, on the yield of fatty acid methyl ester (FAME) has been studied. The Maximum conversion of FAMEs achieved was 77.87% under optimum conditions (solvent free system, reaction time of 24 h, 60 °C, molar ratio of DMC to oil 6:1, catalyst amount 10% Novozym 435 (based on the oil weight)). Moreover, there was no obvious loss in the conversion after lipases were reused for 6 batches under optimized conditions.     

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.     

Effect of water on lipase NS81006-catalyzed alcoholysis for biodiesel production

Biodiesel production catalyzed by free lipase has been drawing attention for its lower cost and faster reaction rate compared to immobilized lipase. It has been found that free lipase NS81006 could efficiently catalyze alkyl esters production and a certain amount of water is demonstrated to be necessary for the catalytic process. The effect of water content on liquid lipase NS81006-mediated methanolysis and ethanolysis for biodiesel production was first explored respectively in this paper. It was found that with water content ranging from 3% to 10% (based on oil weight), there was no significant difference in the final alkyl ester yield either in NS81006-mediated methanolysis or ethanolysis process, while the quality of biodiesel varied obviously. The acid value as well as the contents of monoglyceride and diglyceride were much lower in the lower water-containing system. With the water content decreasing from 10% to 3%, the acid value reduced from 8.24 to 4.89 mg KOH/g oil, and the content of MAG and DAG dropped to 0.31 and 0.22, from 0.62 and 0.74, respectively. Lipase could maintain rather good stability with proper alcohol adding strategy and the gradual reduction in biodiesel yield in the repeated uses resulted from the accumulation of by-product glycerol. The continuous running of lipase-mediated methanolysis of waste cooking oil was successfully realized at 30L reactor and a final methyl ester yield of over 90% could be obtained.     

Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles

A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst was developed for biodiesel fuel production by plant oil methanolysis. Lipase-producing Rhizopus oryzae cells were immobilized within 6 mm × 6 mm × 3 mm cuboidal polyurethane foam biomass support particles (BSPs) during batch cultivation in a 20-l air-lift bioreactor. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. Using a high flow rate for the reaction mixture in the PBR caused exfoliation of the immobilized cells from the BSPs, while the inefficient mixing of the reaction mixture at low flow rates allowed the BSPs to be covered with a hydrophilic layer of high methanol concentration, leading to a significant decrease in lipase activity. A high methyl ester content of over 90% was achieved at a flow rate of 25 l/h in the first cycle of repeated batch methanolysis and a high value of around 80% was maintained even after the tenth cycle. Comparison with methanolysis reaction in a shaken bottle suggested that the PBR enhances repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The process presented here is therefore considered to be promising for industrial biodiesel-fuel production.     

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of pNPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.     

Two-step enzymatic selective synthesis of water-soluble ketoprofen–saccharide conjugates in organic media

Ketoprofen–saccharide conjugates were synthesized by selectively enzymatic hydrolysis and acylation. Firstly, the (S)-ketoprofen vinyl ester was prepared by enzymatic hydrolysis of (R,S)-ketoprofen vinyl ester. Then enzymatic transesterification of (S)-ketoprofen vinyl ester with a series of saccharides were performed by the catalysis of a commercial protease from Bacillus licheniformis (BLP) in organic medium mixture of pyridine and tert-butanol. The ketoprofen was selectively conjugated onto the primary hydroxyl group of saccharides and with high yield after 72 h. Partition coefficient determination showed that all the products have better water solubility than parent ketoprofen. Chemical hydrolysis experiment indicated that 50% ketoprofen could be release from ketoprofen glucoside and maltoside in aqueous buffer (pH 7.4) within 48 h.     

Potential use of whole cell lipase from a newly isolated Aspergillus nomius for methanolysis of palm oil to biodiesel

A highly active whole cell lipase (WCL) for efficient methanolysis of palm oil (PO) to biodiesel (BD) was prepared by isolation, cultivation and immobilization of lipase producing fungi. Fungi were screened from soil and the best isolate (PDA-6) identified as Aspergillus nomius exhibited maximum WCL methanolysis activity (1.4 g h⁻¹ g⁻¹) when inexpensive waste cooking oil was used as carbon source. The maximum BD yield with PDA-6 WCL reached 95.3% after 40 h at a lipase load 10% (w/w) of PO and methanol to PO molar ratio 5:1. The immobilization of PDA-6 cells within biomass suspended particle (BSP) made of polyurethane foam improved the repeated use of WCL and the remaining activity after 10 cycles was 88.2%. The PDA-6 WCL was more active in methanolysis of PO to BD than most WCLs previously reported. The newly isolated A. nomius is not only potential for producing WCL but also utilizing waste cooking oil.     

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from Thermomyces lanuginosus

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.     

Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435

The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production.     

Decanter cake waste as a renewable substrate for biobutanol production by Clostridium beijerinckii

The aim of this study was to determine if decanter cake waste from a palm oil mill could be used as a renewable substrate for biobutanol production. Decanter cake waste was first hydrolyzed to fermentable sugars by nitric acid and detoxified by activated-charcoal. The detoxified hydrolysate supplemented with whey protein and ammonium sulfate as cheap nitrogen sources, was used for butanol production by growing cells of Clostridium beijerinckii. The detoxified hydrolysate was also used as a co-substrate for direct conversion of butyric acid to butanol in a nitrogen-free medium. By these two steps, C. beijerinckii produced 3.42 g/L of butanol with a yield of 0.28 C-mol butanol/C-mol carbon in the first step and produced 6.94 g/L of butanol with a yield of 0.47 C-mol butanol/C-mol carbon in the second step. This study has showed that decanter cake waste could serve as a low-cost substrate for biobutanol production.     

Evaluation of insecticidal activity of the essential oil of Allium chinense G. Don and its major constituents against Liposcelis bostrychophila Badonnel

Water-distilled essential oil from the dried bulbs of Allium chinense (Liliaceae) was analyzed by gas chromatography–mass spectrometry (GC–MS). Eighteen compounds, accounting for 98.4% of the total oil, were identified and the main components of the essential oil of A. chinense were methyl allyl trisulfide (30.7%), dimethyl trisulfide (24.1%), methyl propyl disulfide (12.8%) and dimethyl disulfide (9.6%) followed by methyl allyl disulfide (3.4%) and methyl propyl trisulfide (3.6%). The essential oil exhibited contact toxicity against the booklice (Liposcelis bostrychophila) with an LC₅₀ value of 441.8 μg/cm² while the two major constituents, dimethyl trisulfide and methyl propyl disulfide had LC₅₀ values of 153.0 μg/cm² and 738.0 μg/cm² against the booklice, respectively. The essential oil of A. chinense possessed strong fumigant toxicity against the booklice with an LC₅₀ value of 186.5 μg/l while methyl allyl trisulfide (LC₅₀ = 90.4 μg/l) and dimethyl trisulfide (LC₅₀ = 114.2 μg/l) exhibited stronger fumigant toxicity than methyl propyl disulfide (LC₅₀ = 243.4 μg/l) and dimethyl disulfide (LC₅₀ = 340.8 μg/l) against the booklice. The results indicated that the essential oil and its major constituents have potential for development into natural insecticides or fumigants for control of insects in stored grains.     

Enhancement of Novozym-435 catalytic properties by physical or chemical modification

Physical (ionic exchange of ionic polymers) or chemical (aminoethylamidation, succinylation, hydroxyethylamidation) modifications of Novozym 435 have been performed and the resulting biocatalysts have been assayed in diverse reactions. The coating of the immobilized enzyme with dextran-sulphate via ionic exchange permitted to increase the asymmetric factor of the biocatalyst from A = 13 (ee = 83%) to 24 (ee > 90%) in the hydrolysis of 3-phenylglutaric acid dimethyl diester, producing the (R)-monomethyl ester. The chemical succinylation of Novozym 435 permitted to enhance the biocatalyst enantiospecificity from E = 1 to 13 in the hydrolysis of (±)-mandelic acid methyl ester. In the hydrolysis of (±)-2-O-butyryl-2-phenylacetic acid, the enantiospecificity of Novozym 435 was very high towards the S-enantiomer (E > 100) but it was inverted after the chemical hydroxyethylamidation of the immobilized enzyme (E = 6.6 towards R-enantiomer).Thus, these simple protocols seem to be a very powerful tool to generate a library of biocatalysts from Novozym 435 with very different catalytic properties.     

Optimized synthesis of (Z)-3-hexen-1-yl caproate using germinated rapeseed lipase in organic solvent

(Z)-3-hexen-1-yl esters are important green top-note components of food flavors and fragrances. Effects of various process conditions on (Z)-3-hexen-1-yl caproate synthesis employing germinated rapeseed lipase acetone powder in organic solvent were investigated. Rapeseed lipase catalyzed ester formation more efficiently with non-polar compared to polar solvents despite high enzyme stability in both types of solvents. Maximum ester yield (90%) was obtained when 0.125 M (Z)-3-hexen-1-ol and caproic acid were reacted at 25 °C for 48 h in the presence of 50 g/L enzyme in heptane. Enzyme showed little sensitivity towards a_w with optimum yield at 0.45, while added water did not affect ester yield. Esterification reduced by increasing molecular sieves (>0.0125%, w/v). The highest yields of caproic acid were obtained with isoamyl alcohol (93%) followed by butanol and (Z)-3-hexen-1-o1 (88%) respectively reflecting the enzyme specificity for straight and branched chain alcohols. Secondary alcohols showed low reactivity, while tertiary alcohol had either very low reactivity or not esterified at all. A good relationship has been found between ester synthesis and the solvent polarity (log P value); while no correlation for the effect of solvents on residual enzyme activity was observed. It may be concluded that germinated rapeseed lipase is a promising biocatalyst for the synthesis of valuable green flavor note compound. The enzyme also showed a wide range of temperature stability (5–50 °C).     

Biodiesel production from rice bran oil and supercritical methanol

In this study, production of biodiesel from low cost raw materials, such as rice bran and dewaxed-degummed rice bran oil (DDRBO), under supercritical condition was carried out. Carbon dioxide (CO₂) was employed as co-solvent to decrease the supercritical temperature and pressure of methanol. The effects of different raw materials on the yield of biodiesel production were investigated. In situ transesterification of rice bran with supercritical methanol at 30 MPa and 300 °C for 5 min was not a promising way to produce biodiesel because the purity and yield of fatty acid methyl esters (FAMEs) obtained were 52.52% and 51.28%, respectively. When DDRBO was reacted, the purity and yield were 89.25% and 94.84%, respectively. Trans-FAMEs, which constituted about 16% of biodiesel, were found. They were identified as methyl elaidate [trans-9], methyl linoleaidate [trans-9, trans-12], methyl linoleaidate [cis-9, trans-12], and methyl linoleaidate [trans-9, cis-12]. Hydrocarbons, which constituted about 3% of the reaction product, were also detected.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99-125

In this paper, immobilized lipase catalyzed biodiesel production from lard was studied. Using Candida sp. 99-125, the effect of temperature, water content, enzyme amount, solvent and three-step methanolysis were investigated. The optimal conditions for processing 1 g of lard were: 0.2 g immobilized lipase, 8 ml n-hexane as solvent, 20% water based on the fat weight, temperature 40 °C, and three-step addition of methanol. As a result, the fatty acid methyl esters (FAMEs) yield was 87.4%. The lipase was proved to be stable when used repeatedly for 180 h.     

Preparation of biodiesel from waste oils catalyzed by a Brønsted acidic ionic liquid

Preparation of biodiesel from waste oils catalyzed by a novel Brønsted acidic ionic liquid with an alkane sulfonic acid group was investigated. The acidity and the activity of the ionic liquid were very low at lower temperature when the ionic liquid was crystalloid; they recovered at higher temperature when the crystallized ionic liquid was liquefied. When methanol:oils:catalyst molar ratios were 12:1:0.06, the yield of fatty acid methyl esters can reach 93.5% after the reaction of acidic oil with methanol had taken place at 170 °C for 4 h. In addition, the ionic liquid had a good reusability and can be easily separated from the biodiesel by simple decantation.     

Biochemical characteristics of three feruloyl esterases with a broad substrate spectrum from Bacillus amyloliquefaciens H47

Feruloyl esterases (Faes) are a subclass of the carboxylic esterases that hydrolyze the ester bonds between ferulic acid and polysaccharides in plant cell walls. Until now, the biochemical characteristics of FAEs from Bacillus spp. have not been reported. In this study, a strain with high activity of FAEs, Bacillus amyloliquefaciens H47 was screened from 122 Bacillus – type strains. Finally, three FAEs (BaFae04, BaFae06, and BaFae09) were identified. Comparing with other bacterial FAEs, these novel FAEs exhibited low sequence identities (less than 30%). The profiles of 52 esterase substrates showed that the three FAEs had a broad substrate spectrum and could effectively hydrolyze several common FAE substrates, such as methyl ferulate, ethyl caffeate, methyl p-coumarate, methyl sinapate, and chlorogenic acid. Furthermore, the three FAEs also can release ferulic acid from destarched wheat bran. They showed maximal activity with an optimal pH of 8.0 at 30 °C, 35 °C, and 40 °C, respectively. BaFae04 showed high stability in the temperature range of 25–60 °C for 1 h and retained 59% of its activity at 60 °C. The present study displays some useful characteristics of FAEs for potential industrial application and contributes to our understanding of FAEs.     

Genetic variability studies for morphological and qualitative attributes among Jatropha curcas L. accessions grown under subtropical conditions of North India

Evaluation of Jatropha curcas germplasm comprising seven accessions indicated a wide range of variability in vegetative growth and other qualitative attributes. These characteristics could be harnessed in future improvement programme of Jatropha curcas. Seed yield/plant had a positive and significant correlation with number of branches/plant, oil yield, plant spread (r = 0.806, 0.802, 0.782), plant spread had a highest correlation with plant height (r = 0.840). The seeds analyzed for proximate composition, fatty acid and physiochemical characteristics revealed that fiber and ash content in seed flour were high (16.5% and 4.35%). Oil content varied from 24.5% to 37.9%. The lower value of the viscosity suggests it as diesel oil. Accession JC006 could be an alternative source of linoleic acid (51%) while the accession JC001 could be a source for oleic acid (48%) and linoleic acid 42.4%. Stearic acid was highest in accession JC003 (42.9%).This evaluation has helped to identify cultivar with specific yield and vegetative growth features. Among all the seven accession evaluated accession JC007 is found to be promising which could be taken as productive genotype for commercial exploitation.