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.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology

Utilizing whole cell biocatalyst instead of free or immobilized enzyme is a potential way to reduce the cost of catalyst in lipase-catalyzed biodiesel production. Rhizopus oryzae (R. oryzae) IFO4697 whole cell immobilized within biomass support particles (BSPs) was used for the methanolysis of soybean oil for biodiesel production in this paper. tert-Butanol was demonstrated to be an ideal reaction medium, in which the negative effects caused by substrate methanol could be eliminated effectively. A central composite design was adopted to study the effect of tert-butanol quantity, methanol quantity, water content and dry biomass of the immobilized cell on biodiesel (methyl ester) yield. Each factor was studied in five levels. Using response surface methodology, a quadratic polynomial equation was obtained for methyl ester yield by multiple regression analysis. Biodiesel yield of 72% could be obtained under the optimal conditions and further verification experiments confirmed the validity of the predicted model.     

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.     

复合脂肪酶协同催化制备生物柴油的研究

【目的】探讨复合酶协同催化体系在含水量较高的体系中催化油脂制备生物柴油的工艺条件。【方法】通过基因工程手段在毕赤酵母中分别高效分泌表达南极假丝酵母脂肪酶(CALB)和米根霉脂肪酶(ROL),构建CALB和ROL复合酶协同催化体系制备生物柴油,利用单因素实验优化工艺条件,以甲酯化得率作为复合酶协同催化体系效能的评价标准。【结果】优化工艺条件为:CALB?ROL最佳复合酶配比为7?3,每克大豆油中加入16 U的复合脂肪酶,甲醇与大豆油摩尔比为4?1,并按0 h时2?1醇油摩尔比,12 h和24 h时以1?1醇油摩尔比分批加入甲醇,含水量为30%-60%之间,40°C反应29-34 h,甲酯得率达到93%。【结论】该复合酶协同催化体系对环境友好,与常规酶法制备生物柴油工艺相比对酶的使用量和催化时间减少幅度都在50%以上,本复合酶协同催化体系能有效降低生物柴油制备成本,具有较好的工业化应用前景。     

Biodiesel production from pomace oil by using lipase immobilized onto olive pomace

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto olive pomace. Immobilized support material used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy (SEM). The maximum immobilization of T. lanuginosus was obtained as 18.67 mg/g support and the highest specific activity was 10.31 U/mg protein. The properties of immobilized lipase were studied. The effects of protein concentration, pH and buffer concentration on the immobilization and lipase activity were investigated. Biodiesel production using the immobilized lipase was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 93% at 25 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.     

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

Background

The enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.

Results

The Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.

Conclusions

The presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.     

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.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Dynamic modeling of biodiesel production from simulated waste cooking oil using immobilized lipase

The effectiveness of lipase immobilized on ceramic beads, in the production of biodiesel from simulated waste cooking oil in organic solvent system, was compared to that of free lipase. Experimental determination of the effect of concentrations of methanol on the rate of the enzymatic transesterification was experimentally determined. In addition, the effectiveness of lipases from bacterial and yeast sources for biodiesel production from simulated waste cooking oil was compared. A kinetic model was developed to describe the system, taking into consideration the mass transfer resistances of the reactants. Inhibition effects by both substrates on the interfacial reaction were also considered. The experimental results were used to determine the kinetic parameters of the proposed model and to determine the effect of mass transfer. 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.     

固定化全细胞催化可再生油脂合成生物柴油的稳定性

酶法合成生物柴油具有反应条件温和、醇用量小、无污染物排放、产物易分离回收等优点,越来越得到关注。全细胞催化剂,无需酶的提取和纯化,减少了酶活损失,有望大幅降低生产成本;Rhizopus oryzae IFO4697全细胞可以有效催化植物油脂合成生物柴油,进一步提高全细胞在催化植物油脂甲醇解制备生物柴油过程中的稳定性,对于工业放大具有重要意义。本实验对固定化全细胞Rhizopus oryzae IFO4697催化植物油脂合成生物柴油的稳定性进行了系统地研究,结果表明:反应体系水含量对于全细胞催化剂的反应活性和催化稳定性有重要影响,5%～15%含水量适宜;研究范围内,载体粒度及干燥方式对稳定性影响不显著;经过戊二醛交联后,全细胞催化油脂甲醇解反应的稳定性显著提高,1200h反应后,仍然可以保持75%的生物柴油得率;真空抽滤直接回用的方式有利于稳定性的保持。在优化条件下,回用20个批次,生物柴油得率可维持在80%。     

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.     

Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.     

Combined immobilized lipases for effective biodiesel production from spent coffee grounds

This work describes the enzymatic transesterification of the oil extracted from SCGs for synthesis of biodiesel as a promising alternative to diesel fuels based on petroleum. Biocatalysts from various sources were tested for biodiesel synthesis using coffee oil among which CaCO₃- immobilized Staphylococcus aureus and Bacillus stearothermophilus showed the highest conversion yields (61 ± 2.64% and 64.3 ± 1.53%, respectively) in 4 h. In further optimizing reaction parameters, methanol to oil molar ratio, biocatalyst quantity, water content, as well as incubation time and temperature markedly improved oil-to-biodiesel conversion up to 99.33 ± 0.57 % in a solvent free reaction after 12 h at 55 °C. A mixture of inexpensive CaCO₃-immobilized bacterial lipases at a 1:1 ratio was the best environment-friendly catalyst for biofuel synthesis as well as the ideal trade-off between conversion and cost. Obtained coffee biodiesel remained stable beyond 40 days at ambient storage conditions and its chemical characteristics were comparable to those of other known biodiesels according to the European requirements (EN14214). Collectively, SCGs, after oil extraction, could be an ideal substrate for the production of an environment-friendly biodiesel by using appropriate mixture of CaCO₃-immobilized lipases.     

Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.     

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.     

Alternative Oils Tested as Feedstocks for Enzymatic FAMEs Synthesis: Toward a More Sustainable Process

Previously isolated and characterized Pseudomonas lipases were immobilized in a low‐cost MP‐1000 support by a re‐loading procedure that allowed a high activity per weight of support. Immobilized LipA, LipC, and LipCmut lipases, and commercial Novozym® 435 were tested for fatty acid methyl ester (FAMEs) synthesis using conventional and alternative feedstocks. Triolein and degummed soybean oils were used as model substrates, whereas waste cooking oil and M. circinelloides oil were assayed as alternative, low cost feedstocks, whose free fatty acid (FFA), and acylglyceride profile was characterized. The reaction conditions for FAMEs synthesis were initially established using degummed soybean oil, setting up the best water and methanol concentrations for optimum conversion. These conditions were further applied to the alternative feedstocks and the four lipases. The results revealed that Pseudomonas lipases were unable to use the FFAs, displaying a moderate FAMEs synthesis, whereas a 44% FAMEs production was obtained when M. circinelloides oil was used as a substrate in the reaction catalysed by Novozym® 435, used under the conditions established for degummed soybean oil. However, when Novozym® 435 was tested under previously described optimal conditions for this lipase, promising values of 85 and 76% FAMEs synthesis were obtained for waste cooking oil and M. circinelloides oil, respectively, which might result in promising, nonfood, alternative feedstocks for enzymatic biodiesel production. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1209–1217, 2017     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica–PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 °C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1:15 at 55 °C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production.     

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system

In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with the support surface, resulting in activation of the enzyme catalytic center. Due to the interest in biodiesel production by mild enzymatic transesterification, this heterogeneous biocatalyst has been used in transesterification of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme.