Liquid Lipase Research Articles

Biodiesel preparation from Semen Abutili (Abutilon theophrasti Medic.) seed oil using low-cost liquid lipase Eversa® transform 2.0 as a catalyst

To decrease the cost of biodiesel production, undeveloped nonedible Semen Abutili seed oil (SASO) was first used as a feedstock to prepare biodiesel. Some low-cost liquid lipases were screened and used as green catalysts for biodiesel production via the ethanolysis of SASO. The effect of reaction factors on ethanolysis was optimized by the response surface methodology (RSM). The results demonstrated that SASO was a promising alternative feedstock, and Eversa® Transform 2.0 showed the best performance for biodiesel production from low-quality SASO feedstock (high FFA and phospholipid contents). In addition, Eversa® Transform 2.0 can be reused 3 times without significant loss. The influence of reaction factors on biodiesel yield decreased in the order of reaction temperature > lipase load > reaction time. The maximum biodiesel yield was 94.2 ± 1.3 % under the optimal conditions (lipase load 6%, water content 20 %, 7:1 (mol/mol) ethanol to SASO, 11 h, 37 °C). The kinetic parameters (Vmax and K'm) of the transesterification of SASO were 4.16 × 10−2mol/(L∙min) and 5.27 × 10-1 mol/L, respectively. The Arrhenius equation and activation energy, Ea, of Eversa® Transform 2.0-catalyzed transesterification of SASO were lnV0 = 4.8252–2382.3/T and 19.80 kJ/mol, respectively. The biodiesel produced from SASO was accordance with the ASTM D6751 standard, except for the oxidation stability. Thus, SASO, an undeveloped nonedible low-quality vegetable oil, can be used as a potential feedstock for biodiesel production, and the low-cost liquid lipase Eversa® Transform 2.0 can be used for biodiesel production.

Effects of Imprinting and Water Activity on Transesterification and Thermostability with Lipases in Ionic Liquid

The effect of bio-imprinting and water activity on catalytic activities and the thermostability of lipases was investigated for transesterification using vinyl acetate and benzyl alcohol as substrates in ionic liquid, [Cnmim][PF6] (n=4,6,8), and benzene. The catalytic activities were enhanced by imprinting in benzene and [C4mim][PF6], and the relations between the transesterification activities and the water activity in both solvents were approximately bell shaped. The reactivity of the transesterification in benzene was higher than that in [C4<br /> mim][PF6]. The effects of water activity and imprinting on the kinetic parameters in [C4mim][PF6] were examined. Without controlling the water content, the values of Km,VA and Km,BA (Michaelis constants of vinyl acetate and benzyl alcohol, respectively) decreased, and the values of Vm (maximum rate) increased by imprinting. On the other hand, by controlling the water content in the organic media, the values of Vm, Km,VA, and Km,BA increased by imprinting. The activities of lipase in ionic liquid are more strongly affected by water activity and imprinting than those in benzene. We observed effects of water activity on thermostability but none from imprinting.

Combining technology with liquid-formulated lipases for in-spec biodiesel production.

Enzymatic biodiesel production has been at the forefront of biofuels research in recent decades because of the significant environmental advantages it offers, while having the potential to be as effective as conventional chemically catalyzed biodiesel production. However, the higher capital cost, longer reaction time, and sensitivity of enzyme processes have restricted their widespread industrial adoption so far. It is also posited that the lack of research to bring the biodiesel product into final specification has scuppered industrial confidence in the viability of the enzymatic process. Furthermore, the vast majority of literature has focused on the development of immobilized enzyme processes, which seem too costly (and risky) to be used industrially. There has been little focus on liquid lipase formulations such as the Eversa Transform 2.0, which is in fact already used commercially for triglyceride transesterification. It is the objective of this review to highlight new research that focuses on bringing enzymatically produced biodiesel into specification via a liquid lipase polishing process, and the process considerations that come with it.

Semi-continuous production of biodiesel on pilot scale via enzymatic hydroesterification of waste material: Process and economics considerations

Nowadays, the use of edible oils with elevate purity in the biodiesel production represents around 70% of the chain of feedstocks applied in the process by the main producing countries at world level. This occurs due, mainly, to reaction limitations of the traditional alkaline route employed industrially. Bypassing drawbacks of the chemical process, enzymatic technique is an effective alternative for the biodiesel synthesis. In this sense, this paper reports results to semi-continuous production of biodiesel on pilot scale, from waste cooking oil, via hydroesterification mediated by different commercial lipases in liquid formulation: Eversa® Transform and its novel thermostable successor Eversa® Transform 2.0. Data for yields, residual acidity, productivities, biocatalysts performance, kinetics and economic analysis are presented in this comprehensive research. Using the best reaction conditions found (40 °C, 0.7 wt% of Eversa® Transform 2.0, 4.0 wt% of water, methanol to oil molar ratio of 6.3:1 and 8 h at a two-step reaction system), 96.2% of FAME yield was achieved. A pseudo-first order model presented a good fit of the experimental data, where a kApp of 0.373∙h−1 was obtained for the process with the best performance. Moreover, for this process, an economic analysis indicated the feasibility of the system through a positive net return and operating cost of US$ 0.50 kg−1 of biofuel. This information served as foundation to conclude that enzymatic hydroesterification catalyzed by liquid lipases has the necessary tools to be implemented on industrial biodiesel production.

Biodiesel production catalysed by low-cost liquid enzyme Eversa® Transform 2.0: Effect of free fatty acid content on lipase methanol tolerance and kinetic model

A low-cost liquid lipase from genetically modified Aspergillus oryzae (Eversa® Transform 2.0) was used in this study for biodiesel production. The catalytic performance of this enzyme was evaluated using refined palm oil adjusted with different free fatty acid (FFA) content. The methanol-to-oil molar ratio was varied from 1:1 to 8:1 to determine the reaction rate, ester conversion and methanol tolerance of this enzyme at different FFA content. The reaction was carried out at low temperature (40 °C) for 24 h using very low enzyme concentration (0.2 wt%). The results showed that the enzyme could tolerate a higher dosage of methanol and could increase reaction rate and conversion when higher FFA content was present in the feedstocks. A biodiesel conversion of 97% could be obtained when the feedstocks contained ≥80 wt% FFA. Furthermore, a new semi-empirical model based on the Ping-Pong Bi-Bi mechanism has been developed to estimate the reaction kinetics of biodiesel production from feedstocks containing a mixture of triacylglycerol and FFA. In conclusion, Eversa® Transform 2.0 can be used for the production of biodiesel from low-quality feedstocks containing high FFA content in a sustainable and economical manner.

Developments in the Use of Lipase Transesterification for Biodiesel Production from Animal Fat Waste

Biodiesel constitutes an attractive source of energy because it is renewable, biodegradable, and non-polluting. Up to 20% biodiesel can be blended with fossil diesel and is being produced and used in many countries. Animal fat waste represents nearly 6% of total feedstock used to produce biodiesel through alkaline catalysis transesterification after its pretreatment. Lipase transesterification has some advantages such as the need of mild conditions, absence of pretreatment, no soap formation, simple downstream purification process and generation of high quality biodiesel. A few companies are using liquid lipase formulations and, in some cases, immobilized lipases for industrial biodiesel production, but the efficiency of the process can be further improved. Recent developments on immobilization support materials such as nanoparticles and magnetic nanomaterials have demonstrated high efficiency and potential for industrial applications. This manuscript reviews the latest advances on lipase transesterification and key operational variables for an efficient biodiesel production from animal fat waste.

Esterification using a liquid lipase to remove residual free fatty acids in biodiesel

Lately, the price of liquid formulated lipase enzymes, usable in biodiesel production, has been significantly reduced. This enables one-time use of these enzymes for transesterification, and the process is used industrially. However, the process suffers a drawback by leaving 2−3 % free fatty acids in the crude biodiesel, which reduces the profitability. This article discusses a novel enzymatic FFA esterification reaction utilizing liquid lipase B from Candida antarctica (CALB) along with glycerol at low water concentrations to eliminate the residual FFA. The reaction setup was found able to reduce the free fatty acid concentration to within biodiesel specifications of < 0.25 wt.% FFA. Additionally, two alternative process setups are proposed, which were both found viable through a combination of experiments and simulations, and can be developed into full-scale processes. The resulting two-step enzymatic biodiesel process - transesterification followed by esterification - provides a potential process layout for the industrial production of biodiesel.

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called “combilipases” and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes.

Sustainable biodiesel production from the green microalgae Nannochloropsis: Novel integrated processes from cultivation to enzyme-assisted extraction and ethanolysis of lipids

A novel integrated process from cultivation to enzyme-assisted extraction and ethanolysis of lipids was established and implemented to produce sustainable and clean microalgal biodiesel by three marine Nannochloropsis strains (N. oculata, Nannochloropsis sp. and N. oceanica). Among the selected strains, N. oculata was selected as a model for the evaluation of lipid extraction performance and biodiesel conversion, due to its highest biomass (4.38 g/L) and total fatty acids (TFAs) yield (1.55 g/L). In the lipid extraction, three phase partitioning (TPP) exhibited superior lipid extraction performance when N. oculata cells were treated by four mixed enzymes (cellulase, hemicellulase, papain and pectinase). After optimization, the highest FAMEs recovery (92.59%) was obtained by TPP at 1.5% microalgal biomass, 6% K2HPO4, 65% ethanol content, 80 °C extraction temperature, 1 h incubation time and two extraction cycles. In the enzymatic ethanolysis, the extracted lipids from the ethanol phase of TPP were directly used for biodiesel with liquid lipase from Thermomyces lanuginosus (TL) and results showed that lipase TL performed the greatest biodiesel conversion (90.24%) under the optimized conditions. Lastly, this integrated process was re-evaluated with two Nannochloropsis strains. Results showed that the integrated process attained 85.12% biodiesel conversion for Nannochloropsis sp. and 76.33% for N. oceanica, demonstrating that the lipidic class and fatty acid composition influenced the biodiesel conversion by enzymatic ethanolysis. Taken together, the integrated process mediated with enzymes and ethanol as green media was a promising strategy for cost-effective and low-value bulky biodiesel by oleaginous Nannochloropsis strains.

Conversion of waste frying palm oil into biodiesel using free lipase A from Candida antarctica as a novel catalyst

Biodiesel, also known as fatty acid methyl ester (FAME), is usually prepared using edible vegetable oils by transesterification. In this work, a clean method for biodiesel production was developed using frying palm oil (FPO) and free Candida antarctica lipase A (CALA) as feedstock and catalyst, respectively. The effect of transesterification conditions (water content, lipase load, substrate ratio, time and temperature) on FAME yield was evaluated and optimized by response surface methodology. Results showed that free lipase CALA had the excellent methanol resistance. And lipase CALA can simultaneously catalyze the transesterification of FPO and the esterification of free fatty acid (FFA) in the presence of excess water. Low mass transfer limitation of the system using lipase CALA as catalyst was found. The effect of reaction conditions on FAME yield decreased in order of water content > reaction time > enzyme load. The optimal reaction conditions were 16.6 wt% water content and 5.5 wt% enzyme load with 7:1 molar ratio of methanol to FPO at 30 °C for 22 h. The maximum FAME yield (94.6 ± 1.4%) was achieved under the optimal conditions. Arrhenius equation and Ea of the transesterification were lnV0 = 9.45–3964.4/T and 32.96 kJ/mol, respectively. K’m and Vmax were 6.75 × 10−1 mol/L and 2.38 × 10−2 mol/(L·min), respectively. This work supplied a clean method to produce biodiesel using waste frying oil with high water and FFA contents as feedstock and a green method using cheap free liquid lipase as catalyst.

Hydrophilic Glyceryl Ferulates Preparation Catalyzed by Free Lipase B from Candida antartica.

Ferulic acid (FA), 4-hydroxyl-3-methoxy-2-benzylacrylic acid, has antioxidant, anticancer and ultraviolet absorption activities. However, the low hydrophilicity of FA has limited its application. Glyceryl ferulate (FG), which is an all-natural hydrophilic derivative of FA, can be used as an antioxidant and UV filter in food and cosmetic formulations. However, the applications of FG in these fields are limited due to its low content in nature. In this work, free liquid lipase was firstly used as a catalyst for FG preparation. Several different free liquid lipases (Candida antartica lipase-B, Candida antartica lipase-A, Thermomyces lanuginosus (Lipozyme TL 100L)) were screened and compared. The effects of the transesterification parameters (time, temperature, enzyme load and substrate ratio) were optimized and evaluated by response surface methodology. A reaction thermodynamic investigation was also performed. The results showed that, among the tested free lipases, the maximum FG yield (84.8±1.5%) was achieved using free Candida antartica lipase-B. Under the optimized conditions (an atmospheric system, an enzyme load of 11.1% and a 20:1 molar ratio of glycerol to EF at 70°C for 39.5 h), the FG yield and EF conversion were 84.8±1.5% and 95.7±1.2%, respectively. The activation energies of FG formation and EF conversion were 56.4 and 58.0kJ/mol, respectively.

Biodiesel Production Catalyzed by a Methanol-Tolerant Lipase A from Candida antarctica in the Presence of Excess Water.

In this article, biodiesel was prepared using a novel free liquid lipase A from Candida antarctica (CALA) as a catalyst in the presence of excess water. The methanol tolerance of CALA was investigated. The effect of reaction conditions, including the molar ratio of soybean oil to methanol, water load, CALA load, reaction temperature, and reaction time, was evaluated. Reaction thermodynamics and kinetics were also analyzed. Results showed that free liquid lipase CALA showed excellent methanol tolerance in the reaction system using one-step addition of methanol and can be used to prepare biodiesel with water load of 12–14%. The influence of three transesterification variables on biodiesel yield was water load > temperature > time. The transesterification conditions were optimized by response surface methodology as follows: CALA load 5%, substrate molar ratio (soybean oil/methanol) 1:7, water load 14%, reaction time 26 h, and temperature 38 °C. The maximum biodiesel yield (92.4 ± 0.8%) was obtained under optimal conditions. The activation energy for biodiesel formation was 52.58 kJ/mol. Kinetic parameters Km′ and Vmax were 4.84 × 10–1 mol/L and 6.85 × 10–2 mol/(L·min), respectively. The mechanism of CALA-catalyzed transesterification was also proposed.

Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L

Biodiesel is a green and sustainable energy, which is a fatty acid alkyl ester as an alternative to petroleum diesel. In the work, phoenix tree seed oil, one kind of undeveloped woody plant resource, was used as the novel raw material for biodiesel preparation. Several free liquid lipases were used as biocatalysts to catalyze the transesterification of phoenix tree seed oil (PTSO) with methanol. Effects of transesterification variables (enzyme load, substrate ratio, transesterification time and temperature) on biodiesel preparation were evaluated and optimized using RSM. Results showed that PTSO was a good alternative for biodiesel preparation. Among these tested free liquid lipases, lipozyme TL100L from Thermomyces laguginosus showed the best performance for the transesterification. Transesterification variables were optimized and the maximum biodiesel yield (98.8 ± 1.1%) was achieved under the optimal conditions (enzyme load 10%, transesterification temperature 30 °C, substrate ratio (PTSO to methanol) 1:5 (mol/mol) and reaction time 6.98 h). The activation energy of biodiesel formation was 21.3 kJ/mol. Kinetic parameters K’m and Vmax were 2.55 × 10−1 mol/L and 6.9 × 10−3 mol/(L·min), respectively.

Enzymatic fatty acid ethyl esters synthesis using acid soybean oil and liquid lipase formulation

Acid oils represent a low-cost feedstock for biodiesel synthesis. The aim of this work was to study a single-step enzymatic esterification and transesterification process for fatty acid ethyl esters (FAEEs) production employing acid oils, ethanol, and liquid lipase formulation. The effects of lipase source (CALB, Palatase 20000 L, Lipolase 100 L, and Callera Trans L), temperature and enzyme ratio were studied in the transesterification of refined soybean oil with ethanol. The highest yield esters content (97%) was obtained at 30 °C with 10% vol/vol of Lipolase 100 L. The ethanolysis of acid soybean oil, using soybean oils with different acid contents (acid value (AV): 8.5, 50, and 90), was investigated using 10% vol/vol of lipase at 30 °C. The highest esters content (71%) was obtained employing acid oil with AV 50 and Palatase 20,000 L. The results showed the significant performance of soluble lipases in transesterification reactions of acid oils aiming for FAEEs synthesis.

Feeding strategies of methanol and lipase on eversa® transform‐mediated hydroesterification for FAME production

This work reports the influence of different feeding strategies of methanol and catalysts on fatty acid methyl esters (FAME) yield in a beef tallow hydroesterification reaction for biodiesel production catalyzed by liquid lipase. Soluble lipase from the Thermomyces lanuginosus microorganism, named Eversa® Transform, was selected for the reactions in this work. The feeding of methanol and lipase into the system was evaluated varying the methanol to fat molar ratio of 4.0:1 and 4.5:1 and the lipase load of 1.0 wt% and 1.45 wt% in relation to the mass of beef tallow utilized in each assay. The highest yield (85.08 %) of FAME was reached at 35 °C, methanol to fat molar ratio of 4.5:1, 1.0 wt% of lipase, 6.0 wt% of water in only 8 h of reaction, with a one‐step addition of lipase and methanol fed to the process at a constant flow of 3.0 g · h−1. Moreover, the reutilization process of the liquid lipase after four cycles was evaluated, which is a point rarely addressed in similar works available in the open literature. The findings of this paper demonstrate that the adoption of an appropriate strategy to feed inputs to the system is fundamental in order to take advantage of the maximum catalytic capacity of the lipase and consequently to obtain high yields in the process.

Enzymatic ethanolysis subjected to Schizochytrium biomass: Sequential processing for DHA enrichment and biodiesel production

To valorize oleaginous Schizochytrium sp. biomass, a novel sequential process was addressed and developed to simultaneously produce high value DHA-enriched glycerides and low value bulky biodiesels. In this process, Schizochytrium sp. biomass was first treated by alkaline protease; then the resulting biomass was directly subjected to enzymatic ethanolysis catalyzed by liquid enzymes from C. antarctica (CALA), T. lanuginosus (TL), R. miehei (RM) and A. oryzae (PLA). For all four liquid lipases, the alkaline protease treatment on Schizochytrium sp. biomass significantly enhanced total lipid recovery, biodiesel yield and yield of DHA-enriched glycerides. Under optimized conditions, CALA and TL achieved the highest biodiesel yields 76.28–76.66 wt% based on microalgae TFAs while PLA had the lowest (55.25 wt%). Moreover, CALA attained the highest DHA (77.93 wt%) and LC PUFAs (93.08 wt%) contents in glyceride fraction, suggesting a superior performance to concentrate DHA and LC PUFAs in glycerides. 13C NMR analysis illustrated that non-regiospecificity and unfavorability towards LC PUFAs of CALA made it possible to transesterify saturated fatty acids located in all 3 positions of microalgal oil; thus LC PUFAs were highly enriched in glycerides. Overall, the sequential process using enzymes and ethanol as green media was successfully established for the co-production of high-value microalgal bioproducts rich in LC PUFAs and sustainable biodiesels in a comprehensive manner.

Liquid lipase-catalyzed hydrolysis of gac oil for fatty acid production: Optimization using response surface methodology.

Fatty acids are valuable products because they have wide industrial applications in the manufacture of detergents, cosmetics, food, and various biomedical applications. In enzyme-catalyzed hydrolysis, the use of immobilized lipase results in high production cost. To address this problem, Eversa Transform lipase, a new and low-cost liquid lipase formulation, was used for the first time in oil hydrolysis with gac oil as a triglyceride source in this study. Response surface methodology was employed to optimize the reaction conditions and establish a reliable mathematical model for predicting hydrolysis yield. A maximal yield of 94.16% was obtained at a water-to-oil molar ratio of 12.79:1, reaction temperature of 38.9 °C, enzyme loading of 13.88%, and reaction time of 8.41 h. Under this optimal reaction condition, Eversa Transform lipase could be reused for up to eight cycles without significant loss in enzyme activity. This study indicates that the use of liquid Eversa Transform lipase in enzyme-catalyzed oil hydrolysis could be a promising and cheap method of fatty acid production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

Cost-effective biodiesel production from wet microalgal biomass by a novel two-step enzymatic process

In this study, a novel two-step enzymatic process was firstly established to produce microalgae biodiesel using wet Chlorella biomass. In the first hydrolysis step, to reduce energy consumption and effectively disrupt microalgal cell wall, among cellulase, hemicellulase, papain, lysozyme and pectinase, the highest hydrolysis efficiency (67.52%) was obtained by cellulase at pH 5.0 with enzyme dosage of 200 U/g dry biomass at 40 °C for 12 h. In the second transesterification step, compared with liquid CAL-A/B from Candida antarctica and PLA from Aspergillus oryzae, liquid lipase TL from Thermomyces lanuginosus achieved the highest biodiesel conversion at 81.15:1 (v/w) ethanol/g TFAs ratio in 78–83% water content with 100 PLU/g TFAs lipase loading at 25 °C for 48 h. Moreover, similar results were obtained with three Chlorella species by this process. Overall, this two-step enzymatic process was a green, low-energy and efficient method for cost-effective biodiesel production using wet microalgal biomass.

Soluble lipase-catalyzed synthesis of methyl esters using a blend of edible and nonedible raw materials.

This work investigates the use of blends of edible and nonedible raw materials as an alternative feedstock to fatty acid methyl esters (FAME) production through enzymatic catalysis. As biocatalyst, liquid lipase from Thermomyces lanuginosus (Callera™ Trans L), was used. Under reaction conditions of 35°C, methanol to feedstock molar ratio of 4.5:1 and 1.45% of catalyst load, the best process performance was reached using 9% of water concentration in the medium-yield of 79.9% after 480min of reaction. In terms of use of tallow mixed with soybean oil, the best yield was obtained when 100% of tallow was used in the process-84.6% after 480min of reaction-behavior that was associated with the degree of unsaturation of the feedstock, something by that time, not addressed in papers of the area. The results show that tallow can be used as an alternative to FAME production, catalyzed by soluble lipase.

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

Abstract 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.