Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Enzymatic transesterification of waste vegetable oil to produce biodiesel C.G. Lopresto a,n, S. Naccarato a,b, L. Albo a, M.G. De Paola a, S. Chakraborty b, S. Curcio b, V. Calabrò b a
Department of Mechanical, Energy and Management Engineering (D.I.M.E.G.), University of Calabria, Via Pietro Bucci, 87036 Arcavacata di Rende (CS), Italy Department of Informatics, Modeling, Electronics and Systems Engineering (D.I.M.E.S.), University of Calabria, Via Pietro Bucci, 87036 Arcavacata di Rende (CS), Italy b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 February 2015 Received in revised form 17 March 2015 Accepted 23 March 2015
An experimental study on enzymatic transesterification was performed to produce biodiesel from waste vegetable oils. Lipase from Pseudomonas cepacia was covalently immobilized on a epoxy–acrylic resin support. The immobilized enzyme exhibited high catalytic specific surface and allowed an easy recovery, regeneration and reutilisation of biocatalyst. Waste vegetable oils – such as frying oils, considered not competitive with food applications and wastes to be treated – were used as a source of glycerides. Ethanol was used as a short chain alcohol and was added in three steps with the aim to reduce its inhibitory effect on lipase activity. The effect of biocatalyst/substrate feed mass ratios and the waste oil quality have been investigated in order to estimate the process performances. Biocatalyst recovery and reuse have been also studied with the aim to verify the stability of the biocatalyst for its application in industrial scale. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biodiesel Enzymatic transesterification Immobilized lipase Waste vegetable oil
1. Introduction Biodiesel is a mixture of alkyl esters, produced by catalytic transesterification of glycerides with short chain alcohols (Fukuda et al., 2001). Transesterification is the alcoholysis of triglycerides resulting in a mixture of mono-alkyl esters and glycerol, which is separated and removed to achieve a low-viscosity product similar to conventional diesel fuel. Biodiesel shows a lower viscosity as compared to petro-diesel and it is less polluting since it allows to save 2.4–3.2 kg of CO2 per kg of fuel. Furthermore, it is biodegradable and, during the combustion, a reduced level of particulate, carbon monoxide and nitrogen oxides is produced (Ma and Hanna, 1999). Waste or low quality vegetable oils should be used as substrate for the production of biodiesel, so that the transesterification allows their valorisation and determines significantly reduced posttreatments aimed to a proper disposal. The amount of waste oils is over 15 million tons per year, which, if converted to biodiesel, satisfy the European demand, estimated at 10 million tons (2010). Furthermore, waste vegetable oils as substrate permit to overcome the competition with the food field: the area of different crops that
are needed to meet the 50% of the demand for diesel in the United States of America is estimated in 0.265 billion m3 per year. Since triglycerides represent the most relevant cost of the biodiesel production (almost 71% when fresh oils are used), economic feasibility of the process is obtained by using waste oils (Chisti, 2007). The transesterification process can be performed by different catalysts: alkaline, acid or biological catalysts. The enzymatic process has some advantages, such as a higher yield in esters and a better glycerol recovery, as well as the possibility of using, in the reaction mixture, free fatty acids oils without saponification products (Formo, 1954; Freedman et al., 1986; Srivastava and Prasad, 2000). Moreover, the process is performed at lower temperatures (up to 323 K), lipase can esterify free fatty acids (Ban et al., 2001, 2002). The reaction pattern of bio-catalytic transesterification of triolein in presence of ethanol was described as a sequence of three reactions in series (Al-Zuhair et al., 2007), leading to the production of one mole of ester in each step and of glycerol in the last step, according to the following scheme: Triolein þ ethanol2diolein þethyloleate Diolein þethanol2monoleinþ ethyloleate
n
Corresponding author. E-mail address: [email protected] (C.G. Lopresto).
Monolein þethanol2glycerolþethyloleate
http://dx.doi.org/10.1016/j.ecoenv.2015.03.028 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Table 1 Commercial vegetable oil based on sunflower and fractionated vegetable oils: fatty acids components. Sunflower fatty acid composition
wt%
Palmitoleic (C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2)
6.08 3.26 16.93 73.73
In a previous paper, Calabrò et al. (2009) showed that immobilized-lipase catalysed transesterification could reliably be described by a Ping–Pong bi–bi kinetic mechanism, with ethanol inhibition. Even if an excess of alcohol promotes the transesterification reaction (Ban et al., 2002), it has an inhibitory effect on enzymatic activity. Some studies (Kaieda et al., 1999; Samukawa et al., 2000; Shimada et al., 2000; Ban et al., 2002; Shimada et al., 2002), carried out with methanol as alcohol, showed the possibility to introduce alcohol step by step. Among alcohols, ethanol is less used than methanol, but it lead to high conversions; moreover, it has been also demonstrated that the lipase prefers to exert its activity on long chains of alcohol as compared to short ones (Nelson et al., 1996; Shimada et al., 1997; Mittelbach and Enzelsberger, 1999; Kaieda et al., 2001). As a consequence of the previous observations, an experimental study has been carried out in order to investigate the effect of a key parameter such as the mass ratio biocatalyst/oil, as well as the quality of oil, in a batch bioreactor. In this study, ethanol was used with the aim to realise a completely green bio-process. Ethanol was added step by step and the stoichiometric molar ratio oil/ethanol was reached in three steps. Since the main disadvantage associated with the use of biocatalysts is their high cost, the possibility of reuse them for multiple reaction cycles can be a solution. Nevertheless, it needs to analyse how enzymatic activity varies with time, because a lot of factors can lead to enzyme degradation, such as prolonged use, contact with ethanol and washing procedure. For this reason, stability of biocatalyst, reused for more cycles, was verified in order to set the bases of a proper design of bioreactors, by analysing the effects of process and operating conditions on system performances.
2. Experimental: materials and methods 2.1. Chemicals Commercial vegetable oil based on sunflower and fractionated vegetable oils was used to perform the experimental tests, whose composition is reported in Table 1 in terms of esters of fatty acid. Two kinds of pre-treatments were performed, in order to obtain waste vegetable oils, simulating cooking and frying process: Waste 1 was the commercial oil fried at 190 °C for 30 min, Waste 2 was the vegetable oil baked at 250 °C for 2 h. In Fig.1 chromatograms (HPLC) of all kinds of oils are reported. Ethanol (99.8% grade) from Fluka was used as substrate. HPLC grade acetone and acetonitrile were supplied from Fluka and used as mobile phase in HPLC analysis. Triolein, diolein, monolein and ethyloleate were supplied from SIGMA and used as standard components in HPLC analysis. 2.2. Biocatalyst Epobond Pseudomonas cepacia kindly supplied from SPRIN Technologies (Trieste, Italy) was used as biocatalyst. This lipase from Pseudomanas cepacia was covalently immobilized on a support consisting of an epoxy–acrylic resin, with particle size of
Fig. 1. Chromatograms of commercial and waste oils used during the experimental activity. (a) Fresh commercial oil, (b) Waste 1: commercial oil fried at 190 °C for 30 min, and (c) Waste 2: commercial oil baked at 250 °C for 2 h.
200–500 μm. The activity of the biocatalyst was 161 U/gDRY (unit: 1-phenylethyl acetate). 2.3. Experimental protocol Tests were performed at 37 °C and neutral pH in a mixed batch
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 Initial composition of lipid fractions in commercial and waste oils (% w/w). Components
Commercial fresh oil Fried oil: Waste Baked oil: Waste 1 2
Monoglycerides Diglycerides Triglycerides Esters
0 1.86 98.14 0
0 2.22 97.78 0
2.93 3.71 93.36 0
3
reactor, placed in stirred thermostatic bath (OSL-200, Grant). The stirring rate was 200 rpm: a lower value would lead to a too slow reaction, whereas a higher value could cause a lipase denaturation (De Paola et al., 2009). The reaction mixture was prepared by loading oil and biocatalyst in the reactor; then, they were mixed at 100 rpm and heated up to 37 °C. When final temperature was reached, stirring rate was increased to 200 rpm and ethanol was added. Samples (200 μl) of reaction mixture were collected at different times (0, 10, 20, 30, 60, 90, 120, 150, 180 min). The total
Fig. 2. Time evolution of triglyceride concentration during enzymatic trans-esterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) and quality of oil.
Fig. 3. Time evolution of ester concentration during enzymatic trans-esterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) and quality of oil.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
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amount of collected samples was less than 5% respect to the total volume. Samples are filtered, cooled, diluted in 1000 μl of acetone and analysed by HPLC. Three mass ratios biocatalyst/oil (E0/Oil0) were investigated: 1%, 3% and 5% w/w. The substrate molar ratio oil/ethanol (EtOH0/Oil0) was 3:1, feed of ethanol was carried out step by step with feed molar ratio EtOH0/Oil0 for step 1:1 in anhydrous conditions. Stability tests were also carried out with the aim to verify the possibility of recovering and reusing the biocatalyst for many reaction cycles. Biocatalyst was separated from the reaction mixture by filtration, washed for three times with hexane, which is able to remove the lipid phase from the catalyst, and dried. All tests were performed in duplicate. 2.4. Analytical methods
Fig. 4. Percent yield in esters, in each step, during enzymatic transesterification with Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 1% w/ w, and different quality of oil.
Before each analysis, the biocatalyst and glycerol were removed by filtration and centrifugation, respectively. Concentrations of reactants, e.g. glycerides, and products, i.e. ethyl-esters, were quantitatively measured by high performance liquid chromatography (HPLC) JASCO (pump: PU-980; RI-detector: RI-930; autosampler: AS-1555; column: Alltech Adsorbosphere HS (C18) 5 μm, length 25 mm, inlet diameter 4.6 mm; pre-column: Alltech 7.5 4.6 mm2). Eluent phase was composed of acetone/acetonitrile 70/30 v/v, with a flow rate of 1 ml/min. Temperature was 40 °C. 2.5. Data analysis Experimental data were elaborated to evaluate time evolution of reactants and products as well as the triglycerides conversion. The amount (wi) of components in lipid phase was calculated buy areas under each peak (Areai) in HPLC chromatograms, according the following ratios:
wi =
Areai Areai ⋅100 or %wi = ∑j Area j ∑j Area j
Then, the concentration of each component Ci is calculated as Fig. 5. Comparison among yields in esters, during enzymatic transesterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) for commercial oil.
Ci =
wi ⋅ρtot MWi
where
[mol/l]
ρtot is the density of the lipid phase (g/l) and MWi is the
Fig. 6. Time evolution of monoglyceride concentration during enzymatic transesterification with reused Epobond P. Cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
5
Fig. 7. Time evolution of diglyceride concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
Fig. 8. Time evolution of triglyceride concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
molecular weight of the i-esim component (g/mol). Density varied slightly during reaction and was calculated as average between the initial and the final reaction mixture. Standard components were considered for molecular weight: triolein for triglycerides, diolein for diglycerides, monolein for monoglycerides and ethyloleate for esters. The total yield in esters or conversion, η, is calculated with reference to the total amount of ethanol fed to the system in three steps.
η=
CE 0 3⋅CEtOH/at
[/] or %η = I Step
CE 0 3⋅CEtOH/at
⋅100 [%] I Step
3. Results and discussion During frying, three main reactions occur and involve significant changes in physical and chemical properties of fresh oil (Mittelbach and Enzelsberger, 1999), such as thermolysis, oxidation of free fatty acids and hydrolysis of triglycerides. HPLC analysis of samples of the three oils used as substrates is reported in Table 2 in terms of percent mass of triglycerides, diglycerides, monoglycerides and esters. It is evident that frying process has modified the oil. In fact, the amount of triglycerides decreases, because of hydrolysis and thermolysis, with consequent formation of monoglycerides and
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
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Fig. 9. Time evolution of ester concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 3% w/ w. Six cycles of reaction were carried out with reused biocatalyst.
Fig. 10. Comparison between the yields in esters during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/ oil E0/Oil0 ¼ 3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
diglycerides. This change led to a different performance during transesterification, as confirmed by the comparison shown in Figs. 2 and 3, where concentrations of triglycerides and esters are plotted, respectively. In the same figures, the effect of the amount of biocatalyst – in terms of different biocatalyst/oil mass ratios, 1%, 3% and 5% – was also investigated. As expected, the changes in oil due to frying processes gave worse performances. Specifically, transesterification began in the same way and the quality of oil did not significantly affect the kinetic during the first step, whereas a different trend was observed in the following steps, as shown in Fig. 4, where yield in esters was reported for the test in which mass ratio biocatalyst/oil E0/Oil0 was 1%. With reference to the mass ratio biocatalyst/oil E0/Oil0, it is possible to observe in Figs. 2 and 3 that there is a significant difference among the trends obtained with different biocatalyst E0/Oil0 values. When E0/Oil0 was 1%, the reaction was very slow if compared to the other two cases and, after 9 h, the concentration of esters was 30% lower than concentration obtained with 5%. This
aspect is more evident in Fig. 5, where final conversion is reported for the commercial oil. When E0/Oil0 was 1%, final conversion was 33%, whereas 46–47% was reached in the other two cases. Trends with 3% and 5% were very similar. A E0/Oil0 value of 5% gave only a slight increase in concentration of products, consequently the choice of operating with 3% of biocatalyst respect to initial mass of oil could be considered optimal for an economical and technical point of view. Finally, time evolution of triglycerides, diglycerides, monoglycerides and esters are reported in Figs. 6, 7, 8 and 9, respectively, during six cycles of reaction in sequence. The reuse of the biocatalyst influenced the reaction rate, which gradually decreased. Consequently, the conversion decreased specially in the last cycle (Fig. 10). Prolonged use and washing of biocatalyst, as well as the contact with ethanol, are causes of lipase degradation. The trend observed is equivalent to that obtainable using gradually smaller amounts of biocatalyst in consecutive tests. Since out tests were carried out at the same operating conditions, washing phase could be the critical step.
4. Conclusions The transesterification reaction catalysed by immobilized lipase was studied in this work, in order to verify the possibility of using it for the production of biodiesel from suitable wastes, such as vegetable oils. In particular, laboratory tests were carried out with the aim of evaluating the influence of some factors on reaction yields. Our biocatalyst – Epobond P. cepacia – was found to be very interesting in transesterification of fresh and waste vegetable oils. Moreover, the possible problems that occur in the transition from refined vegetable oils to waste oils were studied and it was observed that the the yields in esters did not vary significantly. Finally, it was verified that the biocatalyst can be used for multiple reaction cycles, even if it requires more investigations in order to evaluate and distinguish the effects of deactivation caused by washing and use in the reaction system.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Acknowledgements The authors are very gratefulto SPRIN Technologies (Trieste, Italy) that kindly supplied the biocatalyst, Epobond Pseudomonas Cepacia. The authors thank also MIUR (Italian Ministry of Education, Universities, and Research) for the financial support by the Research project PRIN 2009 (Code 20095CXMZE).
References Al-Zuhair, S., Ling, F.W., Jun, L.S., 2007. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochem. 42 (6), 951–960. Ban, K., Hama, S., Nishizuka, K., Kaieda, M., Matsumoto, T., Kondo, A., Noda, H., Fukuda, H., 2002. Repeated use of whole cell biocatalysts immobilized within biomass support particles for biodiesel fuel production. J. Mol. Catal. B: Enzym. 17 (3–5), 157–165. Ban, K., Kaieda, M., Matsumoto, T., Kondo, A., Fukuda, H., 2001. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem. Eng. J. 8 (1), 39–43. Calabrò, V., Ricca, E., De Paola, M.G., Curcio, S., Iorio, G., 2009. Kinetics of enzymatic trans-esterification of glycerides for biodiesel production. Bioprocess Biosyst. Eng. 33 (6), 701–710. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. De Paola, M.G., Ricca, E., Calabro’, V., Curcio, S., Iorio, G., 2009. Factor analysis of transesterification reaction of waste oil for biodiesel production. Bioresour. Technol. 100 (21), 5126–5131. Formo, M.W., 1954. Ester reactions of fatty materials. J. Am. Oil Chem. Soc. 31 (11), 548–559.
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Freedman, B., Butterfield, R.O., Pryde, E.H., 1986. Transesterification kinetics of soybean oil. J. Am. Oil Chem. Soc. 63 (10), 1375–1380. Fukuda, H., Kondo, A., Noda, H., 2001. Biodiesel fuel production by transesterification of oils. J. Biosci. Bioeng. 92 (5), 405–416. Kaieda, M., Samukawa, T., Kondo, A., Fukuda, H., 2001. Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J. Biosci. Bioeng. 91 (1), 12–15. Kaieda, M., Samukawa, T., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y., Noda, H., Nomoto, F., Ohtsuka, K., Izumoto, E., Fukuda, H., 1999. Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J. Biosci. Bioeng. 88 (6), 627–631. Ma, F., Hanna, M.A., 1999. Biodiesel production: a review. Bioresour. Technol. 70 (1), 1–15. Mittelbach, M., Enzelsberger, H., 1999. Transesterification of heated rapeseed oil for extending diesel fuel. J. Am. Oil Chem. Soc. 76 (5), 545–550. Nelson, L.A., Foglia, T.A., Marmer, W.N., 1996. Lipase-catalyzed production of biodiesel. J. Am. Oil Chem. Soc. 73 (9), 1191–1195. Samukawa, T., Kaieda, M., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y., Noda, H., Fukuda, H., 2000. Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil. J. Biosci. Bioeng. 90 (2), 180–183. Shimada, Y., Sugihara, A., Nakano, H., Kuramoto, T., Nagao, T., Gemba, M., Tominaga, Y., 1997. Purification of docosahexanoic acid by selective esterification of fatty acids from tuna oil with Rhizopus delemar lipase. J. Am. Oil Chem. Soc. 74 (2), 97–101. Shimada, Y., Watanabe, Y., Samukawa, T., Sugihara, A., Noda, H., Fukuda, H., Tominaga, Y., 2000. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J. Am. Oil Chem. Soc. 76 (7), 789–793. Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B: Enzym. 17 (3–5), 133–142. Srivastava, A., Prasad, R., 2000. Triglycerides-based diesel fuels. Renew. Sustain. Energy Rev. 4 (2), 111–133.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Enzymatic transesterification of waste vegetable oil to produce biodiesel C.G. Lopresto a,n, S. Naccarato a,b, L. Albo a, M.G. De Paola a, S. Chakraborty b, S. Curcio b, V. Calabrò b a
Department of Mechanical, Energy and Management Engineering (D.I.M.E.G.), University of Calabria, Via Pietro Bucci, 87036 Arcavacata di Rende (CS), Italy Department of Informatics, Modeling, Electronics and Systems Engineering (D.I.M.E.S.), University of Calabria, Via Pietro Bucci, 87036 Arcavacata di Rende (CS), Italy b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 February 2015 Received in revised form 17 March 2015 Accepted 23 March 2015
An experimental study on enzymatic transesterification was performed to produce biodiesel from waste vegetable oils. Lipase from Pseudomonas cepacia was covalently immobilized on a epoxy–acrylic resin support. The immobilized enzyme exhibited high catalytic specific surface and allowed an easy recovery, regeneration and reutilisation of biocatalyst. Waste vegetable oils – such as frying oils, considered not competitive with food applications and wastes to be treated – were used as a source of glycerides. Ethanol was used as a short chain alcohol and was added in three steps with the aim to reduce its inhibitory effect on lipase activity. The effect of biocatalyst/substrate feed mass ratios and the waste oil quality have been investigated in order to estimate the process performances. Biocatalyst recovery and reuse have been also studied with the aim to verify the stability of the biocatalyst for its application in industrial scale. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biodiesel Enzymatic transesterification Immobilized lipase Waste vegetable oil
1. Introduction Biodiesel is a mixture of alkyl esters, produced by catalytic transesterification of glycerides with short chain alcohols (Fukuda et al., 2001). Transesterification is the alcoholysis of triglycerides resulting in a mixture of mono-alkyl esters and glycerol, which is separated and removed to achieve a low-viscosity product similar to conventional diesel fuel. Biodiesel shows a lower viscosity as compared to petro-diesel and it is less polluting since it allows to save 2.4–3.2 kg of CO2 per kg of fuel. Furthermore, it is biodegradable and, during the combustion, a reduced level of particulate, carbon monoxide and nitrogen oxides is produced (Ma and Hanna, 1999). Waste or low quality vegetable oils should be used as substrate for the production of biodiesel, so that the transesterification allows their valorisation and determines significantly reduced posttreatments aimed to a proper disposal. The amount of waste oils is over 15 million tons per year, which, if converted to biodiesel, satisfy the European demand, estimated at 10 million tons (2010). Furthermore, waste vegetable oils as substrate permit to overcome the competition with the food field: the area of different crops that
are needed to meet the 50% of the demand for diesel in the United States of America is estimated in 0.265 billion m3 per year. Since triglycerides represent the most relevant cost of the biodiesel production (almost 71% when fresh oils are used), economic feasibility of the process is obtained by using waste oils (Chisti, 2007). The transesterification process can be performed by different catalysts: alkaline, acid or biological catalysts. The enzymatic process has some advantages, such as a higher yield in esters and a better glycerol recovery, as well as the possibility of using, in the reaction mixture, free fatty acids oils without saponification products (Formo, 1954; Freedman et al., 1986; Srivastava and Prasad, 2000). Moreover, the process is performed at lower temperatures (up to 323 K), lipase can esterify free fatty acids (Ban et al., 2001, 2002). The reaction pattern of bio-catalytic transesterification of triolein in presence of ethanol was described as a sequence of three reactions in series (Al-Zuhair et al., 2007), leading to the production of one mole of ester in each step and of glycerol in the last step, according to the following scheme: Triolein þ ethanol2diolein þethyloleate Diolein þethanol2monoleinþ ethyloleate
n
Corresponding author. E-mail address: [email protected] (C.G. Lopresto).
Monolein þethanol2glycerolþethyloleate
http://dx.doi.org/10.1016/j.ecoenv.2015.03.028 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
Table 1 Commercial vegetable oil based on sunflower and fractionated vegetable oils: fatty acids components. Sunflower fatty acid composition
wt%
Palmitoleic (C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2)
6.08 3.26 16.93 73.73
In a previous paper, Calabrò et al. (2009) showed that immobilized-lipase catalysed transesterification could reliably be described by a Ping–Pong bi–bi kinetic mechanism, with ethanol inhibition. Even if an excess of alcohol promotes the transesterification reaction (Ban et al., 2002), it has an inhibitory effect on enzymatic activity. Some studies (Kaieda et al., 1999; Samukawa et al., 2000; Shimada et al., 2000; Ban et al., 2002; Shimada et al., 2002), carried out with methanol as alcohol, showed the possibility to introduce alcohol step by step. Among alcohols, ethanol is less used than methanol, but it lead to high conversions; moreover, it has been also demonstrated that the lipase prefers to exert its activity on long chains of alcohol as compared to short ones (Nelson et al., 1996; Shimada et al., 1997; Mittelbach and Enzelsberger, 1999; Kaieda et al., 2001). As a consequence of the previous observations, an experimental study has been carried out in order to investigate the effect of a key parameter such as the mass ratio biocatalyst/oil, as well as the quality of oil, in a batch bioreactor. In this study, ethanol was used with the aim to realise a completely green bio-process. Ethanol was added step by step and the stoichiometric molar ratio oil/ethanol was reached in three steps. Since the main disadvantage associated with the use of biocatalysts is their high cost, the possibility of reuse them for multiple reaction cycles can be a solution. Nevertheless, it needs to analyse how enzymatic activity varies with time, because a lot of factors can lead to enzyme degradation, such as prolonged use, contact with ethanol and washing procedure. For this reason, stability of biocatalyst, reused for more cycles, was verified in order to set the bases of a proper design of bioreactors, by analysing the effects of process and operating conditions on system performances.
2. Experimental: materials and methods 2.1. Chemicals Commercial vegetable oil based on sunflower and fractionated vegetable oils was used to perform the experimental tests, whose composition is reported in Table 1 in terms of esters of fatty acid. Two kinds of pre-treatments were performed, in order to obtain waste vegetable oils, simulating cooking and frying process: Waste 1 was the commercial oil fried at 190 °C for 30 min, Waste 2 was the vegetable oil baked at 250 °C for 2 h. In Fig.1 chromatograms (HPLC) of all kinds of oils are reported. Ethanol (99.8% grade) from Fluka was used as substrate. HPLC grade acetone and acetonitrile were supplied from Fluka and used as mobile phase in HPLC analysis. Triolein, diolein, monolein and ethyloleate were supplied from SIGMA and used as standard components in HPLC analysis. 2.2. Biocatalyst Epobond Pseudomonas cepacia kindly supplied from SPRIN Technologies (Trieste, Italy) was used as biocatalyst. This lipase from Pseudomanas cepacia was covalently immobilized on a support consisting of an epoxy–acrylic resin, with particle size of
Fig. 1. Chromatograms of commercial and waste oils used during the experimental activity. (a) Fresh commercial oil, (b) Waste 1: commercial oil fried at 190 °C for 30 min, and (c) Waste 2: commercial oil baked at 250 °C for 2 h.
200–500 μm. The activity of the biocatalyst was 161 U/gDRY (unit: 1-phenylethyl acetate). 2.3. Experimental protocol Tests were performed at 37 °C and neutral pH in a mixed batch
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 Initial composition of lipid fractions in commercial and waste oils (% w/w). Components
Commercial fresh oil Fried oil: Waste Baked oil: Waste 1 2
Monoglycerides Diglycerides Triglycerides Esters
0 1.86 98.14 0
0 2.22 97.78 0
2.93 3.71 93.36 0
3
reactor, placed in stirred thermostatic bath (OSL-200, Grant). The stirring rate was 200 rpm: a lower value would lead to a too slow reaction, whereas a higher value could cause a lipase denaturation (De Paola et al., 2009). The reaction mixture was prepared by loading oil and biocatalyst in the reactor; then, they were mixed at 100 rpm and heated up to 37 °C. When final temperature was reached, stirring rate was increased to 200 rpm and ethanol was added. Samples (200 μl) of reaction mixture were collected at different times (0, 10, 20, 30, 60, 90, 120, 150, 180 min). The total
Fig. 2. Time evolution of triglyceride concentration during enzymatic trans-esterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) and quality of oil.
Fig. 3. Time evolution of ester concentration during enzymatic trans-esterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) and quality of oil.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
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amount of collected samples was less than 5% respect to the total volume. Samples are filtered, cooled, diluted in 1000 μl of acetone and analysed by HPLC. Three mass ratios biocatalyst/oil (E0/Oil0) were investigated: 1%, 3% and 5% w/w. The substrate molar ratio oil/ethanol (EtOH0/Oil0) was 3:1, feed of ethanol was carried out step by step with feed molar ratio EtOH0/Oil0 for step 1:1 in anhydrous conditions. Stability tests were also carried out with the aim to verify the possibility of recovering and reusing the biocatalyst for many reaction cycles. Biocatalyst was separated from the reaction mixture by filtration, washed for three times with hexane, which is able to remove the lipid phase from the catalyst, and dried. All tests were performed in duplicate. 2.4. Analytical methods
Fig. 4. Percent yield in esters, in each step, during enzymatic transesterification with Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 1% w/ w, and different quality of oil.
Before each analysis, the biocatalyst and glycerol were removed by filtration and centrifugation, respectively. Concentrations of reactants, e.g. glycerides, and products, i.e. ethyl-esters, were quantitatively measured by high performance liquid chromatography (HPLC) JASCO (pump: PU-980; RI-detector: RI-930; autosampler: AS-1555; column: Alltech Adsorbosphere HS (C18) 5 μm, length 25 mm, inlet diameter 4.6 mm; pre-column: Alltech 7.5 4.6 mm2). Eluent phase was composed of acetone/acetonitrile 70/30 v/v, with a flow rate of 1 ml/min. Temperature was 40 °C. 2.5. Data analysis Experimental data were elaborated to evaluate time evolution of reactants and products as well as the triglycerides conversion. The amount (wi) of components in lipid phase was calculated buy areas under each peak (Areai) in HPLC chromatograms, according the following ratios:
wi =
Areai Areai ⋅100 or %wi = ∑j Area j ∑j Area j
Then, the concentration of each component Ci is calculated as Fig. 5. Comparison among yields in esters, during enzymatic transesterification with Epobond P. cepacia, at 37 °C, 200 rpm, different mass ratio biocatalyst/oil (E0/Oil0) for commercial oil.
Ci =
wi ⋅ρtot MWi
where
[mol/l]
ρtot is the density of the lipid phase (g/l) and MWi is the
Fig. 6. Time evolution of monoglyceride concentration during enzymatic transesterification with reused Epobond P. Cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Fig. 7. Time evolution of diglyceride concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
Fig. 8. Time evolution of triglyceride concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
molecular weight of the i-esim component (g/mol). Density varied slightly during reaction and was calculated as average between the initial and the final reaction mixture. Standard components were considered for molecular weight: triolein for triglycerides, diolein for diglycerides, monolein for monoglycerides and ethyloleate for esters. The total yield in esters or conversion, η, is calculated with reference to the total amount of ethanol fed to the system in three steps.
η=
CE 0 3⋅CEtOH/at
[/] or %η = I Step
CE 0 3⋅CEtOH/at
⋅100 [%] I Step
3. Results and discussion During frying, three main reactions occur and involve significant changes in physical and chemical properties of fresh oil (Mittelbach and Enzelsberger, 1999), such as thermolysis, oxidation of free fatty acids and hydrolysis of triglycerides. HPLC analysis of samples of the three oils used as substrates is reported in Table 2 in terms of percent mass of triglycerides, diglycerides, monoglycerides and esters. It is evident that frying process has modified the oil. In fact, the amount of triglycerides decreases, because of hydrolysis and thermolysis, with consequent formation of monoglycerides and
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
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Fig. 9. Time evolution of ester concentration during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/oil E0/Oil0 ¼ 3% w/ w. Six cycles of reaction were carried out with reused biocatalyst.
Fig. 10. Comparison between the yields in esters during enzymatic transesterification with reused Epobond P. cepacia, at 37 °C, 200 rpm, mass ratio biocatalyst/ oil E0/Oil0 ¼ 3% w/w. Six cycles of reaction were carried out with reused biocatalyst.
diglycerides. This change led to a different performance during transesterification, as confirmed by the comparison shown in Figs. 2 and 3, where concentrations of triglycerides and esters are plotted, respectively. In the same figures, the effect of the amount of biocatalyst – in terms of different biocatalyst/oil mass ratios, 1%, 3% and 5% – was also investigated. As expected, the changes in oil due to frying processes gave worse performances. Specifically, transesterification began in the same way and the quality of oil did not significantly affect the kinetic during the first step, whereas a different trend was observed in the following steps, as shown in Fig. 4, where yield in esters was reported for the test in which mass ratio biocatalyst/oil E0/Oil0 was 1%. With reference to the mass ratio biocatalyst/oil E0/Oil0, it is possible to observe in Figs. 2 and 3 that there is a significant difference among the trends obtained with different biocatalyst E0/Oil0 values. When E0/Oil0 was 1%, the reaction was very slow if compared to the other two cases and, after 9 h, the concentration of esters was 30% lower than concentration obtained with 5%. This
aspect is more evident in Fig. 5, where final conversion is reported for the commercial oil. When E0/Oil0 was 1%, final conversion was 33%, whereas 46–47% was reached in the other two cases. Trends with 3% and 5% were very similar. A E0/Oil0 value of 5% gave only a slight increase in concentration of products, consequently the choice of operating with 3% of biocatalyst respect to initial mass of oil could be considered optimal for an economical and technical point of view. Finally, time evolution of triglycerides, diglycerides, monoglycerides and esters are reported in Figs. 6, 7, 8 and 9, respectively, during six cycles of reaction in sequence. The reuse of the biocatalyst influenced the reaction rate, which gradually decreased. Consequently, the conversion decreased specially in the last cycle (Fig. 10). Prolonged use and washing of biocatalyst, as well as the contact with ethanol, are causes of lipase degradation. The trend observed is equivalent to that obtainable using gradually smaller amounts of biocatalyst in consecutive tests. Since out tests were carried out at the same operating conditions, washing phase could be the critical step.
4. Conclusions The transesterification reaction catalysed by immobilized lipase was studied in this work, in order to verify the possibility of using it for the production of biodiesel from suitable wastes, such as vegetable oils. In particular, laboratory tests were carried out with the aim of evaluating the influence of some factors on reaction yields. Our biocatalyst – Epobond P. cepacia – was found to be very interesting in transesterification of fresh and waste vegetable oils. Moreover, the possible problems that occur in the transition from refined vegetable oils to waste oils were studied and it was observed that the the yields in esters did not vary significantly. Finally, it was verified that the biocatalyst can be used for multiple reaction cycles, even if it requires more investigations in order to evaluate and distinguish the effects of deactivation caused by washing and use in the reaction system.
Please cite this article as: Lopresto, C.G., et al., Enzymatic transesterification of waste vegetable oil to produce biodiesel. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.028i
C.G. Lopresto et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Acknowledgements The authors are very gratefulto SPRIN Technologies (Trieste, Italy) that kindly supplied the biocatalyst, Epobond Pseudomonas Cepacia. The authors thank also MIUR (Italian Ministry of Education, Universities, and Research) for the financial support by the Research project PRIN 2009 (Code 20095CXMZE).
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