Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system

Biocatalytic synthesis is a promising environmentally friendly process for the production of biodiesel, a sustainable alternative fuel from renewable plant resources. In order to develop an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs) were prepared from a commercial enzyme preparation from a recombinant Aspergillus strain expressing Thermomyces lanuginosus lipase and used for synthesis of biodiesel from palm olein by ethanolysis. Reaction parameters, including catalyst loading, temperature, and oil/alcohol molar ratio have been systematically optimized. Addition of tert-butanol was found to markedly increase the biocatalyst activity and stability resulting in improved product yield. Optimized reactions (20%, w/w PCMC-lipase to triacylglycerol and 1:4 fatty acid equivalence/ethanol molar ratio) led to the production of alkyl esters from palm olein at 89.9% yield on molar basis after incubation at 45 °C for 24 h in the presence of tert-butanol at a 1:1 molar ratio to triacylglycerol. Crude palm oil and palm fatty acid distillate were also efficiently converted to biodiesel with 82.1 and 75.5% yield, respectively, with continual dehydration by molecular sieving. Operational stability of PCMC-lipase could be improved by treatment with tert-butanol allowing recycling of the biocatalyst for at least 8 consecutive batches with only slight reduction in activity. This work thus shows a promising approach for biodiesel synthesis with microcrystalline lipase which could be further developed for cost-efficient industrial production of biodiesel.     

A flow-through enzymatic microreactor immobilizing lipase based on layer-by-layer method for biosynthetic process: Catalyzing the transesterification of soybean oil for fatty acid methyl ester production

A simple and convenient method was proposed in this paper to develop a flow-through enzymatic micro-reactor made from polytetrafluoroethylene (PTFE). It consisted of the polydopamine layer (functioned as a primer) and layer by layer (LBL) coatings composed of polyethylenimine (PEI) and lipase. The multiple deposition of PEI and lipase was the key factor of increasing the enzyme loading on microreactor. After 8 PEI/lipase layers, enzyme loading on the inner surface of 5-m microchannel reached a maximum (350 μg to 400 μg), compared with approximately 20 μg in single layer. Microreactor with higher enzyme loading was successfully applied on transesterification of soybean oil for effective fatty acid methyl ester (FAME, biodiesel) production. A 95.2% conversion rate of biodiesel can be achieved in 53 min under optimized conditions, instead of a couple of hours in the traditional batch reaction.     

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.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8 ± 8.60 IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3 ± 1.70 mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50 °C, while for the crude PPL extract were at pH 8.0 and 45 °C. Immobilized PPL exhibited full hydrolytic activity after 2 h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2 h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48 h of reaction for MPKO and 35% after 96 h of reaction for MPPO.     

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.     

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.     

Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system

The monoethyl esters of the long chain fatty acids (biodiesel) were prepared by alcoholysis of Jatropha oil, a non-edible oil, by a lipase. The process optimization consisted of (a) screening of various commercial lipase preparations, (b) pH tuning, (c) immobilization, (d) varying water content in the reaction media, (e) varying amount of enzyme used, and (f) varying temperature of the reaction. The best yield 98% (w/w) was obtained by using Pseudomonas cepacia lipase immobilized on celite at 50 °C in the presence of 4–5% (w/w) water in 8 h. It was found that yields were not affected if analytical grade alcohol was replaced by commercial grade alcohol. This biocatalyst could be used four times without loss of any activity.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification

Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization.     

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.     

Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum

An inexpensive self-made immobilized lipase from Penicillium expansum was shown to be an efficient biocatalyst for biodiesel production from waste oil with high acid value in organic solvent. It was revealed that water from the esterification of free fatty acids and methanol prohibited a high methyl ester yield. Adsorbents could effectively control the concentration of water in the reaction system, resulting in an improved methyl ester yield. Silica gel was proved to be the optimal adsorbent, affording a ME yield of 92.8% after 7 h. Moreover, the enzyme preparation displayed a higher stability in waste oil than in corn oil, with 68.4% of the original enzymatic activity retained after being reused for 10 batches.     

Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase

Experimental determination of the separate effects of palm oil and methanol concentrations on the rate of their enzymatic transesterification was used to propose suitable mechanismic steps and to test the generated kinetic model. The reaction took place in n-hexane organic medium and the lipase used was from Mucor miehei. At a constant methanol concentration of 300 mol m⁻³, it was found that, initially as the palm oil concentration increased, the initial reaction rate increased. However, the initial rate dropped sharply at substrate concentrations larger than 1250 mol m⁻³. Similar behaviour was observed for methanol concentration effect, where at a constant substrate concentration of 1000 mol m⁻³, the initial rate of reaction dropped at methanol concentrations larger than 3000 mol m⁻³. Ping Pong Bi Bi mechanism with inhibition by both reactants was adopted as it best explains the experimental findings. A mathematical model was developed from a proposed kinetic mechanism and was used to identify the regions where the effect of inhibition by both substrates arised. The proposed model equation is essential for predicting the rate of methanolysis of palm oil in a batch or a continuous reactor and for determining the optimal conditions for biodiesel production.     

Biodiesel production from sunflower,soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer

This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology.Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 °C, each lasting 24 h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9–9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.     

Increased production of FAEEs for biodiesel with lipase enhanced Saccharomyces cerevisiae

In this study, we expressed lipase 2 from Candida sp. 99-125 in Saccharomyces cerevisiae, and tried direct biodiesel production. Driven by 3-phosphoglycerate kinase promoter, Lip2 showed high expression level in cytoplasm. SDS-PAGE analysis confirmed the successful lipase expression with a 40 kDa molecular weight. The enzyme assay indicated that lipase 2 had a specific activity of 12.12 μmol/min/mg toward p-nitrophenyl palmitate. Gas chromatography showed that the main fatty acids of S. cerevisiae lipids were palmitoleic acid (31.79%) and oleic acid (29.84%). By three-step addition of 4% ethanol to culture broth, the yield of fatty acid ethyl esters by recombinant S. cerevisiae reached 11.4 mg/g dry cell weight. This work proposed a novel pathway for S. cerevisiae that could be applied for producing biodiesel directly.     

Effect of catalyst additives on the production of biofuels from palm oil cracking in a transport riser reactor

Catalytic cracking of crude palm oil (CPO) and used palm oil (UPO) were studied in a transport riser reactor for the production of biofuels at a reaction temperature of 450 °C, with residence time of 20 s and catalyst-to-oil ratio (CTO) of 5 g g^?1. The effect of HZSM-5 (differrent Si/Al ratios), beta zeolite, SBA-15 and AlSBA-15 were studied as physically mixed additives with cracking catalyst Rare earth-Y (REY). REY catalyst alone gave 75.8 wt% conversion with 34.5 wt% of gasoline fraction yield using CPO, whereas with UPO, the conversion was 70.9 wt% with gasoline fraction yield of 33.0 wt%. HZSM-5, beta zeolite, SBA-15 and AlSBA-15 as additives with REY increased the conversion and the yield of organic liquid product. The transport riser reactor can be used for the continuous production of biofuels from cracking of CPO and UPO over REY catalyst.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.