Article pubs.acs.org/EF
Synthesis of Ethylic Esters for Biodiesel Purposes Using Lipases Naturally Immobilized in a Fermented Solid Produced Using Rhizopus microsporus Erika Zago,† Vanderleia Botton,† Dayane Alberton,‡ Jesús Córdova,§ Carlos Itsuo Yamamoto,∥ Lílian Cristina Côcco,∥ David Alexander Mitchell,⊥ and Nadia Krieger*,† †
Departamento de Química, Universidade Federal do Paraná, Caixa Postal 19081, Centro Politécnico, Curitiba, Paraná 81531-980, Brazil ‡ Departamento de Análises Clínicas, Universidade Federal do Paraná, Jardim Botânico, Curitiba, Paraná 80210-170, Brazil § Departamento de Quimica, Centro Universitario de Ciencias Exactas e Ingenierías (CUCEI), Universidad de Guadalajara, Boulevard Marcelino García Barragán 1421, 44430 Guadalajara, Jalisco, Mexico ∥ Laboratório de Análises de Combustíveis Automotivos (LACAUT), Universidade Federal do Paraná, Centro Politécnico, Curitiba, Paraná 81531-980, Brazil ⊥ Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Caixa Postal 19046, Centro Politécnico, Curitiba, Paraná 81531-980, Brazil ABSTRACT: We grew Rhizopus microsporus CPQBA 312-07 DRM in solid-state fermentation on a 1:1 mixture, by mass, of sugarcane bagasse and sunflower seed meal, to produce a fermented solid containing lipases (tricaprylin-hydrolyzing activity of 91 U g−1) and then used this fermented solid to catalyze the ethanolysis of corn oil. A 23 factorial design was used to optimize the reaction using n-heptane as the solvent. The best conversion was 91% at 48 h, obtained at 44 °C, with a molar ratio of ethanol/oil of 3:1 and the addition of 1.32 g of fermented solids/15 mL of reaction medium. Using these optimized conditions, we studied the effect of increasing the concentration of the reactants in the medium and even the use of a solvent-free system. In these systems, conversions were quite poor when the ethanol was added in a single aliquot at the start of the reaction. However, when the ethanol was added stepwise, with three equal aliquots added at 0, 24, and 48 h, promising conversions were obtained, including an ester yield of 51% at 72 h in the solvent-free medium. An improved fermented solid (tricaprylin-hydrolyzing activity of 183 U g−1) was then used to improve the production of ethylic esters in solvent-free medium, with an ester yield of 68% being obtained at 72 h. These results are promising and justify further optimization studies.
1. INTRODUCTION Lipases can be used to produce biodiesel through the transesterification of oils with short-chain alcohols. The lipase-catalyzed process has several potential advantages over the traditional process of alkaline transesterification: raw materials can contain fatty acids because lipases will simultaneously catalyze their esterification, and both the glycerin and biodiesel that are formed are relatively pure. In contrast, in the alkaline transesterification process, the presence of free fatty acids in the feedstock leads to soap formation and the alkaline catalyst contaminates both the glycerin and the biodiesel and must be removed. However, the lipase-catalyzed process is still not competitive with alkaline transesterification, because of the costs of the biocatalyst and the long reaction times that are necessary, from hours to days for the enzymatic process versus 60 min for the chemical process.1,2 There is currently much research into the production and use of lipases with the aim of reducing costs and decreasing reaction times.3 One of the strategies for cost reduction is to use immobilized lipases, because this allows them to be used in several subsequent batches. To date, most of the studies with immobilized lipases involve lipases that are produced by submerged fermentation, then recovered, and immobilized.4 Our group has developed a process in which the lipases are © 2014 American Chemical Society
produced by solid-state fermentation, and then the end product of the fermentation is directly dried, thereby producing a “fermented solid with lipase activity”. This fermented solid is then added directly to the reaction medium to catalyze esterification, transesterification, and interesterification reactions.5−8 Use of this fermented solid has the potential to reduce costs significantly because it not only avoids the need for recovery and immobilization steps but also avoids the need for expensive materials to act as the support for immobilization. Further, although we initially dried the fermented solids by lyophilization,7,8 which is a relatively expensive process, we have recently shown that the lipase activity is maintained during a simple air drying.5 To date, we have produced the fermented solid for biodiesel production by growing Burkholderia cepacia on sugarcane bagasse supplemented with a source of vegetable oils to induce lipase synthesis. The results that we have obtained using this fermented solid in solvent-free media have been promising. For transesterification of soybean oil with ethanol, we have achieved Received: May 14, 2014 Revised: July 24, 2014 Published: July 24, 2014 5197
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ethylenediaminetetraacetic acid (EDTA), 2.0 g of MnCl2·4H2O, 2.8 g of CoSO4·7H2O, 1.5 g of CaCl2·2H2O, 0.2 g of CuCl2·2H2O, and 0.3 g of ZnSO4·7H2O at pH 4.0]. The pH of the mineral solution was adjusted to 7.0 with 10% HCl (v/v). In both cases, the Erlenmeyer flasks were plugged with cotton wool and autoclaved at 121 °C for 15 min. After cooling, spore suspension was added to give 3 × 107 spores g−1 of dry solid substrate. The initial moisture content, 75−80% (m/m, wet basis), was measured in an infrared moisture balance (Gehaka, model IV 2000, São Paulo, Brazil). The flasks were incubated for 18 h at 40 °C. After fermentation, the fermented solids were lyophilized for 24 h at −45 °C and 0.1 mbar in a Jouan LP3 lyophilizer (Allerød, Frederiksborg, Denmark). For the transesterification reactions, lyophilized FS1 was milled in a knife mill and sieved to obtain particles ranging between 0.80 and 2.0 mm, while lyophilized FS2 was not further processed. Lyophilized FS1 was maintained at 4 °C, while lyophilized FS2 was maintained at −18 °C. 2.4. Determination of Tricaprylin-Hydrolyzing Activity. A tricaprylin emulsion was prepared with gum arabic (3%, m/v), 2 mM CaCl2, 2.5 mM Tris−HCl, and 150 mM NaCl.16 For each assay, 20 mL of this emulsion and 250 mg of the dry fermented solid were mechanically stirred in a thermostated vessel, at 37 °C for 5 min, with the free fatty acids released during the reaction being titrated with 0.05 M NaOH in a pH-Stat (Metrohm, model 718 Stat Titrino, Herisau, Switzerland) set at pH 7.0. A total of 1 unit of activity (U) was defined as the release of 1 μmol of fatty acid/min, under the assay conditions. Lipase activities are expressed on the basis of the grams of dry fermented solid (i.e., U g−1). The tricaprylin-hydrolyzing activities of FS1 and FS2 were 91 ± 1 and 183 ± 6 U g−1, respectively. These activities remained stable during storage over the period of experimentation. 2.5. Transesterification Reactions in n-Heptane with FS1. Reactions were carried out in 250 mL Erlenmeyer flasks (with plastic caps) containing 15 mL of substrate mixture, composed of ethanol and corn oil, at the stated concentrations, dissolved in n-heptane or 17.1 mL of solvent-free reaction mixture (45 mmol of ethanol and 15 mmol of corn oil). The mixture was preincubated for 10 min on a rotary shaker at 180 rpm and at the stated temperature, and the reactions were started by adding 1.32 g of dry FS1. Samples were collected at intervals for gas chromatography (GC) analysis. The conditions of the transesterification reaction in n-heptane were optimized, using FS1, by a 23 factorial design with three central points (see Table 1).17 The contrast coefficients (Ci), which allow for the determination of the effect of each independent variable, were calculated as
95% conversion after 46 h.7 For the esterification with ethanol of a mixture of free fatty acids derived from soybean soapstock acid oil, we have obtained 92% conversion after 31 h.5 Currently, all reports in the literature of the use of fermented solids to produce biodiesel involve strains of B. cepacia5,7,8 and Burkholderia cenocepacia.9,10 However, these processes have only been demonstrated at a small scale, involving fermentations of up to 80 g of dry substrate in Erlenmeyer flasks.5 To have sufficient fermented solids for a large-scale biodiesel production process, it would be necessary to undertake solidstate fermentations involving hundreds of kilograms of solids. Because both B. cepacia and B. cenocepacia belong to the B. cepacia complex, which are opportinustic pathogens,11−13 largescale processes undertaken with these strains would require the implementation of adequate containment systems, thereby increasing process costs significantly. These considerations related to the operation of large-scale processes led us to investigate the possibility of using a different microorganism to produce the fermented solid. We have previously produced a fermented solid using the fungus Rhizopus microsporus and used it to catalyze the interesterification of fats in the production of structured lipids.6 The aim of the current work was to evaluate the potential of this fermented solid in the production of biodiesel esters from corn oil.
2. MATERIALS AND METHODS 2.1. Reagents and Media. The reagents used were ethanol (99.5%) and urea from Synth (São Paulo, Brazil), lactose from Nuclear (São Paulo, Brazil), K2HPO4 from Qhemis (São Paulo, Brazil), nheptane (99.5%) and MgSO4·7H2O from Vetec (Rio de Janeiro, Brazil), and tricaprylin (99%), triolein (65%), and methyl heptadecanoate (99.8%) from Sigma-Aldrich (St. Louis, MO). The sugarcane bagasse used to prepare the solid-state fermentation media was kindly donated by Companhia Melhoramentos Norte do Paraná (Paraná, Brazil). Corn oil and soybean oil (produced by Cargill, São Paulo, Brazil) and sunflower seeds were purchased at a local market. The corn oil, used as a substrate in the transesterification reaction, had a free fatty acid content of 0.075% (m/m) and a density of 0.939 g mL−1. The fatty acid profile of the oil was as follows: 14.8% (m/m) palmitic acid, 8.8% (m/m) stearic acid, 31.8% (m/m) oleic acid, 37.9% (m/m) linoleic acid, and 6.7% (m/m) linolenic acid. All other reagents were of analytical grade and were obtained from Brazilian suppliers. 2.2. Fungal Strain and Inoculum. A strain of Rhizopus, originally isolated in Guadalajara, Mexico, and previously characterized as R. microsporus CPQBA 312-07 DRM14 was used. A spore suspension was prepared by growing the fungus on potato dextrose agar at 30 °C for 7 days and harvesting the spores with a Tween 80 solution (0.01%, m/ v). The spore concentration in the suspension was determined using a Neubauer chamber. 2.3. Production of Fermented Solids with Esterification Activity. Two different fermented solids, denoted FS1 and FS2, were used in this work. To produce FS1,14 10 g of a 1:1 (m/m) mixture of sugarcane bagasse and sunflower seed meal was used. The sugarcane bagasse was neither washed nor sieved, while the sunflower seeds were milled and then sieved to obtain particles between 0.8 and 2 mm. This mixture was placed in a 250 mL Erlenmeyer flask and impregnated with 40 mL of 0.1 M phosphate buffer (pH 7.0). To produce FS2,15 the sugarcane bagasse was washed 3 times with tap water, followed by drying at 80 °C for 24 h and sieving to obtain particles between 0.85 and 2.0 mm. Solid-state fermentation was performed in 250 mL Erlenmeyer flasks, each containing 4 g of sugarcane bagasse impregnated with an emulsion containing 0.8 g of soybean oil and 20 mL of culture medium, which contained (per liter) 4 g of urea, 5 g of lactose, 5 g of K2HPO4, 1 g of MgSO4·7H2O, and 4 mL of oligoelement solution [containing, per liter, 10 g of
Ci =
2(∑ yi + − ∑ yi− ) (1)
n
Table 1. Experimental Conditions and Results for the 23 Factorial Design Experiment Used To Optimize the Transesterification of Corn Oil Using the Fermented Solid (FS1) Produced by R. microsporus CPQBA 312-07 DRM run
T (°C)
MRa
FS1 (g)
conversion (%) at 48 h
1 2 3 4 5 6 7 8 9
30 30 30 44 44 44 30 44 37
3:1 3:1 7:1 7:1 7:1 3:1 7:1 3:1 5:1
0.66 1.32 1.32 1.32 0.66 0.66 0.66 1.32 0.99
60 65 82 76 75 75 62 91 56
a
MR = molar ratio of ethanol/corn oil. Run 9 was performed in triplicate, allowing for calculation of the standard deviation for the central point (1.15).
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where Ci is the contrast coefficient of independent variable i, the yi+ values are the values obtained for the response variable (i.e., percentage conversion) when independent variable i is at its highest value, the yi− values are the values obtained for this response variable when independent variable i is at its lowest value, and n is the number of experiments. This experiment was designed and analyzed using Statistica (Statsoft, Inc.). 2.6. Determination of the Residual Activity during the Transesterification Reaction. A transesterification reaction was undertaken in which the fermented solids were removed at 24 h intervals and washed with n-hexane before determining their tricaprylin-hydrolyzing activity. The error bars represent the standard error of the mean (n = 3). 2.7. Larger Scale Transesterification Reactions with FS2 in Solvent-Free Medium. These assays were performed in 500 mL flasks sealed with Teflon caps, containing 100 mL of reaction mixture (264 mmol of ethanol and 88 mmol of corn oil). The flasks were preincubated for 10 min on a rotary shaker at 225 rpm and 44 °C. Reactions were started by adding 12 g of dry FS2. Ethanol was added in three steps, at 0, 24, and 48 h. Samples were collected at intervals for GC analysis. 2.8. GC Analysis. The ethyl ester content was determined using a GC-2010 (Shimadzu Co., Kyoto, Japan) equipped with a hydrogen flame ionization detector and a SGE HT-5 capillary column (0.32 mm internal diameter, 25 m length, and 0.1 mm film thickness). Prepared sample (5−6 mg) was diluted in 1 mL of an internal standard solution of methyl heptadecanoate (1 mg mL−1) in n-heptane. Then, 1 μL was injected, with a split ratio of 1:50, using N2 as the carrier gas. The injector and detector were set at 250 °C. The oven program was as follows: 120 °C for 2 min, heating at 10 °C min−1 to 180 °C, maintained at 180 °C for 3 min, heating at 5 °C min−1 to 230 °C, and maintained at 230 °C for 2 min. Peaks in the chromatograms were identified by comparison of the retention times to a standard solution. The ester content (in mass percent) was determined relative to the peak area of the internal standard.18
Figure 1. Effect of the ratio of substrates on the ethanolysis of corn oil catalyzed by the fermented solid (FS1) produced by R. microsporus CPQBA 312-07 DRM. Ethanol/oil molar ratios of (○) 3:1, (▲) 5:1, (△) 10:1, and (■) 15:1. Reaction conditions: 1.32 g of dry fermented solids in 15 mL of reaction mixture (70 mM of corn oil dissolved in nheptane and the corresponding ethanol concentration), with incubation at 37 °C and 180 rpm.
3. RESULTS AND DISCUSSION 3.1. Optimization of the Transesterification Reaction in n-Heptane. A preliminary study was carried out at 37 °C in n-heptane, using the first fermented solid (FS1). The corn oil concentration was 70 mmol L−1, with ethanol concentrations of 210, 350, 700, and 1050 mmol L−1, representing ethanol/oil molar ratios of 3:1, 5:1, 10:1, and 15:1, respectively. The best ester yields (69% in 48 h) were reached with the molar ratios of 3:1 and 5:1 (Figure 1). Conversions decreased as the molar ratio increased, with values at 48 h of 58 and 42%, respectively, for the molar ratios of 10:1 and 15:1. In an attempt to understand these results, the experiment was repeated, but this time, the residual activities of the fermented solid were determined. The residual activities decreased over time, with the greatest decrease occurring at the highest ethanol/oil molar ratio (Figure 2). After 72 h, the residual activity for the 15:1 molar ratio was only 45%. With the intention of improving reaction rates, a 23 factorial design with three central points was carried out. The independent variables were the temperature (T), the ethanol/ oil molar ratio (MR), and the loading of fermented solids in the reaction medium (LFS), while the response variable was the percentage conversion of fatty acids into ethylic esters at 48 h (Table 1). The highest conversion, of 91% at 48 h, was obtained at 44 °C, with an ethanol/oil molar ratio of 3:1 and 1.32 g of fermented solid. The contrast coefficients in Table 2 show that the most important variables affecting the percentage conversion were the temperature (Ci = +12) and the loading of fermented solids (Ci = +10). The molar ratio of ethanol/oil was not significant,
Figure 2. Stability of the fermented solid (FS1) produced by R. microsporus CPQBA 312-07 DRM during ethanolysis reactions undertaken with different substrate ratios. Residual activities obtained in ethanol/oil molar ratios of (○) 3:1, (▲) 5:1, (△) 10:1, and (■) 15:1. Reaction conditions: 1.32 g of dry fermented solids in 15 mL of reaction mixture (70 mM of corn oil in n-heptane and the corresponding ethanol concentration), with incubation at 37 °C and 180 rpm. Activities were determined by the titrimetric method, using tricaprylin as the substrate, and expressed relative to the initial activity of the fermented solid (91 U g−1). Values plotted represent the mean of triplicate analyses ± the standard error of the mean.
Table 2. Contrast Coefficients (Ci) Calculated from the Results for the Factorial Experiment variable
contrast coefficient (percentage points for conversion)
Ta MRb LFSc T × MR T × LFS MR × LFS T × MR × LFS
+12.0 +1.0 +10.0 −8.0 −2.0 −0.2 −7.0
a
T = temperature (°C). bMR = molar ratio of ethanol/corn oil. cLFS = loading of fermented solid (FS1) added to the medium. The standard deviation for the central point was 1.15.
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because the Ci of 1.0 was lower than the standard deviation at the central point (1.15). The positive effect of the temperature on the transesterification reaction could be linked to a decrease of the viscosity of the medium, which means that agitation is more effective.7,19,20 On the basis of the results in Table 1 and Figures 1 and 2, the conditions chosen for the experiments in the next section were a molar ratio of ethanol/oil of 3:1, a temperature of 44 °C, and a loading of 1.32 g of dry fermented solids (FS1). This loading of fermented solids is the maximum value that can be used in shake flasks, because higher loadings cause the medium to be so viscous that it is very difficult to remove samples. 3.2. Effects of Increased Substrate Concentrations and Stepwise Addition of Ethanol. Although high conversions were obtained with the 3:1 molar ratio of ethanol/oil in Figure 1, with over 60% conversion in 24 h, this reaction was performed with a relatively dilute reaction medium containing “210 mM ethanol + 70 mM oil”. As a result, the specific productivity of this system, expressed in terms of the amount of ester produced per hour per gram of fermented solid, is relatively low. An attempt was made to increase the specific productivity by increasing the concentrations of the substrates (to “1050 mM ethanol + 350 mM oil” and “2100 mM ethanol + 700 mM oil”) and even using a solvent-free medium, consisting of 45 mmol of ethanol and 15 mmol of corn oil. However, this was not successful. With “1050 mM ethanol + 350 mM oil”, the reaction was quite slow, with only 49% conversion being attained after 96 h (Figure 3). With
same overall amount of ethanol was added but subdivided into three equal aliquots, added at 0, 24, and 48 h. The stepwise addition of ethanol led to improved performance. The percentage conversions that were achieved (Figure 3) were not as high as those obtained with “210 mM ethanol + 70 mM oil” (Figure 1) and fell as the medium became more concentrated. However, not only was the specific productivity significantly higher for the reactions with stepwise addition compared to those with the addition of ethanol only at the beginning but it also increased as the medium became more concentrated (Figure 4). For the solvent-free medium, at 72 h, the conversion was 51% and the specific productivity was 65 mg h−1 g−1.
Figure 4. Specific ester productivities during the ethanolysis of corn oil, catalyzed by the fermented solid (FS1) produced by R. microsporus CPQBA 312-07 DRM, using different substrate concentrations and different ethanol addition strategies. Reactions undertaken with (● and ○) 1050 mM ethanol and 350 mM corn oil in 15 mL of nheptane, (▲ and △) 2100 mM ethanol and 700 mM corn oil in 15 mL of n-heptane, and (■ and □) solvent-free medium containing 45 mmol of ethanol and 15 mmol of corn oil. Closed symbols represent ethanol addition in one step (at 0 h), while open symbols represent stepwise ethanol addition (i.e., equal aliquots at 0, 24, and 48 h). Reaction conditions: 1.32 g of dry fermented solids, with incubation at 44 °C and 180 rpm.
3.3. Scale-up of the Transesterification Reaction in Solvent-Free Medium. To increase the ester productivity in the solvent-free medium, it is necessary to increase the amount of lipase added per millimole of oil. Potentially, this could be performed in either of two ways. First, more fermented solid could be added to the reaction medium. However, as noted above, this option was not available, because loadings greater than that used in Figure 3 (namely, 1.32 g for 15 mL of reaction medium) led to highly viscous solutions from which it was virtually impossible to remove samples. Second, the same mass of fermented solid could be added, but with a higher specific lipase activity. This second strategy was used. Up to this point, the fermented solid (FS1) was produced using a 1:1 mixture (by mass) of sugarcane bagasse and sunflower seed meal.14 An improved fermented solid (FS2) was prepared, following the procedure proposed by Rodriguez et al.15 It had a tricaprylin-hydrolyzing activity of 183 U g−1, which is twice that of FS1 (91 U g−1). It was used in a scaled-up system, involving 100 mL of solvent-free reaction medium with a 3:1 molar ratio of ethanol/oil (264 mmol of ethanol and 88 mmol of corn oil) and 12 g of dry FS2. The temperature was maintained at 44 °C. Stepwise ethanol addition (equal aliquots at 0, 24, and 48 h) was also maintained. A conversion of 68%
Figure 3. Kinetic profile for the ethanolysis of corn oil, catalyzed by the fermented solid (FS1) produced by R. microsporus CPQBA 312-07 DRM, using different substrate concentrations and different ethanol addition strategies. Reactions undertaken with (● and ○) 1050 mM ethanol and 350 mM corn oil in 15 mL of n-heptane, (▲ and △) 2100 mM ethanol and 700 mM corn oil in 15 mL of n-heptane, and (■ and □) solvent-free medium containing 45 mmol of ethanol and 15 mmol of corn oil. Closed symbols represent ethanol addition in one step (at 0 h), while open symbols represent stepwise ethanol addition (i.e., equal aliquots at 0, 24, and 48 h). Reaction conditions: 1.32 g of dry fermented solids, with incubation at 44 °C and 180 rpm.
“2100 mM ethanol + 700 mM oil” and with the solvent-free medium, there was essentially no ester production. In these reactions, the ethanol was added as a single aliquot at the beginning. Given the adverse effect of ethanol on the activity that is shown in Figure 2, it is likely that the poor performance of these reactions was caused by the inactivation of the lipase by high ethanol concentrations. Therefore, the reactions were repeated but this time with stepwise ethanol addition. The 5200
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fermented solid (i.e., FS2) has an important advantage over the fermented solid that we have previously used with B. cepacia (i.e., FS1). It gives relatively high lipolytic activities without the need for using sunflower seed meal, which is not a residue. Previous results of ours suggest that the performance in solvent-free media of the fermented solids produced using R. microsporus can be improved. Our initial studies for solvent-free transesterification with the fermented solids of B. cepacia gave conversions of 21−52% after 96 h.8 Through optimization of the reaction with these fermented solids, we achieved 95% conversion after 46 h.7 Similar improvements are now required with the fermented solids of R. microsporus. Our conversion of 68% in 72 h for the solvent-free ethanolysis of corn oil using this fermented solid is better than the conversion of 70% in 144 h obtained for the solvent-free ethanolysis of rapeseed oil using whole cells of another fungus of the same genus, Rhizopus oryzae23 (Table 3). However, results for the methanolysis of soybean oil with “biomass support particles” (BSPs) of R. oryzae, where 90% conversion was obtained in 72 h24 (Table 3), provide a benchmark that we need to achieve. One of the main problems that will need to be addressed is the deactivation of the enzyme by ethanol. This can be a particular problem in solvent-free media because droplets of alcohol within the oil can inactivate lipases.25 One strategy to minimize such problems is the stepwise addition of ethanol.26 This strategy already proved useful in the current work, with a threestep addition allowing for the conversion in the solvent-free system to increase from 3% in 96 h to values as high as 68% in 72 h. Further improvements in percentage conversions and specific productivities may be possible with the addition of the ethanol in a greater number of smaller aliquots.27,28 Lipases of fungi from the genus Rhizopus are typically sn-1,3specific, removing fatty acids preferentially from the extremities of the glycerol moiety of the triacylglycerol, thereby producing sn-1,2- and sn-2,3-diacylglycerols.29 They also preferentially remove the fatty acid from the extremities of these diacylglycerols, such that the final product of enzymatic hydrolysis is the 2-monoacylglycerol. In fact, this phenomenon has been used to produce a biodiesel-like fuel, with the final product consisting of two molecules of fatty acid ethyl ester per molecule of 2-monoacylglycerol.30 The sn-1,3-specifity of a lipase, in itself, would limit the percentage conversion in the transesterification reaction to a maximum value of 66.6%. However, acyl migration can occur under certain conditions. This phenomenon is independent of enzymatic catalysis and enables conversions above 66.6%, even when sn-1,3-specific
was obtained after 72 h (Figure 5), which is significantly higher than the 51% yield obtained at this time with FS1 (see Figure 3).
Figure 5. Kinetic profile for the ethanolysis of corn oil in a solvent-free medium catalyzed by the improved fermented solid (i.e., FS2) produced by R. microsporus CPQBA 312-07 DRM. Reaction conditions: 12 g of dry fermented solids in 100 mL of a reaction mixture composed of 264 mmol of ethanol and 88 mmol of oil corn, with incubation at 44 °C and 225 rpm. Ethanol was added stepwise (equal aliquots at 0, 24, and 48 h).
3.4. Significance of the Results. Although solid-state fermentation followed by direct drying can be used to produce low-cost immobilized enzymes, there have been relatively few studies of the production of biodiesel esters using such fermented solids as the catalyst (see Table 3). A 91% conversion in 96 h was reported for the ethanolysis of soybean oil using fermented solids obtained with B. cenocepacia.9 However, not only does B. cenocepacia represent a high risk to cystic fibrosis sufferers, but also this system uses tert-butanol as a solvent. It is preferable to use solvent-free systems because reaction volumes are smaller and the need to recover and recycle the solvents is avoided.21 With respect to the use of fermented solids in solvent-free systems, our transesterification results are not as good as those that we have previously obtained with fermented solids of B. cepacia for the ethanolysis of soybean oil using a packed-bed bioreactor, namely, 95% conversion after 46 h.7 However, R. microsporus will be safer for producing fermented solids in large-scale solid-state fermentations, because varieties of this species are already used for the production of several Asian fermented foods.22 Our improved
Table 3. Recent Reports on the Transesterification of Vegetable Oils Catalyzed by Lipases for Biodiesel Synthesis lipase source R. microsporus CPQBA 312-07 DRM R. microsporus CPQBA 312-07 DRM B. cepacia LTEB11 B. cenocepacia R. oryzae R. oryzae IFO 4697
support fermented solids from sugarcane bagasse and sunflower seed meal fermented solids from sugarcane bagasse and nutrient solution fermented solids from sugarcane bagasse and sunflower seed meal fermented solids from sugarcane bagasse and sunflower seed meal whole cells BSPs
conversion (%)
reference
48 h and 44 °C
91
this work
solvent-free
72 h and 44 °C
68
this work
ethanol/soybean oil
solvent-free
46 h and 50 °C
95
7
ethanol/soybean oil
tert-butanol
96 h and 44.2 °C
91
9
ethanol/rapeseed oil methanol/soybean oil
solvent-free solvent-free
144 h and 20 °C 72 h and 35 °C
70 90
23 24
alcohol/oil
solvent
ethanol/corn oil
n-heptane
ethanol/corn oil
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lipases are used.31 Because we obtained values as high as 91% when n-heptane was used as a solvent, acyl migration must have occurred in this system. It remains to be seen whether acyl migration is a rate-limiting step in the solvent-free system.
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4. CONCLUSION We have produced a fermented solid by growing R. microsporus in solid-state fermentation of sugarcane bagasse that was impregnated with a mineral salt solution and contained soybean oil as an inducer of lipases. This fermented solid was able to catalyze the transesterification of corn oil with ethanol in a solvent-free system, with a conversion of 68% in 72 h. Although further improvements in percentage conversion are required, this fermented solid has better potential to be used in largescale processes for lipase-catalyzed biodiesel production than the fermented solids that have been used to date, which involve bacterial strains of the species Burkholderia.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +55-41-33613470. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported financially by Conselho Nacional de ́ e Tecnológico (CNPq), a Brazilian Desenvolvimento Cientifico government agency for the advancement of science. Research scholarships were granted to Erika Zago, Dayane Alberton, Nadia Krieger, and David Alexander Mitchell by CNPq and to Jesús Córdova by Coordenaçaõ de Aperfeiçoamento de Pessoal ́ Superior (CAPES), a Brazilian government agency for de Nivel the development of personnel in higher education, and Consejo Nacional de Ciencia y Tecnologiá (CONACYT), Mexico.
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NOMENCLATURE FS = fermented solid LFS = loading of fermented solids in the reaction medium MR = molar ratio T = temperature U = activity units
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REFERENCES
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