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Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an immobilized Candida antarctica lipase

Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an... Background: Novozym 435, a commercial lipase from Candida antarctica, recombinant, expressed in Aspergillus niger, immobilized on macroporous acrylic resin, has been already described in the obtention of biodiesel. It is here evaluated in the production of a new biofuel that integrates the glycerol as monoglyceride (MG) together with two fatty acid ethyl esters (FAEE) molecules by the application of 1,3-selective lipases in the ethanolysis reaction of sunflower oil. Results: Response surface methodology (RSM) is employed to estimate the effects of main reaction. Optimum conditions for the viscosity, selectivity, and conversion were determined using a multifactorial design of experiments with three factors run by the software Stat Graphics version XV.I. The selected experimental parameters were reaction temperature, oil/ethanol ratio and alkaline environment. On the basis of RSM analysis, the optimum conditions for synthesis were 1/6 oil/EtOH molar ratio, 30°C, and 12.5 μl of NaOH 10 N aqueous solutions, higher stirring than 300 rpm, for 2 h and 0.5 g of biocatalyst. Conclusions: These obtained results have proven a very good efficiency of the biocatalyst in the studied selective process. Furthermore, it was allowed sixteen times the successive reuse of the biocatalyst with good performance. Keywords: Biodiesel; Enzyme biocatalysis; Candida antarctica lipase B (CALB); N435; Response surface methodology; Transesterification Background conditions. In order to shift the equilibrium towards the Currently, fossil fuel is globally the main primary source production of fatty acid methyl esters (FAME), an excess of energy. However, as its availability is becoming in- of methanol is normally utilized in the process to produce creasingly limited, it is accepted and assumed that the biodiesel. However, glycerol is always produced as a con- era of cheap and easily accessible fossil fuel is coming to taminant in addition to alkaline impurities that need to be its end. Thus, the production of biodiesel from renew- removed. The glycerol by-product is the main drawback able raw materials has become very important in recent of this method because it lowers the overall efficacy of the years as a potential alternative to partially satisfy the fu- process and its removal requires several consecutive water ture energy demands in the transport sector [1,2]. washing steps and hence creates a demand for a lot of In this respect, transesterification is currently the most water [4,5]. attractive and widely accepted methodology used for A series of alternative methods are being investigated biodiesel production [3]. This usually involves the use of to avoid the problems associated with the generation of homogeneous base catalysts operating under mild glycerol in the conventional process. They all are based on achieving various glycerol derivatives in the same transesterification process. In this way, the complex and * Correspondence: qo1lumad@uco.es Department of Organic Chemistry, University of Cordoba, Campus de expensive additional separation process of glycerol is Rabanales, Bldg. Marie Curie, 14014, Cordoba, Spain eliminated and the overall yield of the process increased. Seneca Green Catalyst S.A., Bldg Centauro, Technological Science Park of These novel methodologies are able to prepare methyl Cordoba, Rabanales XXI, 14014, Córdoba, Spain Full list of author information is available at the end of the article © 2014 Luna et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 2 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 esters of fatty acids from lipids using different acyl ac- conditions, impurities are not produced, and the biofuel ceptors instead of methanol in the transesterification produced exhibits similar physicochemical properties to process that result in glycerol derivatives as co-products those of conventional biodiesel. Last but not least, MGs [6]. For example, the transesterification reaction of triglyc- enhance biodiesel lubricity, as it was demonstrated by re- erides with dimethyl carbonate (DMC) [7], ethyl acetate cent studies [16-18]. Besides, the ethanol that is not spent [8], or methyl acetate [9] generates a mixture of three mol- in the enzymatic process remains in the reaction mixture, ecules of FAME or fatty acid ethyl esters (FAEE) and one and the blend obtained after the reaction can be used dir- of glycerol carbonate (GC) or glycerol triacetate (triacetin). ectly as fuel. In this respect, very recent studies [19-21] These mixtures, including the glycerol derivative mole- have shown that blends of diesel fuel and biodiesel con- cules, have physicochemical properties similar to biodiesel- taining ethanol reduce power output slightly than regular like biofuel [10]. In the present case, the atom efficiency is diesel. No significant difference in the emissions of CO , also improved because the total number of atoms involved CO, and NO between regular diesel and biodiesel or in the reaction is obtained in the final mixture. ethanol and diesel blends was observed. Furthermore, the On the other hand, we have recently developed a pro- use of these blends resulted in a reduction of particulate tocol for the preparation of a new biodiesel-like biofuel, matter. Consequently, such blends can be used in a diesel which integrates glycerol into monoglycerides via 1,3- engine without any modification despite the slightly re- regiospecific enzymatic transesterification of sunflower oil duced power output compared to pure diesel. Thus, the using free [11] and immobilized [12-14] porcine pancreatic Ecodiesel is currently utilized as a blend of fatty acid alkyl lipase (PPL). The operating conditions of such enzymatic esters with ethanol, alone or with any proportion of diesel process were more efficient compared to the preparation fuel [21,22]. method of conventional biodiesel and did not generate any The high cost of traditional industrial lipases restricted acidic or alkaline impurities. Thus, the Ecodiesel biofuel their use in biofuel production, but the current availability [12-15] obtained through the partial ethanolysis of triglyc- of the recombinant purified in sufficiently high quantities erides with 1,3-selective lipases is constituted by a mixture has helped to achieve the economic viability as the crucial of two parts of FAEE and one of monoacylglyceride (MG). factors affecting productivity of enzymatic biodiesel synthe- These glycerol derivative MGs are soluble components in sis are the suitable raw materials and the selected lipase. the FAEE mixture suitable for use as a biodiesel-like bio- The stability and catalytic efficiency of the latter can be im- fuel. In the current case, ethanol was used as a cheap re- proved by optimizing reaction conditions such as substrate agent, instead of the more expensive substrates such as concentrations, temperature, water activity, and alkaline dimethyl carbonate or methyl acetate. This procedure takes concentration of the enzyme's microenvironment [23]. In advantage of the 1,3-selective nature of the most known li- this respect, although Ecodiesel was initially obtained using pases, which allows to terminate the process in the second pig pancreatic lipases (PPL), remarkable results have been step of the alcoholysis to yield the mixture of 2 mol of also obtained with a low-cost purified microbial lipase, FAEE and 1 mol of MG as products (Figure 1). In this way, Lipopan50BG(NovozymesA/S,Bagsværd, Denmark) the glycerol is kept in the form of monoglyceride, which [15], from Thermomyces lanuginosus microorganism, avoids the production of glycerol as by-product, reducing usually used as bread emulsifier (bread improver) [24]. the environmental impact of the process. To our knowledge, this lipase has not been described as In summary, the enzymatic process to obtain this a biocatalyst in any chemical process. The application of biodiesel-like biofuel operates under much smoother an available lipase on an industrial scale is a significant Figure 1 Representative scheme of Ecodiesel production by application of 1,3-selective enzymatic catalysis. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 3 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 approximation to get an economically feasible biofuel In this respect, additional experiments were conducted production by enzymatic method. However, this lipase has with a pure lipase B from C. antarctica, recombinant a main drawback: it cannot be reused, since the purified from Aspergillus oryzae, powder, from Sigma-Aldrich. lipase extract is meant to be in a soluble form. The specific activity of this commercial lipase in the hy- Theaim of thepresent studyistoevaluateNovozym drolysis of tributyrin was >1,000 U/g. In this way, the ex- 435, a commercial lipase from Candida antarctica, recom- istence of possible differences in the catalytic behavior of binant (CALB), expressed in Aspergillus niger and immobi- CALB lipases by the influence of the support could be lized onto an acrylic macroporous resin [25]. Novozym 435 detected. has previously been used in the synthesis of conventional biodiesel as well as in other transesterification processes Results and discussion such as interesterification [26] and in lipase-catalyzed bio- Comparative chromatograms of standardized reaction diesel production by isopropanolysis of soybean oil [27]. In products this respect, Novozym 435 is also described very recently To identify the most characteristic components of biofuels in lipase-catalyzed simultaneous biosynthesis of biodiesel obtained by enzymatic alcoholysis, as well as to compare and glycerol carbonate, by using dimethyl carbonate as acyl their rheological properties, several commercial standards acceptor to obtain a biodiesel-like biofuel without glycerol of reference for FAME, FAEE, MG, and triacylglycerol generation [28]. Thus, in this study, it is intended to put in (TG) were used, as shown in Figure 2. Here, a representa- value the 1,3-selective behavior of these commercial lipases tive sample of monoglycerides of sunflower oil is also in- to make feasible the profitable production of alternative cluded, which was easily achieved by the substitution of biofuels [5], using an enzymatic approach. methanol or ethanol by glycerol, in a conventional alcoho- lysis process with KOH as homogeneous catalyst following standard experimental conditions. Methods Here, we can see that the different esters of fatty acids In this respect, in order to evaluate the influence of cru- (FAEs), which compose the lipid profile of the sunflower cial parameters (lipase amount, temperature, oil/ethanol oil, display retention times (RTs) slightly higher than those volumetric relationship, and alkaline environment) in of cetane (n-hexane) used as internal standard. Thus, the transesterification reaction, a multifactorial design whereas RT of cetane is around 10 min, all RTs of FAEs of experiments and response surface methodology (RSM) appear in the range of 16 to 26 min. These are composed using a multifactorial design of experiments with three of methyl, ethyl, and glycerol esters (the latter constitute factors run by the software Stat Graphics version XV.I MGs) of palmitic, stearic, linoleic, and oleic acids. Thus, were used. This study has been developed to optimize the palmitic acid (C16:0) derivatives are grouped in a narrow catalytic behavior of this 1,3-selective lipase (N435) in range of RT, 16 to 17 min. Derivatives of oleic (C18:1) and the partial ethanolysis of sunflower oil, to obtain a biofuel linoleic acid (C18:2) are grouped in RT of 19 to 21 min, that integrates glycerol as MG together with the different with the exception of glycerol ester of oleic acid, or what is FAEEs in the enzymatic ethanolysis process as well as with the same, the MG of the oleic acid has a different behavior, the excess of unreacted ethanol. This biofuel mixture cur- with a RT = 26 min. Glycerol RT appears at 5 min, before rently named Ecodiesel is able to directly operate diesel cetane. The absence of this compound in the obtained engines, alone or in whichever mixture with diesel fuel, chromatograms clearly demonstrates the selective nature without anymore separation or purification process. of the studied enzymatic transesterification reaction. Commercial sunflower oil was locally obtained. The In Figure 2, the presence of diacylglycerol (DG) with chromatographically pure ethyl esters of palmitic acid, ste- higher retention times, 40 to 60 min, can also be seen, aric acid, oleic acid, linoleic acid, and linolenic acid were which do not allow its integration into the GC chromato- commercially obtained from AccuStandard (New Haven, gram, so that it is necessary to determine DG together and CT, USA), and hexadecane (cetane) was obtained from TG by using an internal standard such as cetane here Sigma-Aldrich (St. Louis, MO, USA). Other chemicals like employed. It should be noted that the differences in RT absolute ethanol and sodium hydroxide were pure analyt- values between MG and DG are much higher than those ical compounds (99.5%) obtained commercially from existing between MG and FAME or FAEE, such as it is ex- Panreac (Castellar Del Valles, Spain). Novozym 435, the pected by the differences between their corresponding lipase B from C. antarctica (CALB), was kindly provided molecular weights. At the same time, it is clear that the by Novozymes A/S. This commercial lipase is produced FAMEs, FAEEs, and MGs display somewhat higher RT by submerged fermentation of a genetically modified values than cetane, but within the molecular weight range, A. niger microorganism and adsorbed on a macroporous which allows considering similar chemical-physical prop- acrylic resin. The specific activity of this commercial erties between the FAE and the hydrocarbons that consti- lipase in the hydrolysis of tributyrin was 3,000 U/g. tute diesel. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 4 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Retention Time (RT/min) Peak 0-7 Solvent (ethanol + dichloromethane 50:50) 5 Glycerol Internal Standard (n-hexadecane/ Cetane) 9-10 16 Palmitic FAME (16:0) 17 Palmitic MG (16:0) 18 Palmitic FAEE (16:0) 19 Estearic (18:0), Linoleic (18:2), Oleic (18:1) FAME 20 Estearic (18:0), Linoleic (18:2), Oleic (18:1) FAEE Estearic (18:0), Linoleic (18:2) MG 26 Oleic (18:1) MG DG + TG 40-60 Figure 2 Superimposed chromatograms of sunflower oil, FAME, FAEE, and MG. Since the retention times of different derivatives of fatty contain a high proportion of DG molecules, with high mo- acids are considered very closely related to the chemical- lecular weight and high viscosity values. Consequently, a physical properties of these compounds, the great similar- very high selectivity, indicating a very high percentage of ity of RT values obtained is a clear demonstration of the FAEEs and MGs, could promote a viscosity value close to similarity among the rheological properties of the different the petroleum diesel, so that the highest conversion value MGs with their corresponding FAMEs or FAEEs, which is not enough a guaranty of lower viscosity values. Thus, are crucial to allow its use as fuel able to substitute for both parameters will be provided as GC analysis results of petroleum products. Consequently, conversion is a reac- reaction products. tion parameter where all molecules (FAEE, MG, and DG) Taking into account that retention times of a complex obtained in the ethanolysis of TG are included (as %); it mixture of hydrocarbons constituting fossil diesel fuel will be considered as a very different parameter, with re- are ranging from 1 to 25 min, it is used as a reference spect to selectivity, where FAEEs and MGs are only in- value for different biofuels (FAME, FAEE, MG) as select- cluded (as %), all of them having RT values lower than ivity value, all those FAEs that present RT values coinci- 26 min. These molecules exhibit RT values similar to hy- dent with the hydrocarbons constituting diesel, or those drocarbons present in conventional diesel, so that they all with RT lower than 25 min, as it is expected that they could exhibit similar physicochemical and rheological also present similar physicochemical and rheological properties. However, a high conversion, even 100%, could properties to the conventional diesel. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 5 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Analysis of variance and optimization of the reaction ratio (v/v), alkaline environment is obtained by the addition parameters by RSM of different microliters of NaOH 10 N, and T is the reaction Theanalysis of variancemethods hasbecome veryat- temperature, tractive in reaction parameter optimization and in the evaluation of the effects of the parameters in the TG ½ConversionðÞ % ¼ 93:57 þ 5:06  T þ 7:82 transesterification reaction [9,15,27] due to their effect- R−6:42  T − 6:53  T iveness in the analysis of variables. Thus, results ob- R−3:25  R  pH tained operating under the 36 runs - each one with ð1Þ different experimental conditions, selected by the multi- factorial design of experiments with three factors, devel- ½SelectivityðÞ % ¼ 35:97 þ 6:26  T−2:25  pH oped by the software Stat Graphics version XV.I., where þ4:50  R þ 6:93  T  pH two of them are developed at three levels and the other 2 −12:22  T  R þ 6:13  pH at two levels, indicated in Table 1 - are shown in −6:61  pH  R Table 2. ð2Þ The quantity of supported lipase (N435) in all these ex- periments was fixed to 0.5 g. All experiments were dupli- ½ViscosityðÞ cSt ¼ 17 :27 þ 1:65  T−2:32  R cated in order to avoid experimental errors. Data was þ0:97  T þ 1:95  T  R fitted to a quadratic polynomial model using the software. þ1:20  R  pH : The quadratic polynomial model was highly significant ð3Þ and sufficient to explain the relationship between conver- sion/selectivity/kinematic viscosity and important experi- mental variables. Thus, the results of factorial design The surface plots in Figure 3b, described by the regres- suggested that the major factors affecting the transesterifi- sion model, were drawn to display the effects of the inde- cation, for the production of biofuels integrating glycerol pendent variables on conversion, selectivity, and kinematic as monoacylglycerols, were temperature and oil/ethanol viscosity. Here, the influence of the different variables in ratio (v/v). the conversion of the systems can be clearly seen. This The values of correlation coefficients, R , were 0.816 model showed that the optimum values for the parameters for conversion, 0.914 for selectivity, and 0.937 for kine- to maximize transesterification yield (conversion, selectiv- matic viscosity, respectively, which imply a good fit be- ity, kinematic viscosity) were intermediate temperatures tween models and experimental data in Pareto graphics, (30°C), maximum amount of aqueous NaOH 10 N added with respect to conversion, selectivity, and viscosity, as (50 μl), and the maximum oil/ethanol (v/v)ratio=6:1 indicated in Figure 3a. The adjusted correlation coeffi- studied. Conversions up to 100%, selectivities around 70%, 2 2 −1 cients R were 0.762, 0.889, and 0.918 for conversion, se- and values of kinematic viscosity about 10 to 15 mm s lectivity, and kinematic viscosity, respectively. Obtained could be achieved under these conditions, which in theory results pointed out that the temperature and oil/ethanol will render feasible the utilization of the obtained biofuel (v/v) ratios were also important parameters influencing in blends with diesel. For example, by the addition of only the conversion and viscosity in the systems (p < 0.05). 35% of diesel fossil, to this biofuel, a viscosity reduction at 2 −1 The software also allows to obtain equations (Equations 1, 4.8 mm s , a value within the acceptance limits of the 2, and 3), remarkably simpler as compared to initial ones EN 14214, is obtained. after the elimination of non-influent parameters in the model for conversion, selectivity, and kinematic viscosity. Effect of the amount of lipase These equations describe the model created and give solu- The effect of the amount of lipase in the reaction media is tions for the dependent variable based on the independent very important to select the correct reaction conditions. variable combinations, selecting the most significant in the This parameter was evaluated in order to choose the ne- response. Thus, taking into account that R is the oil/ethanol cessary amount of lipase that maximizes yield without mass transfer limitations. Previous optimized values ob- Table 1 Process parameters in factorial design: coded and tained from RSM (12 ml oil/3.5 ml EtOH, 30°C, 12.5 μl actual values NaOH 10 N, stirring speed higher than 300 rpm for 2 h) Variables Unit Levels were chosen. We can see in Figure 4 how the reaction yield and vis- −10 1 cosity were improved, so better values could be obtained Temperature °C 20 30 40 using more quantities of biocatalyst, but larger amounts Oil/ethanol ratio (v/v) ml/ml 12/1,75 - 12/3.5 of enzymes will have a detrimental effect on the eco- Alkaline environment μl (NaOH 10 N) 8 (12.5) 10 (5) 12 (50) nomics of the process. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 6 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Table 2 Experiment matrix of factorial design and the response obtained for conversion, selectivity, and viscosity Run Parameters 2 −1 Temperature Oil/ethanol ratio pH Conversion (%) Selectivity (%) Kinematic viscosity (mm s ) 11 −1 0 81.4 45.99 20.3 21 −1 −1 84.6 46.1 21.3 30 −1 −1 78.6 29.2 21.0 4 −1 −1 0 59.1 11.7 20.8 5 −1 1 1 100 40.4 13.3 6 1 1 1 100 39.6 22.3 7 −1 −1 −1 63.6 23.3 22.1 80 −1 1 89.5 34.1 19.0 9 0 1 0 95.7 31.4 15.2 10 0 −1 0 100 44.2 20.1 11 0 1 1 95.2 37.5 15.7 12 1 −1 1 100 72.5 18.6 13 1 1 −1 100 46.6 18.2 14 1 1 0 86.1 36.1 19.8 15 −1 1 0 94.9 46.8 13.4 16 −11 −1 100 70.9 10.8 17 0 1 −1 100 52.6 12.5 18 −1 −1 1 77.5 17.8 20.2 Repeated experiments 19 0 −1 0 100 43.4 22.5 20 −1 1 1 100 39.7 13.2 21 −1 −1 −1 64.4 22.6 20.1 22 −1 1 0 95.8 46.1 13.4 23 1 1 −1 100 47.5 16.2 24 0 1 −1 100 51.9 14.5 25 −11 −1 100 69.6 10.9 26 0 1 0 96.5 36.5 14.6 27 1 1 1 100 38.8 22.2 28 0 −1 −1 80.1 28.6 19.0 29 1 −1 1 100 71.4 18.5 30 −1 −1 0 60.2 11.0 20.3 31 1 −1 0 82.2 45.2 20.3 32 1 1 0 86.9 33.4 19.5 33 0 −1 1 90.3 33.4 18.1 34 0 1 1 97.0 36.8 15.1 35 1 −1 −1 85.4 43.4 21.6 36 −1 −1 1 69.7 16.3 20.6 Study of enzyme activity in successive reactions method does not provide information on the combined ef- Since we have been doing these successive reactions, the fect of various reaction parameters, but it is useful to de- reaction conditions have been changed such that the pro- termine the influence of isolated variables. Because our cedure enables the study according to OVAT (‘variable at research group had previous information about other lip- one time’) methodology in which initial conditions are set ase enzymes [12-15] and what were the most important and variables to study have been changed one by one. This reaction variables, it was decided to use this experimental Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 7 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 (a) (b) (a.1) (b.1) (a.2) (b.2) (b.3) (a.3) Figure 3 Pareto graphics and response surface plots. (a) Pareto graphics: conversion (a.1), selectivity (a.2), and viscosity (a.3). (b) Response surface plot of more influential parameters: conversion (b.1), selectivity (b.2), and viscosity (b.3). methodology in order to evaluate in more detail the in- environments, temperatures, and relative oil/ethanol ratio) fluence of those variables in the selected intervals for previously determined by the RSM studies. These experi- each study. mental conditions were similar to those previously ob- In this way, reaction tests of N435 lipase systems have tained with T. lanuginosus, Lipopan 50 BG Lipopan [15]. been carried out under the optimum conditions (alkaline In this respect, Table 3 shows a collection of the achieved Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 8 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 results in the transesterification reaction with this biocata- Graphic 2 lyst. Thus, in addition to obtain specific information about the influence of certain parameters (alkaline environment, Visc (cSt) temperature, etc.) on the behavior of the N435, its ability to be reused could be checked. Visc In this sense, from the conversion, given that we start from 0.01 mol of TG, with 2 h as reaction time and 0.5 g 5 of catalyst system (lipase CALB immobilized on acrylic polymer), we can calculate the transformation enzymatic 0,05 0,1 0,2 0,3 0,4 0,5 capacity: turnover frequency (TOF number) expressed in Lipase Weight (g) micromoles of TG transformed per minute and per gram of catalyst (supported lipase). In order to be able to obtain Graphic 1 the enzyme activity of the immobilized CALB, some re- search experiments with a purified powdered commercial Yield (%) Sel 50 lipase B from C. antarctica, recombinant from A. oryzae, Conv have been conducted. Thus, operating under identical ex- perimental conditions with those used in experiments car- ried out with N435, but using only 0.01 g of purified CALB in free form, a conversion of 50.6% was obtained. Excluding the possible influence of support effects in the lipase activity, and taking into account that 0.5 g of CALB 0,05 0,1 0,2 0,3 0,4 0,5 Lipase Weight (g) immobilized enzyme produced a 47.7% conversion, it can be inferred that N435 has a catalytic activity corre- Figure 4 Influence of lipase amount of supported CALB in the performance of ethanolysis reactions, developed under sponding to the 1.9% of the obtained catalytic activity standard conditions. Conversion and selectivity (graphic 1) and by thefreeCALB. kinematic viscosity in cSt (graphic 2). Therefore, to obtain a reference value, and assuming that the activity of the immobilized enzyme is similar to that of the free enzyme, it can be considered that the N435 may contain about 1.9 wt% of immobilized en- zyme. In any case, it works with this equivalent enzyme Table 3 Achieved results in the transesterification reaction with biocatalyst Run number NaOH amount Temperature EtOH/oil Viscosity Selectivity Conversion TOF Enzyme activity 1 25 30 3.5/12 (6:1) 10.5 34.9 47.7 79.5 0.24 2 25 30 3.5/12 (6:1) 13.7 23.6 32.9 54.83 0.35 3 25 30 3.5/12 (6:1) 12.8 24.1 31.8 53.00 0.36 4 25 30 3.5/12 (6:1) 13.3 25.6 33.9 56.50 0.33 5 25 30 3.5/12 (6:1) 12.8 29.1 35.6 59.33 0.32 6 12.5 35 3.5/12 (6:1) 13.8 14.5 20.1 33.50 0.57 7 12.5 30 3.5/12 (6:1) 12.3 45.4 57.9 96.50 0.20 8 12.5 25 3.5/12 (6:1) 14.6 21.4 25.8 43.00 0.44 9 12.5 40 3.5/12 (6:1) 20.9 7.6 4.0 6.67 2.85 10 12.5 30 1.75/12 (3:1) 18,8 17,9 27,4 45.67 0.42 11 12.5 20 3.5/12 (6:1) 18.1 17.5 22.4 37.33 0.51 12 12.5 30 2.3/12 (4:1) 20.0 17.6 21.4 35.67 0.53 13 12.5 30 2.9/12 (5:1) 10.6 34.1 47.7 79.50 0.24 14 12.5 30 3.5/12 (6:1) 10.4 39.1 51.9 86.50 0.22 15 12.5 30 4.1/12 (7:1) 17.9 14.1 16.8 28.00 0.68 16 12.5 30 4.7/12 (8:1) 16.3 17.1 21.8 36.33 0.52 −1 −1 Viscosity (cSt), conversion (%), selectivity (%), turnover frequencies (TOF in μmol min g cat ), and enzyme activities (U/mg lipase) of the obtained biodiesel by using the same biocatalyst (0.5 g of N435) in successive reactions under the evaluated different experimental conditions: temperature (°C), NaOH amount (μl of NaOH 10 N solution), and EtOH/oil ratio (ml/ml or mol/mol). Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 9 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 activity. From this parameter, a reference value of the reaction yield is clearly displayed and we reached simi- catalytic activity of the immobilized enzymes, expressed lar conclusions to those obtained in the analysis of vari- in milligrams of enzyme required to convert 1 μmol of ance (ANOVA) study by RSM; the optimum reaction TG per minute (U/mg), can also be calculated. parameters for the enzymatic ethanolysis reaction with If we subdivide the results in Table 3 in terms of the lipase N435, CALB (C. antarctica lipase B) immobilized variable that has been changed each time in the study on acrylic resin, are 30°C and 12.5 μlof10NNaOH, with OVAT methodology and if we organize these re- with 1:6 oil/ethanol molar ratio. sults in their corresponding graphics (Figure 5), the in- Also, we can obtain from the data in Table 3 that fluence of each variable on the transesterification N435 may be reused repeatedly. Thus, 16 reactions were (a) (b) (a.2) (b.2) Visc Graphic 2 Graphic 2 (cSt) Visc Visc (cSt) (cSt) Visc (cSt) 5 5 20º 25º 30º 35º 40º 3:01 4:01 5:01 6:01 7:01 8:01 Temperature (ºC) Ethanol/Oil Molar Ratio (a.1) (b.1) Graphic 1 Graphic 1 Yield 70 Sel 60 Yield (%) Conv Sel (%) 40 Conv 20º 25º 30º 35º 40º Temperature (ºC) Ethanol/Oil Molar Ratio Figure 5 Reaction temperature and oil/ethanol molar ratio. Influence of the (a) reaction temperature and (b) oil/ethanol molar ratio in the enzymatic ethanolysis reaction performance of supported CALB developed under standard conditions. Conversion and selectivity (graphic 1) and viscosity in cSt (graphic 2). Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 10 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 developed successively without an appreciable loss in cata- to work in the successive reuses, so that centrifugation of lytic activity in the ethanolysis reaction of sunflower oil, op- reaction products is necessary for catalyst recovery to avoid erating under different experimental conditions. However, important losses of catalyst. This additional centrifugation an important drawback is associated to its acrylic poly- step may constitute a major drawback for the application of meric character. Thus, to recover the immobilized lipase on the process on an industrial scale. macroporous resin, after each successive reaction, it is ne- cessary to carry out the centrifugation of reaction products. Experimental Finally, this research, developed in order to improve a Ethanolysis reactions new methodology to integrate the glycerol as different These reactions were performed according to the experi- monoacylglycerol molecules, is in connection with pre- mental procedure previously described [12-15] to deter- cedent researches performed to determine the optimal mine the optimal conditions for obtaining the selective experimental conditions of the selective ethanolysis reac- ethanolysis reaction, such as alkaline environment, amount tion. In this respect, the results here obtained resemble of lipase, the oil/ethanol molar ratio (v/v), and temperature. those described with the commercial PPL lipases [12-14] Thus, enzymatic assays are carried out with 9.4 g (12 ml, and with a low-cost purified lipase, [15] from T. lanugi- 0.01 mol) of commercial sunflower oil at controlled tem- nosus, Lipopan 50 BG (Novozymes A/S), widely used in peratures (20°C to 40°C) in a 25-ml round bottom flask. the bakery industry as an emulsifier [24]. In this respect, Reaction mixtures were stirred with a conventional mag- the possibility of application of commercial immobilized netic stirrer at a stirring speed higher than 300 rpm to lipases in this process is now explored to exploit the op- avoid mass transfer limitations, along a reaction time of portunity of its reuse, thus lowering its operational cost 2 h. Variable oil/alcohol volume ratios at different alkaline and consequently increasing the economic potential of environments and different quantities of lipase are studied. their industrial application. Thus, in this study, the be- The different oil/ethanol ratios (v/v) are obtained by intro- havior of a commercial supported lipase, Novozym 435, ducing absolute ethanol volumes in the range of 1.75 to is determined because this may involve some techno- 3.5 ml, and the influence of different amounts of lipase is logical advantages in its development, so that this study studied in the range of 0.05 to 0.5 g. The influence of alka- has the aim to prove its viability, its yield, and its possi- line environment values was achieved by adding different bility of reuse, what will mean an important advance in volumes (12.5 to 50 μl) of 10 N NaOH aqueous solution. its biotechnological applications in the biofuel enzymatic In this regard, a blank reaction in the presence of the high- production [5]. estquantity ofsolution ofNaOHwas performedtorule Currently, results obtained in this study indicate that out a potential contribution from the homogeneous NaOH this commercial supported lipase is specially efficient in catalyzed reaction. Less than 10% conversion of the starting obtaining 1,3-selective ethanolysis processes, where gly- material was obtained, so that a homogenous base catalysis cerol is maintained as MG in the biofuel mixture, with contribution can be considered as negligible under the in- the different obtained FAEEs and together to the excess vestigated conditions. of unreacted ethanol. In this way, a new kind of biodiesel In this respect, the NaOH solution, to operate as a currently named Ecodiesel is achieved, constituted by a transesterification catalyst, needs usually to be used under mixture of monoacylglycerols and FAEEs mainly (1/2 higher temperatures and under higher catalyst concentra- nominally), which can be used in different blends with tions (1 to 2 wt%), enough to generate sodium methoxide diesel fuel, without anymore separation or purification or ethoxide, the true catalyst of the homogeneous transes- process. This new biofuel can be obtained with the help terification reaction. Thus, the use of a very small concen- of N435 at very short reaction times (1 to 2 h) and tration of NaOH works as a promoter of the process, under soft reaction conditions. Besides, not only a higher through its effect on the lipase biocatalytic activity. Ac- atomic yield is achieved, with respect to the conven- cordingly, NaOH ions are responsible for the pH environ- tional biodiesel reaction (because no glycerol by-product ment that affects the enzyme yield, because depending on is generated), but also a purification step of residual gly- the pH, the enzymatic protein offers different structures, cerol is not necessary, so it can be used directly after its more or less effective in the biocatalytic action. production. All variables were studied and optimized according to Despite the good results obtained in the transesterifica- a factorial experimental design and a response surface tion reaction of sunflower oil with ethanol to produce FAEE methodology. and MG, unfortunately, the main drawback of this com- mercial biocatalyst is the low stability of the organic poly- meric little spheres which constitute the catalytic powder Analytical method that easily disintegrates in the first ethanolysis reaction and Reaction products were monitored by capillary column gas becomes a gelatinous material which is very uncomfortable chromatography, using a Varian 430-GC gas chromatograph Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 11 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 (Varian Inc., Palo Alto, CA, USA), connected to a HT5 ca- Viscosities were determined in a capillary viscometer pillary column (25 m × 0.32 mm ID × 0.1 μm, SGE, Oswald Proton Cannon-Fenske Routine Viscometer 33200, Supelco) with a flame ionization detector (FID) at 450°C size 150. This is based on determining the time needed for and splitless injection at 350°C. Helium is used as carrier a given volume of fluid passing between two points marked gas with a flow of 1.5 ml/min. A heating ramp from 90°C on the instrument. The kinematic viscosity is given by the to 200°C at a rate of 7°C/min has been applied, followed ratio between the dynamic viscosity (h,inPoise,g/cms) 3 2 by another ramp from 200°C to 360°C at a rate of 15°C/ and the density (r,ing/cm ) υ = h/r (in cm /s or centis- min, maintaining the temperature of the oven at 360°C for tokes (cSt), mm /s). Samples, previously centrifuged at 10 min using as internal standard n-hexadecane (cetane) 3,500 rpm for 10 min and filtered at 50°C, are immersed in to quantify the content of ethyl esters and the different a thermostatic bath at 40°C for 15 min, making sure that glycerides (mono-, di-, and triglycerides) with the help of the temperature is stable. Then, samples are introduced several commercial standard fatty acid esters. This method into the viscometer and this, in turn, in the water bath, allows us to make a complete analysis of the sample in a making sure that it is rigorously positioned vertically, with single injection and in a time not longer than 60 min, the bottom end at a minimum distance of 2 cm from the which simplifies the process and increases the speed of floor of the bath [12-15]. analysis [12-15]. Considering that sunflower oil is constituted by a mix- Experimental design ture of fatty acids in variable proportion (mainly linoleic, The effect of process parameters in the enzymatic trans- oleic, palmitic, and stearic acids), the results obtained esterification reaction to obtain the optimum conditions are expressed as the relative amounts of the correspond- for the viscosity, selectivity, and conversion was studied ing ethyl esters (FAEE, fatty acid ethyl esters), monoglyc- using a multifactorial design of experiments with three erides (MG), and diglycerides (DG) that are integrated in factors run by the software Stat Graphics version XV.I. the chromatogram. The amount of triglycerides (TG) Two of them are developed at three levels and the last which has not reacted is calculated from the difference one at two levels, so that it gives 36 runs. The experi- to the internal standard (cetane). Thus, the conversion ments were performed in random order. The experi- includes the total amount of triglyceride transformed mental parameters selected for this study were reaction (FAEE + MG + DG) in the ethanolysis process, and se- temperature, oil/ethanol ratio (v/v), and different alka- lectivity makes reference to the relative amount of FAEE line environments obtained by the addition of variable + MG obtained. The latter are the ones having retention volumes (in μl) of NaOH 10 N. Table 1 shows the coded times close to the cetane standard, which is the refer- and actual values of the process parameters used in the ence hydrocarbon for diesel fuel. design matrix. Viscosity measurements Statistical analysis The transesterification reactions of oils and fats are ba- The experimental data obtained from experimental de- sically carried out to obtain an important reduction in sign were analyzed by response surface methodology the viscosity of these materials, as they share similar (RSM) [9,15,27]. A mathematical model, following a values in all of other chemical-physical significant pa- second-order polynomial equation, was developed to de- rameters with the fossil diesel except the viscosity. In scribe the relationships between the predicted response this respect, most oils exhibit viscosities in the range of variable (viscosity, conversion, and selectivity) and the 30 to 45 mm /s cSt values, while the fossil diesel is in independent variables of reaction conditions, as shown the range of 2.5 to 6 cSt values. Thus, due to the import- in the Equation 4, where Y is the predicted response ance of viscosity for the correct running of diesel en- variable; β , β , β , and β are the intercept, linear, quad- 0 i ii ij gines, this parameter becomes a critical factor to change ratic, and interaction constant coefficients of the model, the chemical-physical properties of vegetable oils before respectively; and X and X (i =1, 3; j =1, 3; i ≠ j) repre- i j their use as biofuel. The transesterification process of sent the coded independent variables. oils and fats is actually developed in order to obtain a 3 3 3 X X XX noticeable lowering of viscosity in oils to employ the Y ¼ β þ β x þ β x þ β x x ð4Þ i i j 0 0 ii ij resulting product as biofuel in current existing diesel en- i¼1 i¼1 i<j¼1 gines. Thus, accurate viscosity measurements are critical to assess the quality of biofuels produced, since unsuit- Response surface plots were developed using the fitted able viscosity values can decisively affect the correct quadratic polynomial equation obtained from regression working conditions of the diesel engine. Therefore, the analysis, holding one of the independent variables at characterization of this parameter is essential to evaluate constant values corresponding to the stationary point the result obtained in the process of ethanolysis. and changing the order of two variables. The quality of Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 12 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 the fit of the polynomial model equation was evaluated 2. Luque R, Herrero-Davila L, Campelo JM, Clark JH, Hidalgo JM, Luna D, Marinas JM, Romero AA (2008) Biofuels: a technological perspective. by the coefficient of determination R , and its regression Energy Environ Sci 1:542–564 coefficient significance was checked with F-test. Con- 3. Oh PP, Lau HLN, Chen JH, Chong MF, Choo YM (2012) A review on firmatory experiments were carried out in order to va- conventional technologies and emerging process intensification (PI) methods for biodiesel production. Renew Sust Energy Rev 16:5131–5145 lidate the model, using combinations of independent 4. Saleh J, Dube MA, Tremblay AY (2011) Separation of glycerol from FAME variables which were not part of the original experimen- using ceramic membranes. Fuel Process Technol 92:1305–1310 tal design but within the experimental region. 5. Calero J, Luna D, Sancho ED, Luna C, Posadillo A, Bautista FM, Romero AA, Berbel J, Verdugo C (2014) Technological challenges for the production of biodiesel in arid lands. J Arid Environ 102:127–138 Conclusions 6. Ganesan D, Rajendran A, Thangavelu V (2009) An overview on the recent Novozym 435 lipase was evaluated in the 1,3-selective advances in the transesterification of vegetable oils for biodiesel production using chemical and biocatalysts. Rev Environ Sci Biotech 8:367–394 ethanolysis of sunflower oil to integrate the glycerol as 7. Ilham Z, Saka S (2010) Two-step supercritical dimethyl carbonate method monoacylglycerol molecules. On the basis of RSM ana- for biodiesel production from Jatropha curcas oil. Bioresour Technol lysis, the optimum conditions for synthesis were 1:6 molar 101:2735–2740 8. Kim SJ, Jung SM, Park YC, Park K (2007) Lipase catalyzed transesterification oil/EtOH ratio, 30°C, and 12.5 μl NaOH 10 N aqueous so- of soybean oil using ethyl acetate, an alternative acyl acceptor. Biotechnol lutions, with stirring speed higher than 300 rpm for 2 h Bioprocess Eng 12:441–445 and 0.5 g of biocatalyst. Accordingly, its ability to be re- 9. Tan KT, Lee KT, Mohamed AR (2011) A glycerol-free process to produce biodiesel by supercritical methyl acetate technology: an optimization study peatedly reused could open a new way for the production via response surface methodology. Bioresour Technol 102:3990–3991 of alternative biodiesel using an enzymatic approach, 10. Casas A, Ruiz JR, Ramos MJ, Perez A (2010) Effects of triacetin on biodiesel which is technically feasible and economically viable. The quality. Energy Fuels 24:4481–4489 11. Verdugo C, Luque R, Luna D, Hidalgo JM, Posadillo A, Sancho ED, Rodriguez S, main drawback is the permanent need for centrifugation Ferreira-Dias S, Bautista F, Romero AA (2010) A comprehensive study of to recover the biocatalyst for the next reuse. reaction parameters in the enzymatic production of novel biofuels integrating glycerol into their composition. Bioresour Technol 101:6657–6662 Abbreviations 12. Caballero V, Bautista FM, Campelo JM, Luna D, Marinas JM, Romero AA, ANOVA: analysis of variance; CALB: Candida antarctica lipase B; Hidalgo JM, Luque R, Macario A, Giordano G (2009) Sustainable preparation DG: diacylglycerol; FAE: fatty acid esters; FAEE: fatty acid ethyl esters; of a novel glycerol-free biofuel by using pig pancreatic lipase: partial 1,3- FAME: fatty acid methyl esters; MG: monoacylglycerol; OVAT: variable at one regiospecific alcoholysis of sunflower oil. Process Biochem 44:334–342 time; RSM: response surface methodology; TG: triacylglycerol; TOF: turnover 13. Luna D, Posadillo A, Caballero V, Verdugo C, Bautista FM, Romero AA, frequency. Sancho ED, Luna C, Calero J (2012) New biofuel integrating glycerol into its composition through the use of covalent immobilized pig pancreatic lipase. Competing interests Int J Mol Sci 13:10091–10112 The authors declare that they have no competing interests. 14. Luna C, Sancho E, Luna D, Caballero V, Calero J, Posadillo A, Verdugo C, Bautista FM, Romero AA (2013) Biofuel that keeps glycerol as Authors' contributions monoglyceride by 1,3-selective ethanolysis with pig pancreatic lipase CL, CV, EDS, DL, JC, AP, FMB, and AAR have made substantive intellectual covalently immobilized on AlPO support. Energies 6:3879–3900 contributions to this study, making substantial contributions to the 15. Verdugo C, Luna D, Posadillo A, Sancho ED, Rodriguez S, Bautista F, Luque conception and design of it as well as to the acquisition, analysis, and R, Marinas JM, Romero AA (2011) Production of a new second generation interpretation of data. All of them have been also involved in the drafting biodiesel with a low cost lipase derived from Thermomyces lanuginosus: and revision of the manuscript. All authors read and approved the final optimization by response surface methodology. Catal Today 167:107–112 manuscript. 16. Xu YF, Wang QJ, Hu XG, Li C, Zhu XF (2010) Characterization of the lubricity of bio-oil/diesel fuel blends by high frequency reciprocating test rig. Energy 35:283–287 Acknowledgements 17. Haseeb A, Sia SY, Fazal MA, Masjuki HH (2010) Effect of temperature on Grants from the Spanish Ministry of Economy and Competitiveness (Project tribological properties of palm biodiesel. Energy 35:1460–1464 ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ2011-28954-C02-02), FEDER funds and Junta de 18. Wadumesthrige K, Ara M, Salley SO, Ng KYS (2009) Investigation of lubricity Andalucía FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully characteristics of biodiesel in petroleum and synthetic fuel. Energy Fuels acknowledged by the authors. We are also grateful to Novozymes A/S, 23:2229–2234 Denmark, for the kind supply of the macroporous resin immobilized lipase 19. Çelikten I (2011) The effect of biodiesel, ethanol and diesel fuel blends on the from Candida antarctica (Novozym 435). performance and exhaust emissions in a diesel engine. GU J Sci 24:341–346 20. Cheenkachorn K, Fungtammasan B (2009) Biodiesel as an additive for Author details diesohol. Int J Green Energy 6:57–72 Department of Organic Chemistry, University of Cordoba, Campus de 21. Jaganjac M, Prah IO, Cipak A, Cindric M, Mrakovcic L, Tatzber F, Ilincic P, Rabanales, Bldg. Marie Curie, 14014, Cordoba, Spain. Crystallographic Studies Rukavina V, Spehar B, Vukovic JP, Telen S, Uchida K, Lulic Z, Zarkovic N Laboratory, Andalusian Institute of Earth Sciences, CSIC, Avda. Las Palmeras, n°4, (2012) Effects of bioreactive acrolein from automotive exhaust gases on 18100, Armilla, Granada, Spain. Department of Microbiology, University of human cells in vitro. Environ Toxicol 27:644–652 Cordoba, Campus de Rabanales, Ed. Severo Ochoa, 14014, Cordoba, Spain. 22. Pang XB, Mu YJ, Yuan J, He H (2008) Carbonyls emission from ethanol-blended Seneca Green Catalyst S.A., Bldg Centauro, Technological Science Park of gasoline and biodiesel-ethanol-diesel used in engines. Atmos Environ Cordoba, Rabanales XXI, 14014, Córdoba, Spain. 42:1349–1358 23. Szczesna-Antczak M, Kubiak A, Antczak T, Bielecki S (2009) Enzymatic Received: 22 April 2014 Accepted: 22 July 2014 biodiesel synthesis - key factors affecting efficiency of the process. Renew Published: 31 August 2014 Energy 34:1185–1194 24. Moayedallaie S, Mirzaei M, Paterson J (2010) Bread improvers: comparison of a range of lipases with a traditional emulsifier. Food Chem 122:495–499 References 1. Demirbas A (2009) Political, economic and environmental impacts of 25. Xu Y, Nordblad M, Woodley JM (2012) A two-stage enzymatic ethanol-based biofuels: a review. Appl Energy 86:108–117 biodiesel production in a packed bed reactor. J Biotechnol 162:407–414 Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 13 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 26. Yara-Varon E, Joli JE, Torres M, Sala N, Villorbina G, Mendez JJ, Canela- Garayoa R (2012) Solvent-free biocatalytic interesterification of acrylate derivatives. Catal Today 196:86–90 27. Chang C, Chen JH, Chang CMJ, Wu TT, Shieh CJ (2009) Optimization of lipase-catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor using response surface methodology. N Biotechnol 26:187–192 28. Min JY, Lee EY (2011) Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate. Biotechnol Lett 33:1789–1796 doi:10.1186/s40643-014-0011-y Cite this article as: Luna et al.: Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an immobilized Candida antarctica lipase. Bioresources and Bioprocessing 2014 1:11. 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Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an immobilized Candida antarctica lipase

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2014 Luna et al. ; Licensee Springer
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Abstract

Background: Novozym 435, a commercial lipase from Candida antarctica, recombinant, expressed in Aspergillus niger, immobilized on macroporous acrylic resin, has been already described in the obtention of biodiesel. It is here evaluated in the production of a new biofuel that integrates the glycerol as monoglyceride (MG) together with two fatty acid ethyl esters (FAEE) molecules by the application of 1,3-selective lipases in the ethanolysis reaction of sunflower oil. Results: Response surface methodology (RSM) is employed to estimate the effects of main reaction. Optimum conditions for the viscosity, selectivity, and conversion were determined using a multifactorial design of experiments with three factors run by the software Stat Graphics version XV.I. The selected experimental parameters were reaction temperature, oil/ethanol ratio and alkaline environment. On the basis of RSM analysis, the optimum conditions for synthesis were 1/6 oil/EtOH molar ratio, 30°C, and 12.5 μl of NaOH 10 N aqueous solutions, higher stirring than 300 rpm, for 2 h and 0.5 g of biocatalyst. Conclusions: These obtained results have proven a very good efficiency of the biocatalyst in the studied selective process. Furthermore, it was allowed sixteen times the successive reuse of the biocatalyst with good performance. Keywords: Biodiesel; Enzyme biocatalysis; Candida antarctica lipase B (CALB); N435; Response surface methodology; Transesterification Background conditions. In order to shift the equilibrium towards the Currently, fossil fuel is globally the main primary source production of fatty acid methyl esters (FAME), an excess of energy. However, as its availability is becoming in- of methanol is normally utilized in the process to produce creasingly limited, it is accepted and assumed that the biodiesel. However, glycerol is always produced as a con- era of cheap and easily accessible fossil fuel is coming to taminant in addition to alkaline impurities that need to be its end. Thus, the production of biodiesel from renew- removed. The glycerol by-product is the main drawback able raw materials has become very important in recent of this method because it lowers the overall efficacy of the years as a potential alternative to partially satisfy the fu- process and its removal requires several consecutive water ture energy demands in the transport sector [1,2]. washing steps and hence creates a demand for a lot of In this respect, transesterification is currently the most water [4,5]. attractive and widely accepted methodology used for A series of alternative methods are being investigated biodiesel production [3]. This usually involves the use of to avoid the problems associated with the generation of homogeneous base catalysts operating under mild glycerol in the conventional process. They all are based on achieving various glycerol derivatives in the same transesterification process. In this way, the complex and * Correspondence: qo1lumad@uco.es Department of Organic Chemistry, University of Cordoba, Campus de expensive additional separation process of glycerol is Rabanales, Bldg. Marie Curie, 14014, Cordoba, Spain eliminated and the overall yield of the process increased. Seneca Green Catalyst S.A., Bldg Centauro, Technological Science Park of These novel methodologies are able to prepare methyl Cordoba, Rabanales XXI, 14014, Córdoba, Spain Full list of author information is available at the end of the article © 2014 Luna et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 2 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 esters of fatty acids from lipids using different acyl ac- conditions, impurities are not produced, and the biofuel ceptors instead of methanol in the transesterification produced exhibits similar physicochemical properties to process that result in glycerol derivatives as co-products those of conventional biodiesel. Last but not least, MGs [6]. For example, the transesterification reaction of triglyc- enhance biodiesel lubricity, as it was demonstrated by re- erides with dimethyl carbonate (DMC) [7], ethyl acetate cent studies [16-18]. Besides, the ethanol that is not spent [8], or methyl acetate [9] generates a mixture of three mol- in the enzymatic process remains in the reaction mixture, ecules of FAME or fatty acid ethyl esters (FAEE) and one and the blend obtained after the reaction can be used dir- of glycerol carbonate (GC) or glycerol triacetate (triacetin). ectly as fuel. In this respect, very recent studies [19-21] These mixtures, including the glycerol derivative mole- have shown that blends of diesel fuel and biodiesel con- cules, have physicochemical properties similar to biodiesel- taining ethanol reduce power output slightly than regular like biofuel [10]. In the present case, the atom efficiency is diesel. No significant difference in the emissions of CO , also improved because the total number of atoms involved CO, and NO between regular diesel and biodiesel or in the reaction is obtained in the final mixture. ethanol and diesel blends was observed. Furthermore, the On the other hand, we have recently developed a pro- use of these blends resulted in a reduction of particulate tocol for the preparation of a new biodiesel-like biofuel, matter. Consequently, such blends can be used in a diesel which integrates glycerol into monoglycerides via 1,3- engine without any modification despite the slightly re- regiospecific enzymatic transesterification of sunflower oil duced power output compared to pure diesel. Thus, the using free [11] and immobilized [12-14] porcine pancreatic Ecodiesel is currently utilized as a blend of fatty acid alkyl lipase (PPL). The operating conditions of such enzymatic esters with ethanol, alone or with any proportion of diesel process were more efficient compared to the preparation fuel [21,22]. method of conventional biodiesel and did not generate any The high cost of traditional industrial lipases restricted acidic or alkaline impurities. Thus, the Ecodiesel biofuel their use in biofuel production, but the current availability [12-15] obtained through the partial ethanolysis of triglyc- of the recombinant purified in sufficiently high quantities erides with 1,3-selective lipases is constituted by a mixture has helped to achieve the economic viability as the crucial of two parts of FAEE and one of monoacylglyceride (MG). factors affecting productivity of enzymatic biodiesel synthe- These glycerol derivative MGs are soluble components in sis are the suitable raw materials and the selected lipase. the FAEE mixture suitable for use as a biodiesel-like bio- The stability and catalytic efficiency of the latter can be im- fuel. In the current case, ethanol was used as a cheap re- proved by optimizing reaction conditions such as substrate agent, instead of the more expensive substrates such as concentrations, temperature, water activity, and alkaline dimethyl carbonate or methyl acetate. This procedure takes concentration of the enzyme's microenvironment [23]. In advantage of the 1,3-selective nature of the most known li- this respect, although Ecodiesel was initially obtained using pases, which allows to terminate the process in the second pig pancreatic lipases (PPL), remarkable results have been step of the alcoholysis to yield the mixture of 2 mol of also obtained with a low-cost purified microbial lipase, FAEE and 1 mol of MG as products (Figure 1). In this way, Lipopan50BG(NovozymesA/S,Bagsværd, Denmark) the glycerol is kept in the form of monoglyceride, which [15], from Thermomyces lanuginosus microorganism, avoids the production of glycerol as by-product, reducing usually used as bread emulsifier (bread improver) [24]. the environmental impact of the process. To our knowledge, this lipase has not been described as In summary, the enzymatic process to obtain this a biocatalyst in any chemical process. The application of biodiesel-like biofuel operates under much smoother an available lipase on an industrial scale is a significant Figure 1 Representative scheme of Ecodiesel production by application of 1,3-selective enzymatic catalysis. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 3 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 approximation to get an economically feasible biofuel In this respect, additional experiments were conducted production by enzymatic method. However, this lipase has with a pure lipase B from C. antarctica, recombinant a main drawback: it cannot be reused, since the purified from Aspergillus oryzae, powder, from Sigma-Aldrich. lipase extract is meant to be in a soluble form. The specific activity of this commercial lipase in the hy- Theaim of thepresent studyistoevaluateNovozym drolysis of tributyrin was >1,000 U/g. In this way, the ex- 435, a commercial lipase from Candida antarctica, recom- istence of possible differences in the catalytic behavior of binant (CALB), expressed in Aspergillus niger and immobi- CALB lipases by the influence of the support could be lized onto an acrylic macroporous resin [25]. Novozym 435 detected. has previously been used in the synthesis of conventional biodiesel as well as in other transesterification processes Results and discussion such as interesterification [26] and in lipase-catalyzed bio- Comparative chromatograms of standardized reaction diesel production by isopropanolysis of soybean oil [27]. In products this respect, Novozym 435 is also described very recently To identify the most characteristic components of biofuels in lipase-catalyzed simultaneous biosynthesis of biodiesel obtained by enzymatic alcoholysis, as well as to compare and glycerol carbonate, by using dimethyl carbonate as acyl their rheological properties, several commercial standards acceptor to obtain a biodiesel-like biofuel without glycerol of reference for FAME, FAEE, MG, and triacylglycerol generation [28]. Thus, in this study, it is intended to put in (TG) were used, as shown in Figure 2. Here, a representa- value the 1,3-selective behavior of these commercial lipases tive sample of monoglycerides of sunflower oil is also in- to make feasible the profitable production of alternative cluded, which was easily achieved by the substitution of biofuels [5], using an enzymatic approach. methanol or ethanol by glycerol, in a conventional alcoho- lysis process with KOH as homogeneous catalyst following standard experimental conditions. Methods Here, we can see that the different esters of fatty acids In this respect, in order to evaluate the influence of cru- (FAEs), which compose the lipid profile of the sunflower cial parameters (lipase amount, temperature, oil/ethanol oil, display retention times (RTs) slightly higher than those volumetric relationship, and alkaline environment) in of cetane (n-hexane) used as internal standard. Thus, the transesterification reaction, a multifactorial design whereas RT of cetane is around 10 min, all RTs of FAEs of experiments and response surface methodology (RSM) appear in the range of 16 to 26 min. These are composed using a multifactorial design of experiments with three of methyl, ethyl, and glycerol esters (the latter constitute factors run by the software Stat Graphics version XV.I MGs) of palmitic, stearic, linoleic, and oleic acids. Thus, were used. This study has been developed to optimize the palmitic acid (C16:0) derivatives are grouped in a narrow catalytic behavior of this 1,3-selective lipase (N435) in range of RT, 16 to 17 min. Derivatives of oleic (C18:1) and the partial ethanolysis of sunflower oil, to obtain a biofuel linoleic acid (C18:2) are grouped in RT of 19 to 21 min, that integrates glycerol as MG together with the different with the exception of glycerol ester of oleic acid, or what is FAEEs in the enzymatic ethanolysis process as well as with the same, the MG of the oleic acid has a different behavior, the excess of unreacted ethanol. This biofuel mixture cur- with a RT = 26 min. Glycerol RT appears at 5 min, before rently named Ecodiesel is able to directly operate diesel cetane. The absence of this compound in the obtained engines, alone or in whichever mixture with diesel fuel, chromatograms clearly demonstrates the selective nature without anymore separation or purification process. of the studied enzymatic transesterification reaction. Commercial sunflower oil was locally obtained. The In Figure 2, the presence of diacylglycerol (DG) with chromatographically pure ethyl esters of palmitic acid, ste- higher retention times, 40 to 60 min, can also be seen, aric acid, oleic acid, linoleic acid, and linolenic acid were which do not allow its integration into the GC chromato- commercially obtained from AccuStandard (New Haven, gram, so that it is necessary to determine DG together and CT, USA), and hexadecane (cetane) was obtained from TG by using an internal standard such as cetane here Sigma-Aldrich (St. Louis, MO, USA). Other chemicals like employed. It should be noted that the differences in RT absolute ethanol and sodium hydroxide were pure analyt- values between MG and DG are much higher than those ical compounds (99.5%) obtained commercially from existing between MG and FAME or FAEE, such as it is ex- Panreac (Castellar Del Valles, Spain). Novozym 435, the pected by the differences between their corresponding lipase B from C. antarctica (CALB), was kindly provided molecular weights. At the same time, it is clear that the by Novozymes A/S. This commercial lipase is produced FAMEs, FAEEs, and MGs display somewhat higher RT by submerged fermentation of a genetically modified values than cetane, but within the molecular weight range, A. niger microorganism and adsorbed on a macroporous which allows considering similar chemical-physical prop- acrylic resin. The specific activity of this commercial erties between the FAE and the hydrocarbons that consti- lipase in the hydrolysis of tributyrin was 3,000 U/g. tute diesel. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 4 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Retention Time (RT/min) Peak 0-7 Solvent (ethanol + dichloromethane 50:50) 5 Glycerol Internal Standard (n-hexadecane/ Cetane) 9-10 16 Palmitic FAME (16:0) 17 Palmitic MG (16:0) 18 Palmitic FAEE (16:0) 19 Estearic (18:0), Linoleic (18:2), Oleic (18:1) FAME 20 Estearic (18:0), Linoleic (18:2), Oleic (18:1) FAEE Estearic (18:0), Linoleic (18:2) MG 26 Oleic (18:1) MG DG + TG 40-60 Figure 2 Superimposed chromatograms of sunflower oil, FAME, FAEE, and MG. Since the retention times of different derivatives of fatty contain a high proportion of DG molecules, with high mo- acids are considered very closely related to the chemical- lecular weight and high viscosity values. Consequently, a physical properties of these compounds, the great similar- very high selectivity, indicating a very high percentage of ity of RT values obtained is a clear demonstration of the FAEEs and MGs, could promote a viscosity value close to similarity among the rheological properties of the different the petroleum diesel, so that the highest conversion value MGs with their corresponding FAMEs or FAEEs, which is not enough a guaranty of lower viscosity values. Thus, are crucial to allow its use as fuel able to substitute for both parameters will be provided as GC analysis results of petroleum products. Consequently, conversion is a reac- reaction products. tion parameter where all molecules (FAEE, MG, and DG) Taking into account that retention times of a complex obtained in the ethanolysis of TG are included (as %); it mixture of hydrocarbons constituting fossil diesel fuel will be considered as a very different parameter, with re- are ranging from 1 to 25 min, it is used as a reference spect to selectivity, where FAEEs and MGs are only in- value for different biofuels (FAME, FAEE, MG) as select- cluded (as %), all of them having RT values lower than ivity value, all those FAEs that present RT values coinci- 26 min. These molecules exhibit RT values similar to hy- dent with the hydrocarbons constituting diesel, or those drocarbons present in conventional diesel, so that they all with RT lower than 25 min, as it is expected that they could exhibit similar physicochemical and rheological also present similar physicochemical and rheological properties. However, a high conversion, even 100%, could properties to the conventional diesel. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 5 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Analysis of variance and optimization of the reaction ratio (v/v), alkaline environment is obtained by the addition parameters by RSM of different microliters of NaOH 10 N, and T is the reaction Theanalysis of variancemethods hasbecome veryat- temperature, tractive in reaction parameter optimization and in the evaluation of the effects of the parameters in the TG ½ConversionðÞ % ¼ 93:57 þ 5:06  T þ 7:82 transesterification reaction [9,15,27] due to their effect- R−6:42  T − 6:53  T iveness in the analysis of variables. Thus, results ob- R−3:25  R  pH tained operating under the 36 runs - each one with ð1Þ different experimental conditions, selected by the multi- factorial design of experiments with three factors, devel- ½SelectivityðÞ % ¼ 35:97 þ 6:26  T−2:25  pH oped by the software Stat Graphics version XV.I., where þ4:50  R þ 6:93  T  pH two of them are developed at three levels and the other 2 −12:22  T  R þ 6:13  pH at two levels, indicated in Table 1 - are shown in −6:61  pH  R Table 2. ð2Þ The quantity of supported lipase (N435) in all these ex- periments was fixed to 0.5 g. All experiments were dupli- ½ViscosityðÞ cSt ¼ 17 :27 þ 1:65  T−2:32  R cated in order to avoid experimental errors. Data was þ0:97  T þ 1:95  T  R fitted to a quadratic polynomial model using the software. þ1:20  R  pH : The quadratic polynomial model was highly significant ð3Þ and sufficient to explain the relationship between conver- sion/selectivity/kinematic viscosity and important experi- mental variables. Thus, the results of factorial design The surface plots in Figure 3b, described by the regres- suggested that the major factors affecting the transesterifi- sion model, were drawn to display the effects of the inde- cation, for the production of biofuels integrating glycerol pendent variables on conversion, selectivity, and kinematic as monoacylglycerols, were temperature and oil/ethanol viscosity. Here, the influence of the different variables in ratio (v/v). the conversion of the systems can be clearly seen. This The values of correlation coefficients, R , were 0.816 model showed that the optimum values for the parameters for conversion, 0.914 for selectivity, and 0.937 for kine- to maximize transesterification yield (conversion, selectiv- matic viscosity, respectively, which imply a good fit be- ity, kinematic viscosity) were intermediate temperatures tween models and experimental data in Pareto graphics, (30°C), maximum amount of aqueous NaOH 10 N added with respect to conversion, selectivity, and viscosity, as (50 μl), and the maximum oil/ethanol (v/v)ratio=6:1 indicated in Figure 3a. The adjusted correlation coeffi- studied. Conversions up to 100%, selectivities around 70%, 2 2 −1 cients R were 0.762, 0.889, and 0.918 for conversion, se- and values of kinematic viscosity about 10 to 15 mm s lectivity, and kinematic viscosity, respectively. Obtained could be achieved under these conditions, which in theory results pointed out that the temperature and oil/ethanol will render feasible the utilization of the obtained biofuel (v/v) ratios were also important parameters influencing in blends with diesel. For example, by the addition of only the conversion and viscosity in the systems (p < 0.05). 35% of diesel fossil, to this biofuel, a viscosity reduction at 2 −1 The software also allows to obtain equations (Equations 1, 4.8 mm s , a value within the acceptance limits of the 2, and 3), remarkably simpler as compared to initial ones EN 14214, is obtained. after the elimination of non-influent parameters in the model for conversion, selectivity, and kinematic viscosity. Effect of the amount of lipase These equations describe the model created and give solu- The effect of the amount of lipase in the reaction media is tions for the dependent variable based on the independent very important to select the correct reaction conditions. variable combinations, selecting the most significant in the This parameter was evaluated in order to choose the ne- response. Thus, taking into account that R is the oil/ethanol cessary amount of lipase that maximizes yield without mass transfer limitations. Previous optimized values ob- Table 1 Process parameters in factorial design: coded and tained from RSM (12 ml oil/3.5 ml EtOH, 30°C, 12.5 μl actual values NaOH 10 N, stirring speed higher than 300 rpm for 2 h) Variables Unit Levels were chosen. We can see in Figure 4 how the reaction yield and vis- −10 1 cosity were improved, so better values could be obtained Temperature °C 20 30 40 using more quantities of biocatalyst, but larger amounts Oil/ethanol ratio (v/v) ml/ml 12/1,75 - 12/3.5 of enzymes will have a detrimental effect on the eco- Alkaline environment μl (NaOH 10 N) 8 (12.5) 10 (5) 12 (50) nomics of the process. Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 6 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 Table 2 Experiment matrix of factorial design and the response obtained for conversion, selectivity, and viscosity Run Parameters 2 −1 Temperature Oil/ethanol ratio pH Conversion (%) Selectivity (%) Kinematic viscosity (mm s ) 11 −1 0 81.4 45.99 20.3 21 −1 −1 84.6 46.1 21.3 30 −1 −1 78.6 29.2 21.0 4 −1 −1 0 59.1 11.7 20.8 5 −1 1 1 100 40.4 13.3 6 1 1 1 100 39.6 22.3 7 −1 −1 −1 63.6 23.3 22.1 80 −1 1 89.5 34.1 19.0 9 0 1 0 95.7 31.4 15.2 10 0 −1 0 100 44.2 20.1 11 0 1 1 95.2 37.5 15.7 12 1 −1 1 100 72.5 18.6 13 1 1 −1 100 46.6 18.2 14 1 1 0 86.1 36.1 19.8 15 −1 1 0 94.9 46.8 13.4 16 −11 −1 100 70.9 10.8 17 0 1 −1 100 52.6 12.5 18 −1 −1 1 77.5 17.8 20.2 Repeated experiments 19 0 −1 0 100 43.4 22.5 20 −1 1 1 100 39.7 13.2 21 −1 −1 −1 64.4 22.6 20.1 22 −1 1 0 95.8 46.1 13.4 23 1 1 −1 100 47.5 16.2 24 0 1 −1 100 51.9 14.5 25 −11 −1 100 69.6 10.9 26 0 1 0 96.5 36.5 14.6 27 1 1 1 100 38.8 22.2 28 0 −1 −1 80.1 28.6 19.0 29 1 −1 1 100 71.4 18.5 30 −1 −1 0 60.2 11.0 20.3 31 1 −1 0 82.2 45.2 20.3 32 1 1 0 86.9 33.4 19.5 33 0 −1 1 90.3 33.4 18.1 34 0 1 1 97.0 36.8 15.1 35 1 −1 −1 85.4 43.4 21.6 36 −1 −1 1 69.7 16.3 20.6 Study of enzyme activity in successive reactions method does not provide information on the combined ef- Since we have been doing these successive reactions, the fect of various reaction parameters, but it is useful to de- reaction conditions have been changed such that the pro- termine the influence of isolated variables. Because our cedure enables the study according to OVAT (‘variable at research group had previous information about other lip- one time’) methodology in which initial conditions are set ase enzymes [12-15] and what were the most important and variables to study have been changed one by one. This reaction variables, it was decided to use this experimental Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 7 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 (a) (b) (a.1) (b.1) (a.2) (b.2) (b.3) (a.3) Figure 3 Pareto graphics and response surface plots. (a) Pareto graphics: conversion (a.1), selectivity (a.2), and viscosity (a.3). (b) Response surface plot of more influential parameters: conversion (b.1), selectivity (b.2), and viscosity (b.3). methodology in order to evaluate in more detail the in- environments, temperatures, and relative oil/ethanol ratio) fluence of those variables in the selected intervals for previously determined by the RSM studies. These experi- each study. mental conditions were similar to those previously ob- In this way, reaction tests of N435 lipase systems have tained with T. lanuginosus, Lipopan 50 BG Lipopan [15]. been carried out under the optimum conditions (alkaline In this respect, Table 3 shows a collection of the achieved Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 8 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 results in the transesterification reaction with this biocata- Graphic 2 lyst. Thus, in addition to obtain specific information about the influence of certain parameters (alkaline environment, Visc (cSt) temperature, etc.) on the behavior of the N435, its ability to be reused could be checked. Visc In this sense, from the conversion, given that we start from 0.01 mol of TG, with 2 h as reaction time and 0.5 g 5 of catalyst system (lipase CALB immobilized on acrylic polymer), we can calculate the transformation enzymatic 0,05 0,1 0,2 0,3 0,4 0,5 capacity: turnover frequency (TOF number) expressed in Lipase Weight (g) micromoles of TG transformed per minute and per gram of catalyst (supported lipase). In order to be able to obtain Graphic 1 the enzyme activity of the immobilized CALB, some re- search experiments with a purified powdered commercial Yield (%) Sel 50 lipase B from C. antarctica, recombinant from A. oryzae, Conv have been conducted. Thus, operating under identical ex- perimental conditions with those used in experiments car- ried out with N435, but using only 0.01 g of purified CALB in free form, a conversion of 50.6% was obtained. Excluding the possible influence of support effects in the lipase activity, and taking into account that 0.5 g of CALB 0,05 0,1 0,2 0,3 0,4 0,5 Lipase Weight (g) immobilized enzyme produced a 47.7% conversion, it can be inferred that N435 has a catalytic activity corre- Figure 4 Influence of lipase amount of supported CALB in the performance of ethanolysis reactions, developed under sponding to the 1.9% of the obtained catalytic activity standard conditions. Conversion and selectivity (graphic 1) and by thefreeCALB. kinematic viscosity in cSt (graphic 2). Therefore, to obtain a reference value, and assuming that the activity of the immobilized enzyme is similar to that of the free enzyme, it can be considered that the N435 may contain about 1.9 wt% of immobilized en- zyme. In any case, it works with this equivalent enzyme Table 3 Achieved results in the transesterification reaction with biocatalyst Run number NaOH amount Temperature EtOH/oil Viscosity Selectivity Conversion TOF Enzyme activity 1 25 30 3.5/12 (6:1) 10.5 34.9 47.7 79.5 0.24 2 25 30 3.5/12 (6:1) 13.7 23.6 32.9 54.83 0.35 3 25 30 3.5/12 (6:1) 12.8 24.1 31.8 53.00 0.36 4 25 30 3.5/12 (6:1) 13.3 25.6 33.9 56.50 0.33 5 25 30 3.5/12 (6:1) 12.8 29.1 35.6 59.33 0.32 6 12.5 35 3.5/12 (6:1) 13.8 14.5 20.1 33.50 0.57 7 12.5 30 3.5/12 (6:1) 12.3 45.4 57.9 96.50 0.20 8 12.5 25 3.5/12 (6:1) 14.6 21.4 25.8 43.00 0.44 9 12.5 40 3.5/12 (6:1) 20.9 7.6 4.0 6.67 2.85 10 12.5 30 1.75/12 (3:1) 18,8 17,9 27,4 45.67 0.42 11 12.5 20 3.5/12 (6:1) 18.1 17.5 22.4 37.33 0.51 12 12.5 30 2.3/12 (4:1) 20.0 17.6 21.4 35.67 0.53 13 12.5 30 2.9/12 (5:1) 10.6 34.1 47.7 79.50 0.24 14 12.5 30 3.5/12 (6:1) 10.4 39.1 51.9 86.50 0.22 15 12.5 30 4.1/12 (7:1) 17.9 14.1 16.8 28.00 0.68 16 12.5 30 4.7/12 (8:1) 16.3 17.1 21.8 36.33 0.52 −1 −1 Viscosity (cSt), conversion (%), selectivity (%), turnover frequencies (TOF in μmol min g cat ), and enzyme activities (U/mg lipase) of the obtained biodiesel by using the same biocatalyst (0.5 g of N435) in successive reactions under the evaluated different experimental conditions: temperature (°C), NaOH amount (μl of NaOH 10 N solution), and EtOH/oil ratio (ml/ml or mol/mol). Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 9 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 activity. From this parameter, a reference value of the reaction yield is clearly displayed and we reached simi- catalytic activity of the immobilized enzymes, expressed lar conclusions to those obtained in the analysis of vari- in milligrams of enzyme required to convert 1 μmol of ance (ANOVA) study by RSM; the optimum reaction TG per minute (U/mg), can also be calculated. parameters for the enzymatic ethanolysis reaction with If we subdivide the results in Table 3 in terms of the lipase N435, CALB (C. antarctica lipase B) immobilized variable that has been changed each time in the study on acrylic resin, are 30°C and 12.5 μlof10NNaOH, with OVAT methodology and if we organize these re- with 1:6 oil/ethanol molar ratio. sults in their corresponding graphics (Figure 5), the in- Also, we can obtain from the data in Table 3 that fluence of each variable on the transesterification N435 may be reused repeatedly. Thus, 16 reactions were (a) (b) (a.2) (b.2) Visc Graphic 2 Graphic 2 (cSt) Visc Visc (cSt) (cSt) Visc (cSt) 5 5 20º 25º 30º 35º 40º 3:01 4:01 5:01 6:01 7:01 8:01 Temperature (ºC) Ethanol/Oil Molar Ratio (a.1) (b.1) Graphic 1 Graphic 1 Yield 70 Sel 60 Yield (%) Conv Sel (%) 40 Conv 20º 25º 30º 35º 40º Temperature (ºC) Ethanol/Oil Molar Ratio Figure 5 Reaction temperature and oil/ethanol molar ratio. Influence of the (a) reaction temperature and (b) oil/ethanol molar ratio in the enzymatic ethanolysis reaction performance of supported CALB developed under standard conditions. Conversion and selectivity (graphic 1) and viscosity in cSt (graphic 2). Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 10 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 developed successively without an appreciable loss in cata- to work in the successive reuses, so that centrifugation of lytic activity in the ethanolysis reaction of sunflower oil, op- reaction products is necessary for catalyst recovery to avoid erating under different experimental conditions. However, important losses of catalyst. This additional centrifugation an important drawback is associated to its acrylic poly- step may constitute a major drawback for the application of meric character. Thus, to recover the immobilized lipase on the process on an industrial scale. macroporous resin, after each successive reaction, it is ne- cessary to carry out the centrifugation of reaction products. Experimental Finally, this research, developed in order to improve a Ethanolysis reactions new methodology to integrate the glycerol as different These reactions were performed according to the experi- monoacylglycerol molecules, is in connection with pre- mental procedure previously described [12-15] to deter- cedent researches performed to determine the optimal mine the optimal conditions for obtaining the selective experimental conditions of the selective ethanolysis reac- ethanolysis reaction, such as alkaline environment, amount tion. In this respect, the results here obtained resemble of lipase, the oil/ethanol molar ratio (v/v), and temperature. those described with the commercial PPL lipases [12-14] Thus, enzymatic assays are carried out with 9.4 g (12 ml, and with a low-cost purified lipase, [15] from T. lanugi- 0.01 mol) of commercial sunflower oil at controlled tem- nosus, Lipopan 50 BG (Novozymes A/S), widely used in peratures (20°C to 40°C) in a 25-ml round bottom flask. the bakery industry as an emulsifier [24]. In this respect, Reaction mixtures were stirred with a conventional mag- the possibility of application of commercial immobilized netic stirrer at a stirring speed higher than 300 rpm to lipases in this process is now explored to exploit the op- avoid mass transfer limitations, along a reaction time of portunity of its reuse, thus lowering its operational cost 2 h. Variable oil/alcohol volume ratios at different alkaline and consequently increasing the economic potential of environments and different quantities of lipase are studied. their industrial application. Thus, in this study, the be- The different oil/ethanol ratios (v/v) are obtained by intro- havior of a commercial supported lipase, Novozym 435, ducing absolute ethanol volumes in the range of 1.75 to is determined because this may involve some techno- 3.5 ml, and the influence of different amounts of lipase is logical advantages in its development, so that this study studied in the range of 0.05 to 0.5 g. The influence of alka- has the aim to prove its viability, its yield, and its possi- line environment values was achieved by adding different bility of reuse, what will mean an important advance in volumes (12.5 to 50 μl) of 10 N NaOH aqueous solution. its biotechnological applications in the biofuel enzymatic In this regard, a blank reaction in the presence of the high- production [5]. estquantity ofsolution ofNaOHwas performedtorule Currently, results obtained in this study indicate that out a potential contribution from the homogeneous NaOH this commercial supported lipase is specially efficient in catalyzed reaction. Less than 10% conversion of the starting obtaining 1,3-selective ethanolysis processes, where gly- material was obtained, so that a homogenous base catalysis cerol is maintained as MG in the biofuel mixture, with contribution can be considered as negligible under the in- the different obtained FAEEs and together to the excess vestigated conditions. of unreacted ethanol. In this way, a new kind of biodiesel In this respect, the NaOH solution, to operate as a currently named Ecodiesel is achieved, constituted by a transesterification catalyst, needs usually to be used under mixture of monoacylglycerols and FAEEs mainly (1/2 higher temperatures and under higher catalyst concentra- nominally), which can be used in different blends with tions (1 to 2 wt%), enough to generate sodium methoxide diesel fuel, without anymore separation or purification or ethoxide, the true catalyst of the homogeneous transes- process. This new biofuel can be obtained with the help terification reaction. Thus, the use of a very small concen- of N435 at very short reaction times (1 to 2 h) and tration of NaOH works as a promoter of the process, under soft reaction conditions. Besides, not only a higher through its effect on the lipase biocatalytic activity. Ac- atomic yield is achieved, with respect to the conven- cordingly, NaOH ions are responsible for the pH environ- tional biodiesel reaction (because no glycerol by-product ment that affects the enzyme yield, because depending on is generated), but also a purification step of residual gly- the pH, the enzymatic protein offers different structures, cerol is not necessary, so it can be used directly after its more or less effective in the biocatalytic action. production. All variables were studied and optimized according to Despite the good results obtained in the transesterifica- a factorial experimental design and a response surface tion reaction of sunflower oil with ethanol to produce FAEE methodology. and MG, unfortunately, the main drawback of this com- mercial biocatalyst is the low stability of the organic poly- meric little spheres which constitute the catalytic powder Analytical method that easily disintegrates in the first ethanolysis reaction and Reaction products were monitored by capillary column gas becomes a gelatinous material which is very uncomfortable chromatography, using a Varian 430-GC gas chromatograph Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 11 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 (Varian Inc., Palo Alto, CA, USA), connected to a HT5 ca- Viscosities were determined in a capillary viscometer pillary column (25 m × 0.32 mm ID × 0.1 μm, SGE, Oswald Proton Cannon-Fenske Routine Viscometer 33200, Supelco) with a flame ionization detector (FID) at 450°C size 150. This is based on determining the time needed for and splitless injection at 350°C. Helium is used as carrier a given volume of fluid passing between two points marked gas with a flow of 1.5 ml/min. A heating ramp from 90°C on the instrument. The kinematic viscosity is given by the to 200°C at a rate of 7°C/min has been applied, followed ratio between the dynamic viscosity (h,inPoise,g/cms) 3 2 by another ramp from 200°C to 360°C at a rate of 15°C/ and the density (r,ing/cm ) υ = h/r (in cm /s or centis- min, maintaining the temperature of the oven at 360°C for tokes (cSt), mm /s). Samples, previously centrifuged at 10 min using as internal standard n-hexadecane (cetane) 3,500 rpm for 10 min and filtered at 50°C, are immersed in to quantify the content of ethyl esters and the different a thermostatic bath at 40°C for 15 min, making sure that glycerides (mono-, di-, and triglycerides) with the help of the temperature is stable. Then, samples are introduced several commercial standard fatty acid esters. This method into the viscometer and this, in turn, in the water bath, allows us to make a complete analysis of the sample in a making sure that it is rigorously positioned vertically, with single injection and in a time not longer than 60 min, the bottom end at a minimum distance of 2 cm from the which simplifies the process and increases the speed of floor of the bath [12-15]. analysis [12-15]. Considering that sunflower oil is constituted by a mix- Experimental design ture of fatty acids in variable proportion (mainly linoleic, The effect of process parameters in the enzymatic trans- oleic, palmitic, and stearic acids), the results obtained esterification reaction to obtain the optimum conditions are expressed as the relative amounts of the correspond- for the viscosity, selectivity, and conversion was studied ing ethyl esters (FAEE, fatty acid ethyl esters), monoglyc- using a multifactorial design of experiments with three erides (MG), and diglycerides (DG) that are integrated in factors run by the software Stat Graphics version XV.I. the chromatogram. The amount of triglycerides (TG) Two of them are developed at three levels and the last which has not reacted is calculated from the difference one at two levels, so that it gives 36 runs. The experi- to the internal standard (cetane). Thus, the conversion ments were performed in random order. The experi- includes the total amount of triglyceride transformed mental parameters selected for this study were reaction (FAEE + MG + DG) in the ethanolysis process, and se- temperature, oil/ethanol ratio (v/v), and different alka- lectivity makes reference to the relative amount of FAEE line environments obtained by the addition of variable + MG obtained. The latter are the ones having retention volumes (in μl) of NaOH 10 N. Table 1 shows the coded times close to the cetane standard, which is the refer- and actual values of the process parameters used in the ence hydrocarbon for diesel fuel. design matrix. Viscosity measurements Statistical analysis The transesterification reactions of oils and fats are ba- The experimental data obtained from experimental de- sically carried out to obtain an important reduction in sign were analyzed by response surface methodology the viscosity of these materials, as they share similar (RSM) [9,15,27]. A mathematical model, following a values in all of other chemical-physical significant pa- second-order polynomial equation, was developed to de- rameters with the fossil diesel except the viscosity. In scribe the relationships between the predicted response this respect, most oils exhibit viscosities in the range of variable (viscosity, conversion, and selectivity) and the 30 to 45 mm /s cSt values, while the fossil diesel is in independent variables of reaction conditions, as shown the range of 2.5 to 6 cSt values. Thus, due to the import- in the Equation 4, where Y is the predicted response ance of viscosity for the correct running of diesel en- variable; β , β , β , and β are the intercept, linear, quad- 0 i ii ij gines, this parameter becomes a critical factor to change ratic, and interaction constant coefficients of the model, the chemical-physical properties of vegetable oils before respectively; and X and X (i =1, 3; j =1, 3; i ≠ j) repre- i j their use as biofuel. The transesterification process of sent the coded independent variables. oils and fats is actually developed in order to obtain a 3 3 3 X X XX noticeable lowering of viscosity in oils to employ the Y ¼ β þ β x þ β x þ β x x ð4Þ i i j 0 0 ii ij resulting product as biofuel in current existing diesel en- i¼1 i¼1 i<j¼1 gines. Thus, accurate viscosity measurements are critical to assess the quality of biofuels produced, since unsuit- Response surface plots were developed using the fitted able viscosity values can decisively affect the correct quadratic polynomial equation obtained from regression working conditions of the diesel engine. Therefore, the analysis, holding one of the independent variables at characterization of this parameter is essential to evaluate constant values corresponding to the stationary point the result obtained in the process of ethanolysis. and changing the order of two variables. The quality of Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 12 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 the fit of the polynomial model equation was evaluated 2. Luque R, Herrero-Davila L, Campelo JM, Clark JH, Hidalgo JM, Luna D, Marinas JM, Romero AA (2008) Biofuels: a technological perspective. by the coefficient of determination R , and its regression Energy Environ Sci 1:542–564 coefficient significance was checked with F-test. Con- 3. Oh PP, Lau HLN, Chen JH, Chong MF, Choo YM (2012) A review on firmatory experiments were carried out in order to va- conventional technologies and emerging process intensification (PI) methods for biodiesel production. Renew Sust Energy Rev 16:5131–5145 lidate the model, using combinations of independent 4. Saleh J, Dube MA, Tremblay AY (2011) Separation of glycerol from FAME variables which were not part of the original experimen- using ceramic membranes. Fuel Process Technol 92:1305–1310 tal design but within the experimental region. 5. Calero J, Luna D, Sancho ED, Luna C, Posadillo A, Bautista FM, Romero AA, Berbel J, Verdugo C (2014) Technological challenges for the production of biodiesel in arid lands. J Arid Environ 102:127–138 Conclusions 6. Ganesan D, Rajendran A, Thangavelu V (2009) An overview on the recent Novozym 435 lipase was evaluated in the 1,3-selective advances in the transesterification of vegetable oils for biodiesel production using chemical and biocatalysts. Rev Environ Sci Biotech 8:367–394 ethanolysis of sunflower oil to integrate the glycerol as 7. Ilham Z, Saka S (2010) Two-step supercritical dimethyl carbonate method monoacylglycerol molecules. On the basis of RSM ana- for biodiesel production from Jatropha curcas oil. Bioresour Technol lysis, the optimum conditions for synthesis were 1:6 molar 101:2735–2740 8. Kim SJ, Jung SM, Park YC, Park K (2007) Lipase catalyzed transesterification oil/EtOH ratio, 30°C, and 12.5 μl NaOH 10 N aqueous so- of soybean oil using ethyl acetate, an alternative acyl acceptor. Biotechnol lutions, with stirring speed higher than 300 rpm for 2 h Bioprocess Eng 12:441–445 and 0.5 g of biocatalyst. Accordingly, its ability to be re- 9. Tan KT, Lee KT, Mohamed AR (2011) A glycerol-free process to produce biodiesel by supercritical methyl acetate technology: an optimization study peatedly reused could open a new way for the production via response surface methodology. Bioresour Technol 102:3990–3991 of alternative biodiesel using an enzymatic approach, 10. Casas A, Ruiz JR, Ramos MJ, Perez A (2010) Effects of triacetin on biodiesel which is technically feasible and economically viable. The quality. Energy Fuels 24:4481–4489 11. Verdugo C, Luque R, Luna D, Hidalgo JM, Posadillo A, Sancho ED, Rodriguez S, main drawback is the permanent need for centrifugation Ferreira-Dias S, Bautista F, Romero AA (2010) A comprehensive study of to recover the biocatalyst for the next reuse. reaction parameters in the enzymatic production of novel biofuels integrating glycerol into their composition. Bioresour Technol 101:6657–6662 Abbreviations 12. Caballero V, Bautista FM, Campelo JM, Luna D, Marinas JM, Romero AA, ANOVA: analysis of variance; CALB: Candida antarctica lipase B; Hidalgo JM, Luque R, Macario A, Giordano G (2009) Sustainable preparation DG: diacylglycerol; FAE: fatty acid esters; FAEE: fatty acid ethyl esters; of a novel glycerol-free biofuel by using pig pancreatic lipase: partial 1,3- FAME: fatty acid methyl esters; MG: monoacylglycerol; OVAT: variable at one regiospecific alcoholysis of sunflower oil. Process Biochem 44:334–342 time; RSM: response surface methodology; TG: triacylglycerol; TOF: turnover 13. Luna D, Posadillo A, Caballero V, Verdugo C, Bautista FM, Romero AA, frequency. Sancho ED, Luna C, Calero J (2012) New biofuel integrating glycerol into its composition through the use of covalent immobilized pig pancreatic lipase. Competing interests Int J Mol Sci 13:10091–10112 The authors declare that they have no competing interests. 14. Luna C, Sancho E, Luna D, Caballero V, Calero J, Posadillo A, Verdugo C, Bautista FM, Romero AA (2013) Biofuel that keeps glycerol as Authors' contributions monoglyceride by 1,3-selective ethanolysis with pig pancreatic lipase CL, CV, EDS, DL, JC, AP, FMB, and AAR have made substantive intellectual covalently immobilized on AlPO support. Energies 6:3879–3900 contributions to this study, making substantial contributions to the 15. Verdugo C, Luna D, Posadillo A, Sancho ED, Rodriguez S, Bautista F, Luque conception and design of it as well as to the acquisition, analysis, and R, Marinas JM, Romero AA (2011) Production of a new second generation interpretation of data. All of them have been also involved in the drafting biodiesel with a low cost lipase derived from Thermomyces lanuginosus: and revision of the manuscript. All authors read and approved the final optimization by response surface methodology. Catal Today 167:107–112 manuscript. 16. Xu YF, Wang QJ, Hu XG, Li C, Zhu XF (2010) Characterization of the lubricity of bio-oil/diesel fuel blends by high frequency reciprocating test rig. Energy 35:283–287 Acknowledgements 17. Haseeb A, Sia SY, Fazal MA, Masjuki HH (2010) Effect of temperature on Grants from the Spanish Ministry of Economy and Competitiveness (Project tribological properties of palm biodiesel. Energy 35:1460–1464 ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ2011-28954-C02-02), FEDER funds and Junta de 18. Wadumesthrige K, Ara M, Salley SO, Ng KYS (2009) Investigation of lubricity Andalucía FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully characteristics of biodiesel in petroleum and synthetic fuel. Energy Fuels acknowledged by the authors. We are also grateful to Novozymes A/S, 23:2229–2234 Denmark, for the kind supply of the macroporous resin immobilized lipase 19. Çelikten I (2011) The effect of biodiesel, ethanol and diesel fuel blends on the from Candida antarctica (Novozym 435). performance and exhaust emissions in a diesel engine. GU J Sci 24:341–346 20. Cheenkachorn K, Fungtammasan B (2009) Biodiesel as an additive for Author details diesohol. Int J Green Energy 6:57–72 Department of Organic Chemistry, University of Cordoba, Campus de 21. Jaganjac M, Prah IO, Cipak A, Cindric M, Mrakovcic L, Tatzber F, Ilincic P, Rabanales, Bldg. Marie Curie, 14014, Cordoba, Spain. Crystallographic Studies Rukavina V, Spehar B, Vukovic JP, Telen S, Uchida K, Lulic Z, Zarkovic N Laboratory, Andalusian Institute of Earth Sciences, CSIC, Avda. Las Palmeras, n°4, (2012) Effects of bioreactive acrolein from automotive exhaust gases on 18100, Armilla, Granada, Spain. Department of Microbiology, University of human cells in vitro. Environ Toxicol 27:644–652 Cordoba, Campus de Rabanales, Ed. Severo Ochoa, 14014, Cordoba, Spain. 22. Pang XB, Mu YJ, Yuan J, He H (2008) Carbonyls emission from ethanol-blended Seneca Green Catalyst S.A., Bldg Centauro, Technological Science Park of gasoline and biodiesel-ethanol-diesel used in engines. Atmos Environ Cordoba, Rabanales XXI, 14014, Córdoba, Spain. 42:1349–1358 23. Szczesna-Antczak M, Kubiak A, Antczak T, Bielecki S (2009) Enzymatic Received: 22 April 2014 Accepted: 22 July 2014 biodiesel synthesis - key factors affecting efficiency of the process. Renew Published: 31 August 2014 Energy 34:1185–1194 24. Moayedallaie S, Mirzaei M, Paterson J (2010) Bread improvers: comparison of a range of lipases with a traditional emulsifier. Food Chem 122:495–499 References 1. Demirbas A (2009) Political, economic and environmental impacts of 25. Xu Y, Nordblad M, Woodley JM (2012) A two-stage enzymatic ethanol-based biofuels: a review. Appl Energy 86:108–117 biodiesel production in a packed bed reactor. J Biotechnol 162:407–414 Luna et al. Bioresources and Bioprocessing 2014, 1:11 Page 13 of 13 http://www.bioresourcesbioprocessing.com/content/1/1/11 26. Yara-Varon E, Joli JE, Torres M, Sala N, Villorbina G, Mendez JJ, Canela- Garayoa R (2012) Solvent-free biocatalytic interesterification of acrylate derivatives. Catal Today 196:86–90 27. Chang C, Chen JH, Chang CMJ, Wu TT, Shieh CJ (2009) Optimization of lipase-catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor using response surface methodology. N Biotechnol 26:187–192 28. Min JY, Lee EY (2011) Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate. Biotechnol Lett 33:1789–1796 doi:10.1186/s40643-014-0011-y Cite this article as: Luna et al.: Production of a biodiesel-like biofuel without glycerol generation, by using Novozym 435, an immobilized Candida antarctica lipase. Bioresources and Bioprocessing 2014 1:11. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com

Journal

"Bioresources and Bioprocessing" – Springer Journals

Published: Dec 1, 2014

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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