Process Biochemistry
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A R T I C L E I N F O A B S T R A C T
Article history: The preparation of a novel biofuel denoted as Ecodiesel-100 from the partial 1,3-regiospecific alcoholysis
Received 31 July 2008 of sunflower oil is reported. Pig pancreatic lipase (PPL) was employed in the reaction as both free and
Received in revised form 29 October 2008 immobilised enzyme on sepiolite. The resulting biofuel is composed of fatty acid ethyl esters and
Accepted 19 November 2008
monoglycerides (FAEE/MG) blended in a molar relation 2/1. The novel biofuel has similar physico-
chemical properties compared to those of conventional biodiesel and/or petrodiesel, avoiding the
Keywords: production of glycerine as by-product.
Biofuels
The biocatalyst was found to be strongly fixed to the inorganic support (87.5%). Nevertheless, the
Ethanolysis
efficiency of the immobilised enzyme was reduced to less than half (42%) compared to that of the free
Pig pancreatic lipase (PPL)
Sepiolite PPL. Quantitative conversions of triglycerides and high yields to FAEE were obtained under mild reaction
Immobilisation conditions (20–80 8C, oil/alcohol 2/1 v:v ratio and PPL 0.01–0.1% w/w of total substrate). The
Fatty acid ethyl ester (FAEE) immobilised enzyme showed a remarkable stability as well as a great reusability (more than 11
Monoglyceride successive reuses) without a significant loss of its initial catalytic activity. Both immobilised and free
enzyme exhibited the same reaction mechanism, according to the coincidental results in the Arrhenius
parameters (Ln A and Ea). The immobilised PPL was found to be very suitable for the continuous
production of biofuel due to its facile recyclability from the reaction mixture.
ß 2008 Elsevier Ltd. All rights reserved.
1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.11.015
V. Caballero et al. / Process Biochemistry 44 (2009) 334–342 335
Table 1
Physico-chemical properties of soybean oil, biodiesel (B100) obtained from soybean
oil and rapeseed oil and No. 2 diesel (D2) [43].
Table 2
1 1
Effect of the pH on the composition, yield, conversion (% by GC) and turn over frequency (TOF, mmol h gPPL ) of the Ecodiesel-100 obtained after the ethanolysis of
sunflower oila.
1 1
pH Time (h) FAEE (%) MG + DG (%) TG (%) Yield (%) Conv. (%) TOF (mmol h gPPL )
Fig. 2. Influence of the pH in the catalytic activity of the free PPL in the ethanolysis of Fig. 3. Effect of the oil/alcohol molar ratio on the catalytic efficiency of the free PPL
sunflower oil at 40 8C. in the transesterification of sunflower oil with ethanol [reaction conditions: 45 8C,
pH 12, 0.01 g free PPL (0.1% w/w of total substrate)].
found for all systems. The use of waste cooking oil implies a FAEE/MG ratio was around 2/1 molar at quantitative triglyceride
decrease in the conversion, as reported using other lipases [71–73]. conversion.
3.2.3. Effect of the different alcohols 3.2.4. Effect of the temperature of reaction
Another important advantage of the enzymatic process is the The influence of the temperature of reaction on the catalytic
possibility of using various alcohols different to methanol or activity of the free enzyme is summarised in Table 4 and Fig. 4. The
ethanol. We have investigated the alcoholysis process of different reaction rate (TOF) gradually increases with temperature having a
short-chain alcohols, obtaining the corresponding fatty acid esters maximum activity (almost 60% FAEE at quantitative conversion) at
(FAE, Table 3). The biofuels could smoothly be produced using the 60 8C. A remarkable decrease in activity was found at temperatures
various alcohols employed, obtaining quantitative triglyceride higher than 60 8C due, most likely, to the denaturalisation of the
conversions and selectivities to FAE higher than 50% in most of the protein structures of the enzyme. Similarly, the reaction rate
cases. The reaction took typically 8–12 h to complete and the values decreased with time, regardless of the level of conversion,
selectivity to FAE increased with the time of reaction as expected. maybe related to the deactivation of the PPL.
The enzyme catalysed biofuel production (Tables 2 and 4) does
not generate any glycerine as a result of the 1,3 selective hydrolysis 3.3. Experiments with immobilised PPL
of the triglycerides in the ethanolysis of sunflower oil. A potentially
useful biofuel blend of FAEE, MG and traces of DG, in varying Table 5 summarises the results obtained employing the
proportions (depending on the conversions) was obtained. The immobilised PPL compared to the free enzyme. The same quantity
338 V. Caballero et al. / Process Biochemistry 44 (2009) 334–342
Table 3
1 1
Effect of the different short-chain alcohols, on composition, yield and conversion (% by GC) and TOF (mmol h gPPL ) of the Ecodiesel-100, obtained in the alcoholysis of pure
and waste frying sunflower oila.
1 1
Alcohol Time (h) FAE (%) MG + DG (%) TG (%) Yield (%) Conv. (%) TOF (mmol h gPPL )
supported biocatalyst was used in each reaction. The number of re- Therefore, an 87.5% of the enzyme was immobilised, in good
uses is an essential parameter to assess the efficiency of the agreement with previously reported results [58–61]. A good
physical entrapment of the PPL into the pores of the demineralised correlation was also obtained between the corresponding TOF
sepiolite. values obtained with the filtrate (53.8), as compared to the free PPL
In principle, the enzymatic activity is proportional to the solution (57.7).
amount of enzyme in solution. Thus, the quantity of immobilised The activities of the free and immobilised PPL (up to 6 reuses)
enzyme can be determined from the differences in activity under identical reaction conditions (Table 5, entries free PPL and 4)
between the PPL in the supernatant (Table 5, entry PPL filtrate) were then investigated. Two different series of reactions were
and the standard quantity (0.01 g) of free PPL (Table 5, entry free carried out. Different temperatures, oil/alcohol ratios and oil/
PPL) [58–61]. The resulting solution (after the enzyme immobi- immobilised PPL ratios have been also investigated and included in
lisation) was filtered off, the reaction flask washed with 6 mL of Tables 5 and 6.
ethanol and its catalytic activity was then tested in the ethanolysis The efficiency of the PPL can be obtained comparing the TOF
process. The filtrate gave a 26.9% yield compared to the 57.7% yield values of free and immobilised PPL (Table 5), both obtained under
obtained using the 0.01 g of free PPL. The calculations showed that the same experimental conditions and temperature. The PPL
only 12.5% of the enzyme (0.005 g of PPL) was in the filtrate. reduced its efficiency to a 42.5% [(24.5/57.7) 100 = 42.5] after
Table 4
1 1
Effect of the temperature on composition, yield, conversion (% by GC) and TOF (mmol h gPPL ) of the Ecodiesel-100 obtained after the ethanolysis of sunflower oila.
1 1
Temp. (8C) Time (h) FAEE (%) MG + DG (%) TG (%) Yield (%) Conv. (%) TOF (mmol h gPPL )
Table 5
1 1
Comparison of activities of the free and immobilised PPL [composition, yield and conversion (% by GC) and TOF (mmol h gPPL )] in the ethanolysis of sunflower oila.
Runb T (8C) t (h) FAEE (%) MG + DG (%) TG (%) Yield (%) Conv. (%) TOF (mmol h 1
gPPL 1
)
Fig. 4. Influence of the temperature in the catalytic activity of the free PPL in the
ethanolysis of sunflower oil under standard experimental conditions.
Table 6
Composition, yield and conversion (% by GC) and TOF (mmol h 1 gPPL 1) of the Ecodiesel-100 obtained after the ethanolysis of sunflower oila. Data corresponds to the number
of runs of the biocatalyst, as a continuation of this table, under different reaction conditions.
1 1
Run T (8C) t (h) FAEE (%) MG + DG (%) TG (%) Yield (%) Conv. (%) TOF (mmol h gPPL )
7 25 27 – – 100.0 – – –
8 35 15 5.2 56.1 38.7 5.2 62.2 17.5
9 40 6 13.8 17.8 68.4 13.8 25.8 36.8
10 45 12 63.5 36.5 – 63.5 100.0 169.4
11 50 15 26.5 53.3 20.1 26.5 76.6 176.8
a
Reaction conditions: 48 mL sunflower oil (0.04 mol), 4.8 mL ethanol (0.09 mol), pH 12, 0.5 g of demineralised sepiolite containing 0.01 g of immobilised PPL (0.1% w/w of
total substrate).
340 V. Caballero et al. / Process Biochemistry 44 (2009) 334–342
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