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Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock
Accepted Manuscript Title: Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock Authors: Silvia Cesarini, Pilar Diaz, Per Munk Nielsen PII: DOI: Reference:
S1359-5113(13)00040-8 doi:10.1016/j.procbio.2013.02.001 PRBI 9755
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
20-8-2012 12-12-2012 3-2-2013
Please cite this article as: Cesarini S, Diaz P, Nielsen PM, Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock, Process Biochemistry (2010), doi:10.1016/j.procbio.2013.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exploring a new, soluble lipase for FAMEs production in water-containing systems using
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crude soybean oil as a feedstock
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Silvia Cesarini[a,b], Pilar Diaz[a]*, Per Munk Nielsen[b]
[a]
S. Cesarini, Dr. P. Diaz: Department of Microbiology, University of Barcelona.
Av. Diagonal 643, 08028 Barcelona Spain [b]
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Senior Science Manager P.M. Nielsen. Dept. Bioenergy opportunities
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NOVOZYMES A/S. Krogshoejvej 36, 2880 Bagsvaerd Denmark
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*Author for correspondence: Pilar Diaz. Tel: +34-934034627, e-mail: [email protected]
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ABSTRACT Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized
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preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a
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cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides
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(TAGs, DAGs, MAGs) variation during 24h reaction. An excellent 96% FAMEs release was achieved when low (3-5%) water concentrations were used in the conversion of crude soybean oil.
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Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs,
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DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.
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Keywords: Biodiesel, crude oil, soluble lipase, transesterification, esterification, water systems
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INTRODUCTION Biodiesel is defined as the mono alkyl ester of long chain fatty acids derived from vegetable oils or animal fats, for use in compression-ignition engines [1]. Nowadays, essentially all industrial
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biodiesel is produced using chemical catalysts. However, biotechnological production of biodiesel with lipases has received great consideration in recent years and is undergoing a rapid development
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[2]. Employment of lipases as biocatalysts in the transesterification of triglycerides allows mild reaction conditions and easy recovery of glycerol, without need for purification or chemical waste
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production. In addition, the enzymatic process tolerates the common water content of oil and increases the biodiesel yield by avoiding the typical soap formation due to alkaline
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transesterification [3]. Nevertheless, still many drawbacks like the high costs remain to be solved
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for the industrial use of lipases in biodiesel production. The main raw materials used to produce biodiesel are vegetable oils extracted from oleaginous plants, which currently account for over 85%
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of biodiesel production costs [4]. Therefore, the use of low-value, non-edible feedstocks for
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biodiesel synthesis is a goal for innovation among biodiesel-producing partners. Besides waste cooking oils or animal fats, the biodiesel fuel market can also utilize unrefined oils [5, 6] like crude
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soybean oil, used in this work. However, crude vegetable oils, also referred to as non-degummed oils, contain relatively high amounts of free fatty acids, plus certain impurities such as phosphorous compounds known as hydratable phospholipids or phosphatides. During a chemical biodiesel production, FFAs react with the catalyst to produce soaps that induce formation of emulsions, making the separation process difficult. Furthermore, soaps bind to the catalyst, and addition of extra catalyst is necessary. This is one of the main reasons why waste oils are not so prevalent as feedstocks, since e.g. used cooking oil contains as much as 15% FFAs that would correspond to an important loss in the process yield [7]. In contrast, enzyme catalysis is insensitive to high FFAs concentrations [8] which can also be used as substrates and converted into fatty acid alkyl esters (FAAEs) through esterification [9]. In this case, the enzyme-catalyzed esterification rate and yield depends on the lipase employed [10]. Concerning phosphatides, their 3
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content in crude soybean oils may vary from 0.5-3% w/w, corresponding to a phosphorous content in the range of 250-1200ppm. Phospholipids can cause oil storage problems, since they precipitate in the form of a gum and promote accumulation of water, thus being a problem for alkaline
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biodiesel production, but not for the enzyme-catalyzed process. The phosphatides found in crude oils have also been described as inhibitory substances in transesterification reactions catalyzed by
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immobilized lipases. Inhibition is due to formation of a film around the support of the enzyme, thus preventing proper interaction of the lipase with the substrate [11]. Phosphorous can be removed
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from crude oils by means of an enzymatic degumming pre-treatment involving a phospholipasecatalyzed step [12]. While alkaline-catalyzed biodiesel synthesis requires raw materials of high and
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uniform quality containing mostly TAGs, in general it is not necessary to completely refine crude
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oils for enzymatic biodiesel production [13]. According to Watanabe and coworkers (2002), degumming would be the only required pre-treatment for enzymatic transformation of vegetable oils
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into biodiesel, with no further refining steps being required in lipase-catalyzed transesterifications.
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The authors reported that, using immobilized Candida antarctica lipase, only degummed soybean oil was efficiently converted into its corresponding FAMEs, whereas crude oil did not undergo
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methanolysis [11].
The use of immobilized lipases for FAMEs production has been extensively reported [14, 15]. But one of the main drawbacks for using immobilized lipases relies on the high costs of the enzyme, the use of expensive supports, and the immobilization process itself. For istance, the price of immobilized Novozym 435 is very high and would require many re-uses (tipically more than 100 cycles) to become cost-efficient. Liquid lipases can be produced and sold at a much lower price (3050 times lower; P.M.Nielsen, personal communication) and can also be re-used for several times after recovery from the glycerin phase, which makes them cost-efficient compared to immobilized lipases. However, only a few reports can be found in the literature concerning the use of soluble lipases for transesterification [16-18]. Moreover, the use of unrefined, low price oils would result in a dramatic reduction of the global costs of enzyme-catalyzed biodiesel production. This would 4
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launch the enzymatic FAMEs synthesis process into the range of the most competitive chemical systems for fuel production [16]. In addition, the use of soluble lipases in water systems would allow combination in one pot of the required steps for enzyme-catalyzed synthesis of biodiesel:
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phospholipase-catalyzed oil degumming followed by lipase-mediated synthesis of FAMEs. As shown here, the use of water, required in the degumming step, would not inhibit transesterification,
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as water is essential to maintain the specific tridimensional structure of the lipase, especially if used in soluble form [7]. In this work we show that it is possible to obtain FAMEs from crude, non-
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degummed soybean oil through the use of a new soluble lipase (Callera Trans L, Novozymes)
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instead of an immobilized enzyme.
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MATERIALS AND METHODS
Crude soybean oil, with high content of FFAs (7.8%) and phosphatides (1.9%, equivalent to 690
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ppm of phosphorus), was obtained from Emmelev Biodiesel (Otterup, Denmark). All solvents and
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solutions were purchased from Sigma-Aldrich. Soluble lipase Callera Trans L (Thermomyces lanuginosus, EP 258068), with a hydrolytic activity of 100KLU/g, was kindly provided by
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Novozymes (Denmark).
FAMEs synthesis reactions were carried out in 100mL squared bottles at 35°C with 200rpm agitation in a horizontal shaker. The reaction mixtures consisted of 20g crude soybean oil, 1% w/w Callera Trans L lipase solution, and different amounts of water: 3%, 5%, 10%, 15% (w/w of oil). Total methanol (MeOH) per reaction was 16% (w/w of oil), that was added stepwise (4% each time) at the reaction times 0h, 3h, 5h and 7h to prevent enzyme inhibition. Samples (800µL) were taken from the reaction mixture at specified times and evaporated in a Heto Vacuum concentrator at 60°C for 1½h to remove excess of methanol. The upper layer, obtained by centrifugation during the same evaporation process, was prepared for FFAs titration, GC and HPLC analysis. For FFAs determination, evaporated samples were weighed into a conical flask and dissolved in 2propanol (5mL). Indicator (0.5mL 1% phenolphthalein in ethanol) was added, and the solution 5
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titrated with 0.05M aqueous NaOH to a purple color. Percent FFAs (expressed as oleic acid) was calculated as c (NaOH) x V (NaOH) x MW (oleic acid) / m (sample) x 100%. Determination of FAMEs (%) was performed according to the EN14103 standard method on a
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Varian Chrompack CP-3800 gas chromatographer (GC) with two flame ionization detectors (FID), equipped with a Varian “Select Biodiesel for FAMEs” (30m, 0.32 i.d.) column. Methyl
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heptadecanoate was used as internal standard, as indicated by EN14103. The solution was prepared at a concentration of 10mg/ml in acetone. Evaporated oil samples were weighed (50 mg) in a vial
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and mixed by vortex with 1ml of internal standard solution. Samples were then diluted 10-fold with acetone to reach a final concentration of 1mg/ml of internal standard and were transferred to a glass
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vial sealed with a septum cover prior to being injected into the gas chromatographer.
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Evaporated oil samples were diluted for subsequent analysis by HPLC. 55 μl from each sample were pipetted from the oil phase and added to 500 μl n-heptane. Samples were then centrifuged at
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14,500 rpm for 10 min to separate the remaining catalyst and glycerol and to prevent them from
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entering the column. Supernatants were diluted 100-fold, thus preparing the samples for HPLC analysis. 40 μl of the prepared samples were injected in an Ultimate 3000, Dionex A/S, HPLC
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(Hvidovre, Denmark) for analysis of the composition of FAMEs, FFAs, TAGs, DAGs and MAGs. The HPLC was equipped with a U3000 autosampler, TCC-3000SD column oven and U3400A quaternary pump modules. A Corona® Charged Aerosol Detector (Thermo Scientific Dionex, Chelmsford, MA, USA) was used for detection, using nitrogen at a flow pressure of 35.0 psi. Sample separation was done on a 250 mm × 4.0 mm cyanopropyl column, 5µm particle size (Discovery® Cyano) from Sigma–Aldrich (Steinheim, Germany) at a flow-rate of 0.75 mL min-1. A binary gradient program was applied using phase A: 99.6% n-heptane, 0.4% acetic acid; and phase B: 99.6% t-butyl methyl ether, 0.4% acetic acid.
RESULTS AND DISCUSSION
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Much attention is being paid nowadays to soluble lipase-mediated methanolysis for biodiesel production [13-16, 19, 20]. Although the use of liquid lipases in transesterification has been scarcely reported [16-18], they have been described as cost-effective biocatalysts compared to
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immobilized lipases [2, 20]. In this work, crude (non-degummed) soybean oil was successfully converted by soluble Callera Trans L into FAMEs (96% w/w) after 24h reaction at 35°C in 200rpm
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agitation in the presence of water (Figure 1). As shown in Table 1, a very high FAMEs production was achieved for every water condition tested. This demonstrates the efficiency of this new lipase
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towards transesterification of triglycerides. Free fatty acids reduction was also analyzed by determining the FFAs concentration in the final product. Considering the original high FFAs
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content of crude soybean oil (7.8%), a general good reduction was reached, especially in the two
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lower (3-5%) water systems (Table 1). These results show not only the capacity of soluble Callera Trans L for FAMEs production but also its esterification ability in water systems.
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Callera Trans L was assayed here for FAMEs production considering two fundamental aspects of
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the industrial process: a drastic cost reduction if no enzyme immobilization is required and the possibility to perform a one pot reaction including transesterification after an enzymatic degumming
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process carried out by a phospholipase, the latter requiring water in the reaction mixture. Water is also essential to maintain the three dimensional structure of the enzyme during FAMEs production, especially if used in soluble form [7, 16] and, as shown above, it does not inhibit the transesterification reaction. The optimum water content for FAMEs production varies among lipases as it should contribute to minimize substrate hydrolysis and to maximize transesterification [15]. In contrast to immobilized lipases, when soluble lipases are used, a larger amount of water is necessary for methanolysis [17, 19]. To discover the water requirement of soluble Callera Trans L for FAMEs production, a 24h time-course transesterification reaction was performed using crude soybean oil and different water content conditions. A significantly lower FAMEs production was obtained at higher water contents (94.5% and 88.2% FAMEs release at 10 and 15% water content, respectively), with lipase Callera Trans L being more efficient at 3 and 5% water concentrations, 7
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where a FAMEs production over 95% was achieved (Figure 1a). The effect of water on FAMEs production from refined soybean oil using immobilized lipases from several microorganisms showed that transesterification was low in the absence of water but displayed a considerable
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increase in ester production with increased addition of water [21]. This supports the idea that water is required to activate the enzyme for FAMEs release. Callera Trans L was more efficient at the
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lowest water concentrations assayed, providing an exceptional 96.3% and 95.6% FAMEs release for reactions using 3 and 5% water, respectively, a transformation never found before for non-
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degummed oils. At higher water contents (10-15%), the methylester release was significantly reduced, down below the international biodiesel standards (EN14214:2003). After 3h reaction in 3-
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5% water content, almost complete transformation of methanol (0.375 eqs, corresponding to 4%
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w/w of oil) into FAMEs (29%) was achieved (Figure 1a).
For immobilized lipases, sometimes triglycerides undergo a three-step reversible transesterification
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that directly produces FAMEs, whereas in some other cases TAGs suffer a three-step hydrolysis
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that generates FFAs, which then turn into FAMEs through esterification [16, 22-24]. To evaluate the mode of action of Callera Trans L, variation of FFAs, FAMEs and acyl-glycerides during the
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reaction was determined by titration, GC and HPLC. A clear increase of FFAs during the first 3 hours was observed (Figure 1b), suggesting that the reaction proceeds through a first step of enzyme-catalyzed TAGs hydrolysis that releases FFAs. As the reaction proceeds, the concentration of FFAs decreases (Figure 1b), suggesting that once the TAGs have been hydrolyzed to FFAs, these are then esterified by the enzyme into the corresponding FAMEs. In that case FFAs would be considered the intermediates of the reaction [16], which probably proceeds through an initial hydrolysis step where TAGs are reduced to DAGs, MAGs and FFAs, followed by a subsequent esterification step of the FFAs derived from the complete hydrolysis of all these glycerides, as previously suggested [16]. The previous hypothesis could be confirmed after HPLC analysis of all species found in the reaction mixture at each reaction time and for every water concentration (Figure 2). In all samples assayed, a progressive TAGs reduction was observed during the time8
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course of the reaction. This TAGs reduction was accompanied by an increase in FAMEs production (Figure 2). Evidence was obtained that TAGs are hydrolyzed by Callera Trans L into DAGs, MAGs and FFAs during the first 5 h of reaction at all water concentrations (Figure 2). This hydrolysis
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process was also favored by the low initial MeOH concentration, added step-wise during the reaction. The release of FAMEs during the first 5 h is probably due to a true transesterification
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activity of the enzyme, whereas at longer reaction times, when TAGs have almost disappeared, esterification activity is predominant and FAMEs formation derives from the FFAs generated by the
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complete hydrolysis of TAGs, DAGs and MAGs. This effect was particularly evident at 3-5% water
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concentrations, where FAMEs production by Callera Trans L was more effective.
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CONCLUSIONS
The good transesterification rate and the esterification activity shown by the enzyme in the presence
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of water suggest that Callera Trans L lipase is a suitable biocatalyst for further investigations in the
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industrial enzymatic biodiesel production under the conditions tested here. Therefore, we propose Callera Trans L as a good, convenient, soluble catalyst for enzymatic biodiesel production in water-
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requiring systems, even when using crude oils. The reaction, driven in the presence of water, revealed the specific mode of action of the enzyme, with a first hydrolysis step followed by esterification of the released FFAs with methanol. In further investigations, the combined transesterification/esterification activity studied here in the presence of water will be coupled to a previous enzymatic degumming process, eventually carried out by one or more phospholipases, performed in one-pot. Biodiesel-producing partners could benefit from this new, industriallyfeasible and cost-reduced process.
ACKNOWLEDGEMENTS We thank Novozymes (Denmark) for kindly providing soluble lipase Callera Trans L and research facilities. Dr. Mathias Nordblad from Technical University of Denmark is also acknowledged for 9
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HPLC analysis. This work was financed by the Scientific and Technological Research Council (MINECO, Spain), grant CTQ2010-21183-C02-02/PPQ, by the IV Pla de Recerca de Catalunya, grant 2009SGR-819, by PCI-AECID project A203563511, and by the Generalitat de Catalunya to
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the “Xarxa de Referència en Biotecnologia” (XRB). S. Cesarini acknowledges doctoral and
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travelling fellowships from the Spanish Ministry of Science and Education (AP2008-04579).
LEGENDS TO THE FIGURES
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Figure 1: FAMEs (a) and FFAs (b) variations during Callera Trans L transesterification reaction. a) FAMEs concentration registered by GC analysis: progressive FAMEs release was recorded. A
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lower concentration of FAMEs at high water content (10 and 15%) could be noticed. b) FFAs
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content analyzed by titration: during the first 3h, an increase of FFAs concentration, directly depending on the water content, was observed suggesting the first TAGs hydrolysis step. After 24h,
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FFAs concentration in the product reaction was lower than in the raw material (7.8%), suggesting
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also a good esterification activity of the lipase. Data are the mean of two determinations. Figure 2: HPLC analysis of total reaction components during the time-course reaction of Callera
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Trans L-mediated conversion of crude soybean oil into FAMEs at different water concentrations. FAMEs, FFAs, TAGs, DAGs, MAGs mass proportions for each condition are graphically represented. Multiple calibrations indicate an error of ±5%.
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Table 1: FAMEs production and FFAs concentration found after 24h reaction at 35°C in 200rpm agitation with 16% MeOH, using soluble Callera Trans L lipase on crude soybean oil at different water
5%
10%
15%
FAMEs (%)
96.3 ± 0.8
95.6 ± 0.5
94.5 ± 0.04
88.2 ± 2.4
FFAs (%)
2.5 ± 0.13
2.8 ± 0.15
3.8 ± 0.06
5.3 ± 0.22
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3%
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Water concentration
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concentrations. Data are the mean of two determinations.
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Figure 1: 100
3% water 60
5% water
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FAME (%)
80
10% water
40
15% water 20
0
5
10
15
20
25
Time (h)
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30
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FFA ( %)
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3% water 5% water
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10% water
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15
20
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Time (h)
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15 % water
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Figure 2 3% H2O
5% H2O
60%
60%
40%
40%
20%
20% 15% H2O
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80%
0%
0%
0h
3h
80%
5h
7h
0h
24h
10% H2O
3h
5h
7h
24h
15% H2O
100%
100% 60%
40%
80%
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80%
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100%
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80%
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100%
100%
20%
60%
60%
0%
5h 40%
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3h
20%
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0% 0h
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FAME
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TAG
DAG
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REFERENCES
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[1] Al-Zuhair S. Production of biodiesel: possibilities and challenges. Biofuels Bioproducts & Biorefining-Biopr 2007;1:57-66.
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[2] Ghaly AE, Dave, D., Brooks, M.S., Budge, S. Production of biodiesel by enzymatic transesterification: review. American Journal of Biochemistry and Biotechnology
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2010;6:54-76.
[3] Dizge N, Aydiner C, Imer DY, Bayramoglu M, Tanriseven A, Keskinler B. Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using
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lipase immobilized onto a novel microporous polymer. Bioresource Technology 2009;100:1983-1991.
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[4] Moser BR. Influence of Blending Canola, Palm, Soybean, and Sunflower Oil Methyl Esters on Fuel Properties of Biodiesel. Energy Fuels 2008;22:4301-4306. [5] Encinar JM, Sanchez, N., Martinez, G., Garcia, L. Study of biodiesel production from animal
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fats with high free fatty acid content. Bioresour Technol 2011;102:10907-10914.
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[6] Encinar JM, González JF, Rodríguez-Reinares A. Ethanolysis of used frying oil. Biodiesel preparation and characterization. Fuel Processing Technology 2007;88:513-522.
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[7] Nielsen PM, Brask, J., Fjerbaek, L. Enzymatic biodiesel production: Technical and economical considerations. European Journal of Lipid Science and Technology 2008;110:692-700.
[8] Halim SFAK, Azlina Harun. Catalytic studies of lipase on FAME production from waste cooking palm oil in a tert-butanol system Process Biochemistry 2008;43:1436-1439.
[9] Brask JD, M. L.; Nielsen, P. M.; Holm, H. C.; Maes, J.; De Greyt, W. . Combining Enzymatic Esterification with Conventional Alkaline Transesterification in an Integrated Biodiesel Process Applied Biochemistry and Biotechnology 2011;163:918-927.
[10] Christensen MW, Abo M, Du W, Liu D. Enzymes in Biodiesel Production. World Congress on Industrial Biotechnology and Bioprocessing. 2006. [11] Watanabe Y, Shimada Y, Sugihara A, Tominaga Y. Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. Journal of Molecular Catalysis B Enzymatic 2002;17:151-155. [12] Holm HC, Nielsen PM, Christensen MW. Production of fatty acid alkyl esters. Patent Pub No 2008/0199924 A1 2008.
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[13] Watanabe Y, Nagao T, Nishida Y, Takagi Y, Shimada Y. Enzymatic production of fatty acid methyl esters by hydrolysis of acid oil followed by esterification. J Am Oil Chem Soc 2007;84:1015-1021. [14] Fukuda H, Hama S, Tamalampudi S, Noda H. Whole-cell biocatalysts for biodiesel fuel production. Trends in Biotechnology 2008;26:668-673.
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[15] Noureddini H, Gao X, Philkana RS. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol 2005;96:769-777.
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[16] Lv D, Du, W., Zhang, G., Liu, D. Mechanism study on NS81006-mediated methanolysis of triglyceride in oil/water biphasic system for biodiesel production. Process Biochemistry
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2010;45:446-450.
[17] Chen X, Du W, Liu D. Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems. World J Microbiol Biotechnol
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2008;24:2097-2102.
[18] Tan T, Xu J, Asano Y. Biocatalysis in biorefinery: A green and highly efficient way to convert
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renewables Preface. J Mol Catal B-Enzym 2009;56:77-77.
[19] Chen X, Du W, Liu D, Ding F. Lipase-mediated methanolysis of soybean oils for biodiesel production. Journal of Chemical Technology and Biotechnology 2008;83:71-76.
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[20] Fjerbaek L, Christensen KV, Norddahl B. A Review of the Current State of Biodiesel 2009;102:1298-1315.
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Production Using Enzymatic Transesterification. Biotechnology and Bioengineering
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[21] Kaieda M, Samukawa T, Kondo A, Fukuda H. Effect of Methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. Journal of Bioscience and Bioengineering 2001;91:12-15.
[22] Al-Zuhair S, Ling FW, Jun LS. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochemistry 2007;42:951-960.
[23] Dossat V, Combes D, Marty A. Lipase-catalysed transesterification of high oleic sunflower oil. Enzyme Microb Technol 2002;30:90-94.
[24] Miller DA, Prausnitz JM, Blanch HW. Kinetics of Lipase-Catalyzed Interesterification of Triglycerides in Cyclohexane. Enzyme Microb Technol 1991;13:98-103.
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Highlights New soluble Callera Trans L lipase successfully assayed for FAMEs production. Cost-effective biodiesel synthesis using crude oils and a non-immobilized lipase. Synthesis of FAMEs more efficient in the presence of 3-5% water. An initial hydrolysis releases FFAs, which are further esterified to FAMEs. System allowing degumming plus transesterification in one-pot-reaction.
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S1359-5113(13)00040-8 doi:10.1016/j.procbio.2013.02.001 PRBI 9755
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
20-8-2012 12-12-2012 3-2-2013
Please cite this article as: Cesarini S, Diaz P, Nielsen PM, Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock, Process Biochemistry (2010), doi:10.1016/j.procbio.2013.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exploring a new, soluble lipase for FAMEs production in water-containing systems using
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crude soybean oil as a feedstock
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Silvia Cesarini[a,b], Pilar Diaz[a]*, Per Munk Nielsen[b]
[a]
S. Cesarini, Dr. P. Diaz: Department of Microbiology, University of Barcelona.
Av. Diagonal 643, 08028 Barcelona Spain [b]
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Senior Science Manager P.M. Nielsen. Dept. Bioenergy opportunities
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NOVOZYMES A/S. Krogshoejvej 36, 2880 Bagsvaerd Denmark
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*Author for correspondence: Pilar Diaz. Tel: +34-934034627, e-mail: [email protected]
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ABSTRACT Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized
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preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a
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cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides
us
(TAGs, DAGs, MAGs) variation during 24h reaction. An excellent 96% FAMEs release was achieved when low (3-5%) water concentrations were used in the conversion of crude soybean oil.
an
Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs,
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DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.
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Keywords: Biodiesel, crude oil, soluble lipase, transesterification, esterification, water systems
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INTRODUCTION Biodiesel is defined as the mono alkyl ester of long chain fatty acids derived from vegetable oils or animal fats, for use in compression-ignition engines [1]. Nowadays, essentially all industrial
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biodiesel is produced using chemical catalysts. However, biotechnological production of biodiesel with lipases has received great consideration in recent years and is undergoing a rapid development
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[2]. Employment of lipases as biocatalysts in the transesterification of triglycerides allows mild reaction conditions and easy recovery of glycerol, without need for purification or chemical waste
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production. In addition, the enzymatic process tolerates the common water content of oil and increases the biodiesel yield by avoiding the typical soap formation due to alkaline
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transesterification [3]. Nevertheless, still many drawbacks like the high costs remain to be solved
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for the industrial use of lipases in biodiesel production. The main raw materials used to produce biodiesel are vegetable oils extracted from oleaginous plants, which currently account for over 85%
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of biodiesel production costs [4]. Therefore, the use of low-value, non-edible feedstocks for
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biodiesel synthesis is a goal for innovation among biodiesel-producing partners. Besides waste cooking oils or animal fats, the biodiesel fuel market can also utilize unrefined oils [5, 6] like crude
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soybean oil, used in this work. However, crude vegetable oils, also referred to as non-degummed oils, contain relatively high amounts of free fatty acids, plus certain impurities such as phosphorous compounds known as hydratable phospholipids or phosphatides. During a chemical biodiesel production, FFAs react with the catalyst to produce soaps that induce formation of emulsions, making the separation process difficult. Furthermore, soaps bind to the catalyst, and addition of extra catalyst is necessary. This is one of the main reasons why waste oils are not so prevalent as feedstocks, since e.g. used cooking oil contains as much as 15% FFAs that would correspond to an important loss in the process yield [7]. In contrast, enzyme catalysis is insensitive to high FFAs concentrations [8] which can also be used as substrates and converted into fatty acid alkyl esters (FAAEs) through esterification [9]. In this case, the enzyme-catalyzed esterification rate and yield depends on the lipase employed [10]. Concerning phosphatides, their 3
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content in crude soybean oils may vary from 0.5-3% w/w, corresponding to a phosphorous content in the range of 250-1200ppm. Phospholipids can cause oil storage problems, since they precipitate in the form of a gum and promote accumulation of water, thus being a problem for alkaline
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biodiesel production, but not for the enzyme-catalyzed process. The phosphatides found in crude oils have also been described as inhibitory substances in transesterification reactions catalyzed by
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immobilized lipases. Inhibition is due to formation of a film around the support of the enzyme, thus preventing proper interaction of the lipase with the substrate [11]. Phosphorous can be removed
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from crude oils by means of an enzymatic degumming pre-treatment involving a phospholipasecatalyzed step [12]. While alkaline-catalyzed biodiesel synthesis requires raw materials of high and
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uniform quality containing mostly TAGs, in general it is not necessary to completely refine crude
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oils for enzymatic biodiesel production [13]. According to Watanabe and coworkers (2002), degumming would be the only required pre-treatment for enzymatic transformation of vegetable oils
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into biodiesel, with no further refining steps being required in lipase-catalyzed transesterifications.
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The authors reported that, using immobilized Candida antarctica lipase, only degummed soybean oil was efficiently converted into its corresponding FAMEs, whereas crude oil did not undergo
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methanolysis [11].
The use of immobilized lipases for FAMEs production has been extensively reported [14, 15]. But one of the main drawbacks for using immobilized lipases relies on the high costs of the enzyme, the use of expensive supports, and the immobilization process itself. For istance, the price of immobilized Novozym 435 is very high and would require many re-uses (tipically more than 100 cycles) to become cost-efficient. Liquid lipases can be produced and sold at a much lower price (3050 times lower; P.M.Nielsen, personal communication) and can also be re-used for several times after recovery from the glycerin phase, which makes them cost-efficient compared to immobilized lipases. However, only a few reports can be found in the literature concerning the use of soluble lipases for transesterification [16-18]. Moreover, the use of unrefined, low price oils would result in a dramatic reduction of the global costs of enzyme-catalyzed biodiesel production. This would 4
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launch the enzymatic FAMEs synthesis process into the range of the most competitive chemical systems for fuel production [16]. In addition, the use of soluble lipases in water systems would allow combination in one pot of the required steps for enzyme-catalyzed synthesis of biodiesel:
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phospholipase-catalyzed oil degumming followed by lipase-mediated synthesis of FAMEs. As shown here, the use of water, required in the degumming step, would not inhibit transesterification,
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as water is essential to maintain the specific tridimensional structure of the lipase, especially if used in soluble form [7]. In this work we show that it is possible to obtain FAMEs from crude, non-
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degummed soybean oil through the use of a new soluble lipase (Callera Trans L, Novozymes)
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instead of an immobilized enzyme.
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MATERIALS AND METHODS
Crude soybean oil, with high content of FFAs (7.8%) and phosphatides (1.9%, equivalent to 690
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ppm of phosphorus), was obtained from Emmelev Biodiesel (Otterup, Denmark). All solvents and
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solutions were purchased from Sigma-Aldrich. Soluble lipase Callera Trans L (Thermomyces lanuginosus, EP 258068), with a hydrolytic activity of 100KLU/g, was kindly provided by
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Novozymes (Denmark).
FAMEs synthesis reactions were carried out in 100mL squared bottles at 35°C with 200rpm agitation in a horizontal shaker. The reaction mixtures consisted of 20g crude soybean oil, 1% w/w Callera Trans L lipase solution, and different amounts of water: 3%, 5%, 10%, 15% (w/w of oil). Total methanol (MeOH) per reaction was 16% (w/w of oil), that was added stepwise (4% each time) at the reaction times 0h, 3h, 5h and 7h to prevent enzyme inhibition. Samples (800µL) were taken from the reaction mixture at specified times and evaporated in a Heto Vacuum concentrator at 60°C for 1½h to remove excess of methanol. The upper layer, obtained by centrifugation during the same evaporation process, was prepared for FFAs titration, GC and HPLC analysis. For FFAs determination, evaporated samples were weighed into a conical flask and dissolved in 2propanol (5mL). Indicator (0.5mL 1% phenolphthalein in ethanol) was added, and the solution 5
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titrated with 0.05M aqueous NaOH to a purple color. Percent FFAs (expressed as oleic acid) was calculated as c (NaOH) x V (NaOH) x MW (oleic acid) / m (sample) x 100%. Determination of FAMEs (%) was performed according to the EN14103 standard method on a
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Varian Chrompack CP-3800 gas chromatographer (GC) with two flame ionization detectors (FID), equipped with a Varian “Select Biodiesel for FAMEs” (30m, 0.32 i.d.) column. Methyl
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heptadecanoate was used as internal standard, as indicated by EN14103. The solution was prepared at a concentration of 10mg/ml in acetone. Evaporated oil samples were weighed (50 mg) in a vial
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and mixed by vortex with 1ml of internal standard solution. Samples were then diluted 10-fold with acetone to reach a final concentration of 1mg/ml of internal standard and were transferred to a glass
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vial sealed with a septum cover prior to being injected into the gas chromatographer.
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Evaporated oil samples were diluted for subsequent analysis by HPLC. 55 μl from each sample were pipetted from the oil phase and added to 500 μl n-heptane. Samples were then centrifuged at
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14,500 rpm for 10 min to separate the remaining catalyst and glycerol and to prevent them from
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entering the column. Supernatants were diluted 100-fold, thus preparing the samples for HPLC analysis. 40 μl of the prepared samples were injected in an Ultimate 3000, Dionex A/S, HPLC
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(Hvidovre, Denmark) for analysis of the composition of FAMEs, FFAs, TAGs, DAGs and MAGs. The HPLC was equipped with a U3000 autosampler, TCC-3000SD column oven and U3400A quaternary pump modules. A Corona® Charged Aerosol Detector (Thermo Scientific Dionex, Chelmsford, MA, USA) was used for detection, using nitrogen at a flow pressure of 35.0 psi. Sample separation was done on a 250 mm × 4.0 mm cyanopropyl column, 5µm particle size (Discovery® Cyano) from Sigma–Aldrich (Steinheim, Germany) at a flow-rate of 0.75 mL min-1. A binary gradient program was applied using phase A: 99.6% n-heptane, 0.4% acetic acid; and phase B: 99.6% t-butyl methyl ether, 0.4% acetic acid.
RESULTS AND DISCUSSION
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Much attention is being paid nowadays to soluble lipase-mediated methanolysis for biodiesel production [13-16, 19, 20]. Although the use of liquid lipases in transesterification has been scarcely reported [16-18], they have been described as cost-effective biocatalysts compared to
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immobilized lipases [2, 20]. In this work, crude (non-degummed) soybean oil was successfully converted by soluble Callera Trans L into FAMEs (96% w/w) after 24h reaction at 35°C in 200rpm
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agitation in the presence of water (Figure 1). As shown in Table 1, a very high FAMEs production was achieved for every water condition tested. This demonstrates the efficiency of this new lipase
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towards transesterification of triglycerides. Free fatty acids reduction was also analyzed by determining the FFAs concentration in the final product. Considering the original high FFAs
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content of crude soybean oil (7.8%), a general good reduction was reached, especially in the two
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lower (3-5%) water systems (Table 1). These results show not only the capacity of soluble Callera Trans L for FAMEs production but also its esterification ability in water systems.
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Callera Trans L was assayed here for FAMEs production considering two fundamental aspects of
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the industrial process: a drastic cost reduction if no enzyme immobilization is required and the possibility to perform a one pot reaction including transesterification after an enzymatic degumming
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process carried out by a phospholipase, the latter requiring water in the reaction mixture. Water is also essential to maintain the three dimensional structure of the enzyme during FAMEs production, especially if used in soluble form [7, 16] and, as shown above, it does not inhibit the transesterification reaction. The optimum water content for FAMEs production varies among lipases as it should contribute to minimize substrate hydrolysis and to maximize transesterification [15]. In contrast to immobilized lipases, when soluble lipases are used, a larger amount of water is necessary for methanolysis [17, 19]. To discover the water requirement of soluble Callera Trans L for FAMEs production, a 24h time-course transesterification reaction was performed using crude soybean oil and different water content conditions. A significantly lower FAMEs production was obtained at higher water contents (94.5% and 88.2% FAMEs release at 10 and 15% water content, respectively), with lipase Callera Trans L being more efficient at 3 and 5% water concentrations, 7
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where a FAMEs production over 95% was achieved (Figure 1a). The effect of water on FAMEs production from refined soybean oil using immobilized lipases from several microorganisms showed that transesterification was low in the absence of water but displayed a considerable
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increase in ester production with increased addition of water [21]. This supports the idea that water is required to activate the enzyme for FAMEs release. Callera Trans L was more efficient at the
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lowest water concentrations assayed, providing an exceptional 96.3% and 95.6% FAMEs release for reactions using 3 and 5% water, respectively, a transformation never found before for non-
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degummed oils. At higher water contents (10-15%), the methylester release was significantly reduced, down below the international biodiesel standards (EN14214:2003). After 3h reaction in 3-
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5% water content, almost complete transformation of methanol (0.375 eqs, corresponding to 4%
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w/w of oil) into FAMEs (29%) was achieved (Figure 1a).
For immobilized lipases, sometimes triglycerides undergo a three-step reversible transesterification
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that directly produces FAMEs, whereas in some other cases TAGs suffer a three-step hydrolysis
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that generates FFAs, which then turn into FAMEs through esterification [16, 22-24]. To evaluate the mode of action of Callera Trans L, variation of FFAs, FAMEs and acyl-glycerides during the
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reaction was determined by titration, GC and HPLC. A clear increase of FFAs during the first 3 hours was observed (Figure 1b), suggesting that the reaction proceeds through a first step of enzyme-catalyzed TAGs hydrolysis that releases FFAs. As the reaction proceeds, the concentration of FFAs decreases (Figure 1b), suggesting that once the TAGs have been hydrolyzed to FFAs, these are then esterified by the enzyme into the corresponding FAMEs. In that case FFAs would be considered the intermediates of the reaction [16], which probably proceeds through an initial hydrolysis step where TAGs are reduced to DAGs, MAGs and FFAs, followed by a subsequent esterification step of the FFAs derived from the complete hydrolysis of all these glycerides, as previously suggested [16]. The previous hypothesis could be confirmed after HPLC analysis of all species found in the reaction mixture at each reaction time and for every water concentration (Figure 2). In all samples assayed, a progressive TAGs reduction was observed during the time8
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course of the reaction. This TAGs reduction was accompanied by an increase in FAMEs production (Figure 2). Evidence was obtained that TAGs are hydrolyzed by Callera Trans L into DAGs, MAGs and FFAs during the first 5 h of reaction at all water concentrations (Figure 2). This hydrolysis
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process was also favored by the low initial MeOH concentration, added step-wise during the reaction. The release of FAMEs during the first 5 h is probably due to a true transesterification
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activity of the enzyme, whereas at longer reaction times, when TAGs have almost disappeared, esterification activity is predominant and FAMEs formation derives from the FFAs generated by the
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complete hydrolysis of TAGs, DAGs and MAGs. This effect was particularly evident at 3-5% water
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concentrations, where FAMEs production by Callera Trans L was more effective.
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CONCLUSIONS
The good transesterification rate and the esterification activity shown by the enzyme in the presence
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of water suggest that Callera Trans L lipase is a suitable biocatalyst for further investigations in the
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industrial enzymatic biodiesel production under the conditions tested here. Therefore, we propose Callera Trans L as a good, convenient, soluble catalyst for enzymatic biodiesel production in water-
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requiring systems, even when using crude oils. The reaction, driven in the presence of water, revealed the specific mode of action of the enzyme, with a first hydrolysis step followed by esterification of the released FFAs with methanol. In further investigations, the combined transesterification/esterification activity studied here in the presence of water will be coupled to a previous enzymatic degumming process, eventually carried out by one or more phospholipases, performed in one-pot. Biodiesel-producing partners could benefit from this new, industriallyfeasible and cost-reduced process.
ACKNOWLEDGEMENTS We thank Novozymes (Denmark) for kindly providing soluble lipase Callera Trans L and research facilities. Dr. Mathias Nordblad from Technical University of Denmark is also acknowledged for 9
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HPLC analysis. This work was financed by the Scientific and Technological Research Council (MINECO, Spain), grant CTQ2010-21183-C02-02/PPQ, by the IV Pla de Recerca de Catalunya, grant 2009SGR-819, by PCI-AECID project A203563511, and by the Generalitat de Catalunya to
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the “Xarxa de Referència en Biotecnologia” (XRB). S. Cesarini acknowledges doctoral and
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travelling fellowships from the Spanish Ministry of Science and Education (AP2008-04579).
LEGENDS TO THE FIGURES
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Figure 1: FAMEs (a) and FFAs (b) variations during Callera Trans L transesterification reaction. a) FAMEs concentration registered by GC analysis: progressive FAMEs release was recorded. A
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lower concentration of FAMEs at high water content (10 and 15%) could be noticed. b) FFAs
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content analyzed by titration: during the first 3h, an increase of FFAs concentration, directly depending on the water content, was observed suggesting the first TAGs hydrolysis step. After 24h,
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FFAs concentration in the product reaction was lower than in the raw material (7.8%), suggesting
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also a good esterification activity of the lipase. Data are the mean of two determinations. Figure 2: HPLC analysis of total reaction components during the time-course reaction of Callera
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Trans L-mediated conversion of crude soybean oil into FAMEs at different water concentrations. FAMEs, FFAs, TAGs, DAGs, MAGs mass proportions for each condition are graphically represented. Multiple calibrations indicate an error of ±5%.
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Table 1: FAMEs production and FFAs concentration found after 24h reaction at 35°C in 200rpm agitation with 16% MeOH, using soluble Callera Trans L lipase on crude soybean oil at different water
5%
10%
15%
FAMEs (%)
96.3 ± 0.8
95.6 ± 0.5
94.5 ± 0.04
88.2 ± 2.4
FFAs (%)
2.5 ± 0.13
2.8 ± 0.15
3.8 ± 0.06
5.3 ± 0.22
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3%
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Water concentration
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concentrations. Data are the mean of two determinations.
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Figure 1: 100
3% water 60
5% water
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FAME (%)
80
10% water
40
15% water 20
0
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Figure 2 3% H2O
5% H2O
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20% 15% H2O
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100%
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REFERENCES
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[1] Al-Zuhair S. Production of biodiesel: possibilities and challenges. Biofuels Bioproducts & Biorefining-Biopr 2007;1:57-66.
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[2] Ghaly AE, Dave, D., Brooks, M.S., Budge, S. Production of biodiesel by enzymatic transesterification: review. American Journal of Biochemistry and Biotechnology
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[3] Dizge N, Aydiner C, Imer DY, Bayramoglu M, Tanriseven A, Keskinler B. Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using
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[4] Moser BR. Influence of Blending Canola, Palm, Soybean, and Sunflower Oil Methyl Esters on Fuel Properties of Biodiesel. Energy Fuels 2008;22:4301-4306. [5] Encinar JM, Sanchez, N., Martinez, G., Garcia, L. Study of biodiesel production from animal
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[6] Encinar JM, González JF, Rodríguez-Reinares A. Ethanolysis of used frying oil. Biodiesel preparation and characterization. Fuel Processing Technology 2007;88:513-522.
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[9] Brask JD, M. L.; Nielsen, P. M.; Holm, H. C.; Maes, J.; De Greyt, W. . Combining Enzymatic Esterification with Conventional Alkaline Transesterification in an Integrated Biodiesel Process Applied Biochemistry and Biotechnology 2011;163:918-927.
[10] Christensen MW, Abo M, Du W, Liu D. Enzymes in Biodiesel Production. World Congress on Industrial Biotechnology and Bioprocessing. 2006. [11] Watanabe Y, Shimada Y, Sugihara A, Tominaga Y. Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. Journal of Molecular Catalysis B Enzymatic 2002;17:151-155. [12] Holm HC, Nielsen PM, Christensen MW. Production of fatty acid alkyl esters. Patent Pub No 2008/0199924 A1 2008.
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[13] Watanabe Y, Nagao T, Nishida Y, Takagi Y, Shimada Y. Enzymatic production of fatty acid methyl esters by hydrolysis of acid oil followed by esterification. J Am Oil Chem Soc 2007;84:1015-1021. [14] Fukuda H, Hama S, Tamalampudi S, Noda H. Whole-cell biocatalysts for biodiesel fuel production. Trends in Biotechnology 2008;26:668-673.
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[15] Noureddini H, Gao X, Philkana RS. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol 2005;96:769-777.
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[16] Lv D, Du, W., Zhang, G., Liu, D. Mechanism study on NS81006-mediated methanolysis of triglyceride in oil/water biphasic system for biodiesel production. Process Biochemistry
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[17] Chen X, Du W, Liu D. Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems. World J Microbiol Biotechnol
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[18] Tan T, Xu J, Asano Y. Biocatalysis in biorefinery: A green and highly efficient way to convert
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[20] Fjerbaek L, Christensen KV, Norddahl B. A Review of the Current State of Biodiesel 2009;102:1298-1315.
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Production Using Enzymatic Transesterification. Biotechnology and Bioengineering
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[21] Kaieda M, Samukawa T, Kondo A, Fukuda H. Effect of Methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. Journal of Bioscience and Bioengineering 2001;91:12-15.
[22] Al-Zuhair S, Ling FW, Jun LS. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochemistry 2007;42:951-960.
[23] Dossat V, Combes D, Marty A. Lipase-catalysed transesterification of high oleic sunflower oil. Enzyme Microb Technol 2002;30:90-94.
[24] Miller DA, Prausnitz JM, Blanch HW. Kinetics of Lipase-Catalyzed Interesterification of Triglycerides in Cyclohexane. Enzyme Microb Technol 1991;13:98-103.
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Highlights New soluble Callera Trans L lipase successfully assayed for FAMEs production. Cost-effective biodiesel synthesis using crude oils and a non-immobilized lipase. Synthesis of FAMEs more efficient in the presence of 3-5% water. An initial hydrolysis releases FFAs, which are further esterified to FAMEs. System allowing degumming plus transesterification in one-pot-reaction.
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