Molar Equivalent Of Methanol Research Articles

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.

Interaction of methanol with the flexible metal-organic framework MIL-53(Fe) observed by inelastic neutron scattering

In situ powder X-ray diffraction and thermogravimetry show that the iron(III) carboxylate material MIL-53(Fe) expands in two steps upon exposure to methanol vapour to give a ‘half open’ phase and then a fully expanded structure; these correspond to the uptake of one and two molar equivalents of methanol, respectively. This knowledge allows interpretation of inelastic neutron scattering (INS) spectra, measured using TOSCA at ISIS, of the solid loaded with various amounts of water, methanol or d3-methanol (CD3OH), aided also by DFT simulation of INS spectra of the analogous MIL-53(Al) structure. The key signature of the expansion and contraction of the structure is the shift of bands in the 400cm−1 region: these are assigned as due to the librations of the terephthalate rings, involving motions that result in distortion of the aromatic ring and a rocking motion of the aromatic ring about the bonds to the carboxylate groups.

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.

The Enzymatic Production of Biodiesel from Pomace Oil Using Immobilized Thermomyces lanuginosus

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto styrene-divinylbenzene polyglutaraldehyde copolymer. Immobilized support material was used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy. Biodiesel production was carried out with a semi-continuous operation system. Methanol was added into the reactor by three successive additions of 1:6 molar equivalent of methanol to avoid enzyme inhibition. The maximum methyl esters yield was obtained as 98%.

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 catalyzed by Rhizomucor miehei lipase-displaying Pichia pastoris whole cells in an isooctane system

A recombinant Pichia pastoris displaying Rhizomucor miehei Lipase (RML) on the cell surface was shown to be an efficient biocatalyst in biodiesel production from soybean oil. Various reaction parameters affecting the transesterification catalyzed by RML-displaying P. pastoris whole cells were studied. The optimal reaction conditions were: temperature 55°C, water activity 0.66, the ratio of isooctane to oil 2:1 (v/v) and the whole-cell biocatalyst 40% (w/w, soybean oil). To reduce the inactivation of displaying lipases by methanol, each 1.5 molar equivalent of methanol (mol/mol, soybean oil) were added in a three-step procedure at the reaction time of 0h, 24h and 48h, respectively. Under these optimized conditions, a methyl esters (ME) yield reached 83.14% after 72h, which significantly enhanced the ME yield catalyzed by the displaying lipase reported previously. The RML-displaying P. pastoris whole-cell biocatalyst exhibited quite a good operational stability with more than 80.46% of its original activity remained even after 10 batches reaction as well as storage stability. Thus, it showed promising potential in biodiesel production.

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.

Enzymatic Production of Bio-Diesel from Waste Cooking Oil Using Lipase

The applications of lipase immobilized on ceramic beads and entrapped in sol-gel matrix, in the production of bio-diesel from waste cooking oil, are compared to that of free lipase. Experimental determination of the effect of molar equivalent of methanol, to moles of ester bond in the triglyceride, on the rate of the enzymatic trans-esterification was experimentally determined. It was found that for the same weight of lipase used, the production of bio-diesel was much higher using lipase immobilized on ceramic beads in comparison to that using lipase entrapped in sol-gel and in free form. Substrates inhibition effect was observed in all cases, which agrees with previous results found in literature. The optimum methanol:oil molar ratio was found to be 0.87 for immobilized lipase from yeast source, C. antartica and 1.00 for free lipase from the same yeast source and immobilized lipase from bacterial source, P. cepacia. On the other hand, it was shown that biodieasel can be produced in considerable amounts, with yield reaching 40%, in absence of organic solvent using immobilized lipase, from P. cepacia, on ceramic beads. The results of this study can be used to determine the kinetics parameters of mathematical models which describe the system.

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.

Lipase-catalyzed methanolysis of palm oil in presence and absence of organic solvent for production of biodiesel

Enzymatic production of methyl esters (biodiesel) by methanolysis of palm oil in presence and absence of organic solvent was investigated using Candida antarctica lipase immobilized on acrylic resin as a biocatalyst. Although, at least molar equivalent of methanol (methanol–palm oil ratio 3:1) is required for the complete conversion of palm oil to methyl esters, lipase catalyzed methanolysis of palm oil in absence of organic solvent was poisoned by adding more than 1/3 molar equivalent of methanol. The use of polar organic solvents prevented the lipase to be poisoned in methanolysis with a molar equivalent of methanol, and tetrahydrofuran (THF) was found to be the most effective. The presence of water in methanolysis of palm oil both in presence and absence of THF inhibited the reaction rate but this inhibition was considerably low in THF containing system. The palm oil-lipase (w/w) ratio significantly influenced the activity of lipase and the optimal ratio in presence and absence of THF was 100 and 50, respectively.

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.

Methyl Esterification of Waste Fatty Acids with Immobilized Candida antarctica Lipase.

Enzymatic production of a docosahexaenoic acid-rich oil from tuna oil creates considerable amounts of free fatty acids (FFA), which are treated as an industrial waste but the handling is difficult because of their solid state. We thus attempted to convert the waste FFA (Tuna-FFA) to their methyl esters through an organic solvent-free enzymatic process. When waste Tuna-FFA were esterified at 30°C with two molar equivalents of methanol (MeOH) using 1.0 wt% immobilized Candida antarctica lipase, the esterification degree reached 95% after 24 h. The reaction was recycled by transferring the enzyme into a fresh substrate mixture every 24 h, but the esterification degree maintained 95% during 45 cycles. To further increase the esterification degree, the esterification was repeated with 5 molar equivalents of MeOH against the remaining FFA after dehydration of the mixture obtained by the single reaction. The repeated reaction increased the esterification degree to 97-98%.

Enzymatic conversion of waste edible oil to biodiesel fuel in a fixed‐bed bioreactor

AbstractThe conversion of waste edible oil to biodiesel fuel in a fixed‐bed bioreactor was investigated. Three‐step methanolysis of waste oil was conducted using three columns packed with 3 g of immobilized Candida antarctica lipase. A mixture of waste oil and 1/3 molar equivalent of methanol against total fatty acids in the oil was used as substrate for the first‐step reaction, and mixtures of the first‐ and second‐step eluates and 1/3 molar equivalent of methanol were used for the second‐ and third‐step reactions, respectively. Ninety percent of waste oil was converted to the corresponding methyl esters (ME) by feeding substrate mixtures into the first, second, and third reactors at flow rates of 6, 6 and 4 mL/h, respectively. We also attempted one‐step methanolysis of waste oil. When a mixture of waste oil and 90% ME‐containing eluate (1∶3, wt/wt) and an equimolar amount of methanol against total fatty acids in the waste oil was fed into a reactor packed with 3 g of immobilized C. antarctica lipase at a flow rate of 4 mL/h, the ME content in the eluate reached 90%. The immobilized biocatalyst could be used for 100 d in the two reaction systems without significant decrease in its activity. Waste oil contained 1980 ppm water and 2.5% free fatty acids, but these contaminants had little influence on enzymatic production of biodiesel fuel.

Effect of Methanol and Water Contents on Production of Biodiesel Fuel from Plant Oil Catalyzed by Various Lipases in a Solvent-Free System.

Methyl esters synthesized from plant oil and methanol by the methanolysis reaction are potentially important as a biodiesel fuel. The methanolysis of soybean oil by lipases from various microorganisms was investigated. Several of the lipases were found to catalyze methanolysis in a water-containing system without an organic solvent. The lipases from Candida rugosa, Pseudomonas cepacia, and Pseudomonas fluorescens displayed particularly high catalytic ability. The reaction rates of methanolysis catalyzed by the C. rugosa and P. fluorescens lipases decreased significantly when the water content was low, showing that water prevents the inactivation of these lipases by methanol. On the other hand, the methanolysis reaction rate catalyzed by the P. cepacia lipase remained high even under a low water content. In addition, the P. cepacia lipase gave high methyl ester contents in the reaction mixture up to 2 or 3 molar equivalents of methanol to oil, which is attributed to the P. cepacia lipase having substantial methanol resistance. For the same methanol content, the reaction rates of methanolysis catalyzed by the P. cepacia lipase increased with decreasing water content, and hence lipases strongly resistant to high methanol, such as that from P. cepacia, are desirable for use in methanolysis reaction processes.

Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase

AbstractCandida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three‐step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two‐step methanolysis of the oil TAG: The first‐step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second‐step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two‐step methanolysis achieved more than 95% of conversion. When two‐step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two‐step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three‐step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final‐step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.

Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil

The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25–0.4 h intervals.

Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase

AbstractBiodiesel derived from vegetable oils has drawn considerable attention with increasing environmental consciousness. We attempted continuous methanolysis of vegetable oil by an enzymatic process. Immobilized Candida antarctica lipase was found to be the most effective for the methanolysis among lipases tested. The enzyme was inactivated by shaking in a mixture containing more than 1.5 molar equivalents of methanol against the oil. To fully convert the oil to its corresponding methyl esters, at least 3 molar equivalents of methanol are needed. Thus, the reaction was conducted by adding methanol stepwise to avoid lipase inactivation. The first step of the reaction was conducted at 30°C for 10 h in a mixture of oil/methanol (1:1, mol/mol) and 4% immobilized lipase with shaking at 130 oscillations/min. After more than 95% methanol was consumed in ester formation, a second molar equivalent of methanol was added and the reaction continued for 14 h. The third molar equivalent of methanol was finally added and the reaction continued for 24 h (total reaction time, 48 h). This three‐step process converted 98.4% of the oil to its corresponding methyl esters. To investigate the stability of the lipase, the three‐step methanolysis process was repeated by transferring the immobilized lipase to a fresh substrate mixture. As a result, more than 95% of the ester conversion was maintained even after 50 cycles of the reaction (100 d).