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.     

A two-step acid-catalyzed process for the production of biodiesel from rice bran oil

A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceride was hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzed methanolysis process was employed for the efficient conversion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 degrees C. Depending on the initial FFA content of oil, 55-90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzed methanolysis at 100 degrees C. By this two-step methanolysis reaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as gamma-oryzanol (16-18%), mixture of phytosterol, tocol and steryl ester (19-21%).     

游离脂肪酶催化蓖麻油制备蓖麻油酸

游离脂肪酶与固定化脂肪酶相比具有成本低、反应速率快等优势,是油脂化工中新的研究方向。前期研究表明,游离脂肪酶NS81006能高效催化多种油脂水解,进一步研究其对含独特羟基的绿色石油材料蓖麻油的水解过程,对于促进游离脂肪酶在新能源领域的应用具有重要意义。本文对影响游离脂肪酶NS81006催化蓖麻油水解过程的主要因素,温度、酶用量、水用量和搅拌速率进行了研究和优化,在优化后的条件下48 h水解率可达94.8%,且发现通过离心分离可有效实现NS81006的重复使用,连续回用5个批次,游离脂肪酶仍能有效催化水解反应。而对比高温高压法水解蓖麻油,发现游离脂肪酶NS81006具有明显优势。     

Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida sp. 99–125 in organic solvent system

In this study, hydrolysis and methanolysis of glycerol trioleate (TG) by lipase Candida sp. 99–125 were investigated under different water conditions. Both the reaction rates were relatively low without water, while increasing water content to 5 wt.% (or more, from 10–20%) based on the TG amount caused remarkable higher TG conversion for both reactions. Moreover, comparing the time course curves of the hydrolysis and methanolysis, it could be concluded that the methanolysis reaction catalyzed by this Candida sp. 99–125 appeared to accord with the successive reaction mechanism. TG was first hydrolyzed to partial glycerides and oleic acid (OA), then oleic acid methyl ester (OAME) was produced by esterification of the OA with methanol. This water effect was also confirmed by the experiments that water substitutions such as t-butanol and some surfactants added into the system did not get such high yields as that of the water included system. So these results showed that water took part in the methanolysis reaction, and successive hydrolysis–esterification process might be the catalytic mechanism of this lipase.     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

A newly isolated fungal strain used as whole‐cell biocatalyst for biodiesel production from palm oil

A strain of Aspergillus niger isolated from atmospherically exposed bread and Jatropha curcas seed was utilized as a whole‐cell biocatalyst for palm oil methanolysis to produce fatty acid methyl esters (FAME), or biodiesel. The A. niger strain had a lipase activity of 212.58 mU mL^?1 after 144 h incubation at 25 °C with an initial pH value of 6.5, using 7% polypeptone (w/w on basal medium) as the nitrogen source and 3% olive oil (w/w on basal medium) as a carbon source. The A. niger cells spontaneously immobilized within polyurethane biomass support particles (BSPs) during submerged fermentation. Thereafter, the methanolysis of palm oil was achieved via a three‐step addition of methanol in the presence of BSPs‐immobilized with A. niger cells. The influence of water content, reaction temperature and enzyme concentration on reaction rate was investigated. An 8% water content and a temperature of 40 °C in the presence of 30 immobilized BSPs, resulted in an 87% FAME yield after 72 h.     

Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one‐step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole‐cell Candida antarctica lipase B‐expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase‐expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.     

Kinetics of sunflower oil methanolysis at low temperatures

The kinetics of the sunflower oil methanolysis process was studied at lower temperatures (10-30 degrees C). The sigmoidal kinetics of the process was explained by the mass transfer controlled region in the initial heterogenous regime, followed by the chemical reaction controlled region in the pseudo-homogenous regime. A simple kinetic model, which did not require complex computation of the kinetic constants, was used for simulation of the TG conversion and the FAME formation in the latter regime: the fast irreversible second-order reaction was followed by the slow reversible second-order reaction close to the completion of the methanolysis reaction. The mass transfer was related to the drop size of the dispersed (methanol) phase, which reduced rapidly with the progress of the methanolysis reaction. This was attributed to the formation of the emulsifying agents stabilizing the emulsion of methanol drops into the oil.     

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides.Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.List of Symbols a_w thermodynamic activity of water - DG diglyceride (s) - FAME fatty acid methyl ester (s) - FFA free fatty acid (s) - FID flame ionization detector Gly glycerol - MG monoglyceride (s) - TG triglyceride (s) - TLC thin layer chromatography The authors are grateful to Profs. P. Adlercreutz, Technical University of Lund, Sweden, and J.M.S. Cabral, Instituto Superior Técnico, Lisbon, Portugal, for inspiring discussions and advice, to Prof. Helena Pereira, Instituto Superior de Agronomia (ISA), Lisbon, Portugal, for the use of GC equipment and to Mrs. Marlene Dionísio, ISA, for invaluable help with some of the experimental work.     

Performance of heterogeneous ZrO2 supported metaloxide catalysts for brown grease esterification and sulfur removal

In order to achieve a viable biodiesel industry, new catalyst technology is needed which can process a variety of less expensive waste oils, such as yellow grease and brown grease. However, for these catalysts to be effective for biodiesel production using these feedstocks, they must be able to tolerate higher concentrations of free fatty acids (FFA), water, and sulfur. We have developed a class of zirconia supported metaloxide catalysts that achieve high FAME yields through esterification of FFAs while simultaneously performing desulfurization and de-metallization functions. In fact, methanolysis, with the zirconia supported catalysts, was more effective for desulfurization than an acid washing process. In addition, using zirconia supported catalysts to convert waste grease, high in sulfur content, resulted in a FAME product that could meet the in-use ASTM diesel fuel sulfur specification (<500 ppm). Possible mechanisms of desulfurization and de-metallization by methanolysis were proposed to explain this activity.     

Lipase catalyzed methanolysis to produce biodiesel: Optimization of the biodiesel production

A lipase from Candida sp., suitable for transesterification of fats and oils to produce fatty acid methyl ester (FAME), was immobilized on a cheap cotton membrane, in this paper. The conversion ratio of salad oil to biodiesel could reach up to 96% with the optimal reaction conditions. Continuous reaction in a fixed bed reactor was also investigated. A three-step transesterification with methanol (methanolysis) of oil was conducted by using a series of nine columns packed with immobilized Candida sp. 99–125 lipase. As substrate of the first reaction step, plant or waste oil was used together with 1/3 molar equivalent of methanol against total fatty acids in the oil. Mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-reaction steps. A hydrocyclone was used in order to on-line separate the by-product glycerol after every 1/3 molar equivalent of methanol was added. Petroleum ether was used as solvent (3/2, v/v of oil) and the pump was operated with a flow rate of 15 L/h giving an annual throughput of 100 t. The final conversion ratio of the FAME from plant oil and waste oil under the optimal condition was 90% and 92%, respectively. The life of the immobilized lipase was more than 10 days. This new technique has many strongpoints such as low pollution, environmentally friendly, and low energy costs.     

Enrichment of polyunsaturated fatty acids from tuna oil using immobilized Pseudomonas fluorescens lipase

Immobilized Pseudomonas fluorescens lipase enzyme was used to enrich the important polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna oil. Hydrolysis, esterification, and transesterification reactions were studied in detail to find out the fractionation pattern of DHA and EPA during these processes due to preferential selectivity for or against these PUFA. Hydrolysis with P. fluorescens biotype I lipase with stoichiometric amount of water content gave more than 80% of DHA and EPA in the free fatty acid (FFA) form after around 60% of hydrolysis. After some preferential specificity during the early stages of hydrolysis, P. fluorescens lipase exhibits nonselective characteristics on extended hydrolysis. Esterification of FFA extracted from the completely hydrolyzed mixture of tuna oil was found to be better with long chain fatty alcohol like octanol which lead to good enrichment (44.5% for DHA and 11.3% for EPA) and yields of the PUFA in the FFA form. Transesterification (ethanolysis) with immobilized P. fluorescens lipase enzyme resulted in good enrichment and recovery of DHA and EPA in the glyceride mixture. After around 60% of ester synthesis, 74% of (DHA + EPA) enrichment was achieved with yields of more than 90% in the glyceride mixture.     

Production of biodiesel by lipase-catalyzed transesterification of vegetable oils: a kinetics study

Kinetics of production of biodiesel by enzymatic methanolysis of vegetable oils using lipase has been investigated. A mathematical model taking into account the mechanism of the methanolysis reaction starting from the vegetable oil as substrate, rather than the free fatty acids, has been developed. The kinetic parameters were estimated by fitting the experimental data of the enzymatic reaction of sunflower oil by two types of lipases, namely, Rhizomucor miehei lipase (RM) immobilized on ion-exchange resins and Thermomyces lanuginosa lipase (TL) immobilized on silica gel. There was a good agreement between the experimental results of the initial rate of reaction and those predicted by the proposed model equations, for both enzymes. From the proposed model equations, the regions where the effect of alcohol inhibition fades, at different substrate concentrations, were identified. The proposed model equation can be used to predict the rate of methanolysis of vegetable oils in a batch or a continuous reactor and to determine the optimal conditions for biodiesel production.     

Continuous and consecutive conversion of free fatty acid in rice bran oil to triacylglycerol using immobilized lipase

Free fatty acid (FFA), monoacylglycerol (MG) and diacylglycerol (DG) in high-FFA rice bran oil were continuously converted with glycerol (G) to form triacylglycerol (TG), using lipase fromRhizomucor miehei immobilized on anion-exchange resin. The reaction was continued for more than 1 month by a reactor with two circulation loops, each being connected to a fixed-bed reactor and a dehydrator. The reaction of 2 FFA + G DG + 2H₂O appeared to occur until the glycerol was exhausted; the reaction of FFA + DG TG + H₂O then followed. The consecutive esterificaion continued in the presence of 2–8 ppm water and the TG content reached 74%–88%. The industrial feasibility of this process was assessed from the standpoints of enzyme cost and value added by esterification.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

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.     

Hydrolysis of crambe oil by enzymatic catalysis: An evaluation of the operational conditions

Abstract

In this work, the enzymatic hydrolysis of the crambe oil by using a commercial immobilized lipase Lipozyme RM IM was evaluated. The effect of the operational conditions, such as temperature, water/oil molar ratio, enzyme/substrate mass ratio and stirring speed were assessed based on the experimental designs. The experiments were performed in a closed and batch system with controlled temperature and stirring speed. In addition, the kinetics of the process was studied in the best operational conditions, wherein the experimental data were obtained and described by a mathematical model. The influence of the operational conditions was assessed based on the measured values of the free fatty acids (FFA) produced by the enzymatic hydrolysis. In 4?h of reaction, a yield of 42.6% was observed and the most significant operational conditions were the enzyme/substrate mass ratio and stirring speed. By the kinetic investigation, an initial reaction rate of 3.5?×?10⁴?mol?mL^?1?h^?1 and a maximum yield of 74% were observed after 40?h of reaction (in the equilibrium condition). The mathematical model was not only able to adequately describe the experimental data of FFA concentrations profiles but also showed predictive capacity to independents assays in different operational conditions. Therefore, based on the simulation analysis of the enzymatic hydrolysis of the crambe oil, the model can be useful for process optimization and phenomenological studies.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Response surface modelling of the production of ω-3 polyunsaturated fatty acids-enriched fats by a commercial immobilized lipase

The aim of this study was to model the production of fats, enriched with ω-3 polyunsaturated fatty acids (ω-3 PUFA) for nutraceutical purposes, via the response surface methodology. These fats were obtained by transesterification of palm oil stearin (POS) with a concentrate (EPAX 2050TG) of triglycerides enriched with ω-3 PUFA and soybean oil, catalysed by a commercial immobilized Candida antarctica lipase (“Novozym 435”).

The initial water activity (a_w) of the biocatalyst, POS and EPAX 2050TG concentrations, time and temperature showed a significant effect on the transesterification reaction, as well as on the competing reactions of hydrolysis and lipid oxidation.

Depending on the factors included, the transesterification reaction was described either by first- or second-order models.

The production of free fatty acids, which is ascribed both to the hydrolytic reaction and the mechanism of lipase-catalysed transesterification, showed a second-order dependence on the initial a_w of the biocatalyst.     

Exploring a new,soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock

Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides (TAGs, DAGs, MAGs) variation during 24 h reaction. An excellent 96% FAMEs release was achieved when low (3–5%) water concentrations were used in the conversion of crude soybean oil. Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs, DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.