Bioresource Technology 192 (2015) 54–59
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Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes Ting-Chun Kuo a, Jei-Fu Shaw b, Guan-Chiun Lee a,⇑ a b
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan
h i g h l i g h t s Recombinant CRL2 isozyme efficiently converted crude Jatropha oil into biodiesel. Optimum reaction parameters of the liquid CRL2-based process were determined. CRL2 isozyme achieved 95.3% of FAME yield in a methanol feed-batch process. Liquid CRL2 retained 53.0% of FAME yield after 3 reuse cycles for 6 days.
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Article history: Received 3 April 2015 Received in revised form 3 May 2015 Accepted 4 May 2015 Available online 19 May 2015 Keywords: Biodiesel Jatropha curcas Crude seed oil Candida rugosa lipase (CRL) isozyme Soluble lipase
a b s t r a c t The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1–CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24 h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, commonly called fatty acid methyl ester (FAME) and synthesized using the transesterification of edible, non-edible, and waste oils, is a renewable alternative to diesel fuel (Park et al., 2008). Commercially, biodiesel is typically produced using efficient chemical processes that exhibit rapid reaction times and high yields; however, this process also presents various drawbacks, such as soap formation, difficult recovery of glycerol, the need to remove salt residues, the production of a large amount of wastewater, and high energy costs. Therefore, lipase-catalyzed biodiesel production under mild conditions is considered a more environmental friendly process than the use of other chemical processes to produce biodiesel (Wang et al., 2014).
⇑ Corresponding author at: Department of Life Science, National Taiwan Normal University, Taipei 11677, Taiwan. Tel.: +886 2 7734 6351; fax: +886 2 2931 2904. E-mail address: [email protected] (G.-C. Lee). http://dx.doi.org/10.1016/j.biortech.2015.05.008 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
The various types of vegetable oils used for biodiesel synthesis are high-cost refined edible oils, such as soybean oil, rapeseed oil, cotton seed oil, and sunflower oil (Ranganathan et al., 2008). However, the primary raw materials used to produce biodiesel account for over 85% of biodiesel production costs (Moser, 2008). Moreover, biodiesel production is expensive in numerous developing countries because of the lack of edible oils. Therefore, the use of non-edible plant feedstocks, such as Jatropha (Jatropha curcas), microalgae, Neem (Azadirachta indica), Karanja (Pongamia pinnata), and rubber seed, for biodiesel synthesis is a more economical process than the use of edible oils (Abdulla et al., 2011). Unlike the chemical process, the enzymatic process for biodiesel production allows the use of less expensive and more varied types of feedstock with a free fatty acid content as high as 100% including non-edible oils (Hama and Kondo, 2013). Many studies used non-edible oils as feedstocks for enzymatic biodiesel productions. For example, nowadays at least eight sources of lipase were identified to convert Jatropha oil into biodiesel with yields ranging from 70% to 98% (Abdulla et al., 2011; Li et al., 2014; You et al.,
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2013). Immobilized lipases have been extensively used in previous studies. However, the use of immobilized lipases is often affected by their high costs; the price of biocatalysts used in the industrial enzymatic biodiesel production process should be reduced. Therefore, liquid enzymatic biodiesel processing has proven technically and economically viable recently. An increasing interest has been shown in the application of low-cost liquid lipase formulations (e.g., Callera™ Trans L) for biodiesel production (Cesarini et al., 2013; Toftgaard Pedersen et al., 2014). Liquid lipase can be reused multiple times, making the production cost per gallon significantly lower compared to that of traditional biodiesel (Huang et al., 2015). Candida rugosa lipase (CRL) is a well-known and widely used enzyme in biotransformation reactions. It has been used to produce valuable materials for foods, flavors, fragrances, cosmetics, pharmaceuticals, and other industrial applications (Akoh et al., 2004). Previous studies have shown that commercial CRL displayed particularly high catalytic activity toward alcoholysis of soybean oil (Kuo et al., 2013), rapeseed oil (Linko et al., 1998), and sunflower oil (Sagiroglu, 2008). However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL. Data have shown that commercial CRL has not been able to efficiently catalyze FAME production from Jatropha oil (Shah and Gupta, 2007). Several studies have indicated that commercial CRL samples are crude enzyme preparations which contain various types of isozymes. The isozyme profiles vary among suppliers and may be affected by fermentation conditions (Dalmau et al., 2000). Therefore, commercial CRL used as a biocatalyst typically exhibits remarkable variation in catalytic efficiency, regioselectivity, and stereospecificity (Dominguez de Maria et al., 2006). We successfully expressed individual recombinant CRL isozymes (CRL1 to CRL5) in Pichia pastoris and demonstrated that they exhibited distinct substrate preferences and catalytic activities (Chang et al., 2006a,b; Lee et al., 2002, 2011; Tang et al., 2001), which suggests a new opportunity for biodiesel production using specific recombinant CRL isozymes. In our previous study, we explored the transesterification of soybean oil with various recombinant CRL isozymes (CRL1–CRL4), and demonstrated that CRL1 exhibits a superior performance with a FAME conversion rate of 61.5% (Chang et al., 2014). However, individual recombinant CRL isozymes have not been used in biodiesel synthesis from non-edible plant oils. In this study, the catalytic efficiencies of the four recombinant CRL isozymes for biodiesel production using three non-edible oils (i.e., Jatropha, Karanja, and castor oils) as feedstocks were studied. The optimal combination of recombinant CRL isozyme and feedstock with the highest catalytic efficiency was identified and investigated to identify optimal biodiesel production conditions, such as water content, reaction temperature, molar ratio of substrates, method of methanol addition, and time course of FAME production. Moreover, the reusability of liquid lipase solution was also investigated.
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2.2. Strains and lipase production Recombinant CRL isozymes from P. pastoris were used in this study. Four recombinant P. pastoris strains harboring recombinant CRL1, CRL2, CRL3, and CRL4 expression vectors were established in our previous studies (Chang et al., 2006a,b; Lee et al., 2002; Tang et al., 2001). An extracellular CRL isozyme was produced by culturing P. pastoris in a shaking flask containing a 50-mL glycerol medium (2% glycerol, 1% yeast extract, and 0.5% ammonium sulfate), comprising 100 lg/mL Zeocin at 20 °C and at 200 rpm for 5 days. At the end of the incubation period, the fermentation supernatant containing CRL was collected using centrifugation at 7000g for 10 min, and was concentrated using ultrafiltration with an Amicon Ultra-4 10 kDa cut-off centrifugal filter (Merk KGaA, Darmstadt, Germany). The concentrated enzyme samples were used in the synthesis of FAME. 2.3. Enzyme assay Lipase activity was determined using p-nitrophenyl butyrate as a substrate in a microplate spectrophotometer (Multiskan FC Microplate Photometer, Thermo Scientific). The reaction contained a 10-lL concentrated fermentation supernatant, 100-lL 20 mM phosphate buffer (pH 7.0), 0.25% Triton X-100, and 0.5 mM substrate. The reaction was performed at 37 °C. The hydrolysis product p-nitrophenol was measured using absorbance at 405 nm. The increase in absorbance was recorded for 10 min to calculate the initial rate of lipase. One unit of activity was defined as the quantity of enzyme necessary to release 1 lmol p-nitrophenol per minute under the stated conditions. 2.4. Enzymatic synthesis of fatty acid methyl ester To identify specific CRL isozymes for converting crude non-edible oils into biodiesel, a enzymatic transesterification reaction was performed in 20-mL screw-cap glass vials on an incubator shaker at 250 rpm. During the reaction, 0.5 g of crude Jatropha, Karanja, or castor oil was mixed with 1 equivalent (eq.) amount of methanol (1 eq. = 3 mol of alcohol per mole of triglycerides), lipase solution, and demineralized water. Water and lipase solution were first mixed and then added to each reaction in weight percent relative to the mass of oil used. The lipase solution contributed to the water content (e.g., 50 lL of lipase solution equivalently contributed 50 mg of water). The reaction was incubated at 37 °C for 24 h. To optimize the biodiesel production condition with the identified CRL isozyme, the effects of water content, enzyme amount, reaction temperature, various substrate molar ratios, and stepwise addition of methanol were studied using single-factor experimental design (i.e., when one variable range was studied, other variables were kept constant). 2.5. Sampling and analysis
2. Methods 2.1. Materials Jatropha seeds were used in this study that were gifts from Bioptik Technology Inc. (Miaoli County, Taiwan) and Shan-Hai-Guan riding club (New Taipei City, Taiwan). Karanja seeds and castor seeds were collected from wild species in the north and south of Taiwan, respectively. Crude oils were extracted using n-hexane in a Soxhlet apparatus (de Oliveira et al., 2009). Standard fatty acid esters were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other types of reagents and solvents used in this study were of analytical reagent grade.
Samples were collected from the reaction mixture at specified time points for the FAME assay using gas chromatography. The reaction mixture was immediately centrifuged at 8000g for 1 min. The upper layer containing FAME was then transferred to clean bottles for further analysis. Ten-milligram samples were added to 600 lL of methyl heptadecanoate (1 mg/mL in n-hexane) which is an internal standard for quantitative analysis. The quantification of FAME contents was performed according to the European Standard Method, EN 14103. Analysis of the FAME was performed by a Thermo TRACE™ 1300 gas chromatograph equipped with a flame-ionization detector, programmable temperature vaporizing injector, and a TR-BioDiesel (F) column
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3. Results and discussion 3.1. Screening of CRL isozymes for the conversion of non-edible oils Four recombinant enzymes (CRL1–CRL4) were expressed in P. pastoris and collected from the fermentation supernatant. Forty units (U) of each concentrated CRL were used to determine the biocatalytic efficiency on FAME production by using Jatropha, Karanja, or castor oils. As shown in Table 1, the catalytic efficiencies of CRLs on Jatropha oil were superior to Karanja and castor oils. The FAME yield from Jatropha oil catalyzed by CRL2 (36.0 ± 2.5%) was similar to that catalyzed by CRL4 (36.9 ± 0.3%), and was higher than that catalyzed by CRL1 (17.3 ± 1.7%) or CRL3 (24.3 ± 0.9%). Based on these results, CRL2 and CRL4 were the superior types of isozymes for biodiesel production using Jatropha oil, and CRL2 was then used as a biocatalyst to study the optimal conditions for transesterification. Phylogenetic analysis based on the amino acid sequence alignment of the four CRL isozymes revealed that CRL2 and CRL4 shared a high homology and CRL1, CRL3, and CRL5 displayed a high degree of homology (Lotti et al., 1993). Sequence similarity may reflect similar protein structure and function. In our previous studies, a comparison of the modeled structures of CRL2 and CRL4 revealed high degrees of similarity, and both showed higher amounts of activity toward medium- and long-chain fatty acid esters (C12– C18) (Lee et al., 2002). In addition, the favorable substrates of CRL1 and CRL3 were medium-chain fatty acid esters, C8–C12 and C8–C14, respectively (Chang et al., 2006a,b). The fatty acid composition of Jatropha oil is 14.6% palmitic acid (16:0), 6.9% stearic acid (18:0), 46.2% oleic acid (18:1), and 30.8% linoleic acid (18:2) (Kawakami et al., 2011), which indicates that long-chain fatty acids are predominant in the Jatropha oil and may account for the preferable conversion of this oil by CRL2 and CRL4. Previous studies have shown that commercial crude CRLs displayed no transesterification activity for Jatropha oil (Shah and Gupta, 2007; Shah et al., 2004). The CRL isozyme composition of commercial CRL samples depended on the suppliers, and varied among lots within the same supplier. It has been reported that CRL1 is the major component in most commercial preparations, and that CRL3 and CRL2 range only from approximately 0–25% (Dominguez de Maria et al., 2006). Our results confirmed that individual CRL isozymes possess distinctive substrate specificities, and that no activity on the transesterification of Jatropha oil by commercial CRL preparations may be due to the lack of CRL2 and CRL4 isozymes. Our research revealed an important new finding
Table 1 Comparison of CRL isozyme transesterification activities for three non-edible oils. Reaction conditions were 0.5 g oil, 1 eq. methanol, 30% water, 40 U of each isozyme, 250 rpm, and 37 °C for 24 h. Lipase activities of each recombinant isozyme were calculated as 2857 U/mL for CRL1, 674 U/mL for CRL2, 307 U/mL for CRL3, and 586 U/ mL for CRL4. FAME yield (%)
CRL1 CRL2 CRL3 CRL4
that instead of commercial crude CRL, the specific recombinant CRL isozymes could provide a promising solution for the biodiesel synthesis from Jatropha oil.
3.2. Optimization of reaction conditions 3.2.1. Effects of water content and enzyme dosage During the transesterification reaction, water is necessary to maintain enzyme configuration and to increase the available interfacial area between water and oil. However, excess water may dilute the amount of methanol available and reverse the esterification reaction into the hydrolysis reaction. The optimal water content depends on the lipase, feedstock oil, and enzyme-immobilized support (You et al., 2013). Therefore, appropriate water content is an essential parameter in enzymatic biodiesel synthesis. The influence of water content on the initial rates and FAME yields that were catalyzed by various CRL2 concentrations (20 U, 40 U, and 80 U per 0.5 g of oil) was investigated (Figs. 1 and 2), which showed that 20% water content was not sufficient for the reactions catalyzed by any CRL2 loads. Sufficient interfacial area was provided by 30% water content for 20 U CRL2, and further increases in water content did not increase or decrease the initial rate due to the dilution of enzymes. A similar situation was observed in 40 U CRL2 loading with 40% water content. In this study, 80 U CRL2 loading with 50% water content achieved the highest initial rate (3.25% h1) (Fig. 1). Moreover, after a reaction lasting 24 h, the FAME yield of 20 U CRL2 loading with 30% water content was 34%, which was higher than 20 U CRL2 loading with 40% and 50% water content (Fig. 2B–D). The 24-h FAME yield of 40 U CRL2 loading with 40% water content reached 49.1%, which was higher than 40 U CRL2 loading with 50% water content (Fig. 2C and D). The 24-h FAME yield of 80 U CRL2 loading was particularly enhanced by increasing water content, and reached 62.9% when water content was 50% (Fig. 2D). These results suggested that the FAME production of each enzyme loading was strongly affected by various percentages of water content. As previous studies have shown, the biodiesel production which was catalyzed using a higher enzyme concentration benefited more from the larger interfacial area provided by the higher level of water content (Nordblad et al., 2014). Although 50% water content provided the maximum interfacial area in this study and achieved the highest 24-h FAME yield when 80 U CRL2 was used, excessive volume of the polar phase occupied part of the reactor capacity and reduced
4 -1
(30 m 0.25 mm; 0.25-lm film thickness). A sample volume of 1 lL was injected into the column using the split mode (1:100 split ratio). The type of carrier gas used was high purity nitrogen at a flow rate 1 mL/min. The oven temperature was increased by 2 °C/min from 200 °C to 220 °C, and maintained at 260 °C for 10 min. The injector and the detector temperature were set at 260 °C and 270 °C, respectively.
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2.30% ± 0.07 1.42% ± 0.04 0.68% ± 0.02 0.28% ± 0.14
1.51% ± 0.11 1.47% ± 0.04 19.08% ± 0.29 0.22% ± 0.10
Fig. 1. Effects of water content and enzyme dosage on the initial rate of FAME production. Lipase activity of the concentrated CRL2 was calculated to be 674 U/mL, and the CRL2 solution also contributed to the water content (wt% based on oil weight). Reaction conditions were 0.5 g Jatropha oil, 1 eq. methanol, 250 rpm, and 37 °C for 4 h.
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Fig. 2. Time courses of FAME yield for the effects of water content and enzyme dosage. (A–D) indicate the effects of 20%, 30%, 40%, and 50% water contents on 24-h FAME yield, respectively. Reaction conditions were 0.5 g Jatropha oil, 1 eq. methanol, and 250 rpm, and 20 U (d), 40 U (s), or 80 U (.) of CRL2 solution was loaded to start the reaction at 37 °C for 24 h.
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volumetric efficiency (i.e., the ratio of oil to polar phase) of the process. Therefore, the FAME yield should be a compromise between the water content and the enzyme loading amount. 3.2.2. Effect of temperature To examine the effect of temperature on the biodiesel production catalyzed by CRL2, the experiments in this study were performed between 10 °C and 50 °C (Fig. 3). The reaction mixture
included 0.5 g Jatropha oil, 66 mg methanol (molar ratio of oil to methanol was 1:3), 50% water, and 80 U CRL2; the reaction lasted for 24 h. FAME yield increased with the increasing temperatures from 10 °C to 37 °C and reached a maximum at 37 °C (56.9% FAME yield); however, they rapidly decreased at 50 °C. When the reaction temperature was at 10 °C, 20 °C, and 30 °C, CRL2 remained around 57.7%, 84.8% and 88.4% of the activity, respectively it had at 37 °C. In our previous study, the CRL2-catalyzed conversion of soybean oil reached a maximum at 40 °C (40.2% FAME yield) and remained above 80% of the activity it had at 40 °C when the temperature was between 10 °C and 40 °C (Chang et al., 2014). Our results implied that CRL2 can be applied in biodiesel production by using refined oil or crude non-edible oil. Notably, the catalytic efficiency on Jatropha oil was even higher than that on soybean oil. In addition, the mild and broad-range optimal production temperatures may reduce the energy cost for the industrial process, and thus reduce the production cost of biodiesel.
3.2.3. Effect of substrate molar ratio A stoichiometric amount (1 eq.) of methanol is typically required for the complete conversion of triacylglycerols into FAMEs. Usually, adding more alcohol could increase the transesterification yield. However, excessive amount of alcohol can also inhibit the enzyme activity and thus decrease biodiesel production (Noureddini et al., 2005). In this study, the optimal substrate molar ratio (oil: methanol) was investigated in a range from 1:3 to 1:6 (i.e., 1–2 eq.) (Fig. 4). The reactions containing 50% water content, 80 U CRL2 per 0.5 g of oil, and various substrate ratios were
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performed at 37 °C for 72 h. As shown in Fig. 4, the oil to methanol molar ratios of 1:3 and 1:4 resulted in higher FAME yields (93.5% for 1:3 and 88.8% for 1:4); however, methanol amounts of 1:5 or 1:6 decrease the FAME production. The methanol amounts of 1:5 and 1:6 are equivalent to 23.0% and 26.4% (w/w) in the water phase, respectively. The effect of methanol on the activity of recombinant CRL2 was investigated previously (Chang et al., 2011). After incubation at 37 °C in 0.1 M Tris/HCl buffer containing 30% methanol for 1 h, the activity of CRL2 decreased significantly. Therefore, the higher amounts of methanol in the water phase may inactivate CRL2, and thus decrease the FAME production.
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To prevent the inactivation of enzymes by methanol, previous studies have attempted to add methanol stepwise into the reaction system or to maintain the oil to methanol ratio below 1:5 (Wang et al., 2014). Based on the optimal oil to methanol molar ratio of 1:3 (1 eq. of methanol), we investigated the time courses of FAME yields for various stepwise methanol feeding profiles; 1 eq. of methanol was initially added to the reaction, and then 1 eq. or 0.5 eq. of methanol was additionally added at various reaction times (8 h, 16 h, or 24 h). As shown in Fig. 5A, an additional 1 eq. feeding of methanol at 8 h would inactivate CRL2, and thus reduce the FAME yield; additional feeding at 16 h or 24 h resulted in a similar 72-h FAME yield when compared with a reaction without additional methanol feeding. However, 91.6% of a 48-h FAME yield could be achieved for the additional 1 eq. feeding at 24 h. The amount of methanol does not exceed 1.4 eq. (i.e., 3 3 60% + 3 = 4.2 mol = 1.4 eq. for about 60% conversion at 24 h) when an additional 1 eq. feeds at 24 h, and this substrate molar ratio would not inactivate CRL2, as described in the results and discussion section. As shown in Fig. 5B, an additional 0.5 eq. feeding of methanol at 8 h, 16 h, or 24 h achieved 94.5%, 94.8%, or 95.3%, respectively, of 48-h FAME yields, which were all higher than the yield from no additional methanol feeding. These results suggested that an initial 1 eq. of methanol feeding followed by an additional 0.5 eq. feeding of methanol at 24 h was the optimal methanol feeding profile because of no inhibition of CRL2. In the previous published reports about enzymatic biodiesel production from Jatropha oil, they all used high-cost immobilized biocatalysts. In the present study, we showed that the low-cost liquid enzyme-based process can achieve a FAME yield which is comparable to those best yields of the immobilized enzyme-based studies (Shah and Gupta, 2007; Su et al., 2009; You et al., 2013).
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Reaction time (hr) Fig. 5. Time courses of FAME yield for various stepwise methanol feeding profiles. (A) 1 eq. methanol was initially added to the reaction and an additional 1 eq. methanol was added at 8 h (d), 16 h (s), or 24 h (.) during biodiesel production. (B) 1 eq. methanol was initially added to the reaction and an additional 0.5 eq. methanol was added at 8 h (d), 16 h (s), or 24 h (.) during biodiesel production. Symbol (4) indicates the reaction without additional methanol feeding.
We used the liquid recombinant CRL2 directly from fermentation medium for the transesterification of Jatropha oil without enzyme preparation process. P. pastoris is a well-established system for recombinant protein expression, which can produce a large amount of recombinant protein through high-density fermentation, and thus may be used to reduce the cost of enzymes in industrial applications. Using the P. pastoris expression system, a large amount of recombinant CRL2 and CRL4 isozymes could be obtained, implying that this enzymatic process may provide a solution for the biodiesel synthesis of Jatropha oil on an industrial scale.
3.4. Reusability of liquid lipase
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Reaction time (hr) Fig. 4. Time courses of FAME yield for the effect of substrate molar ratio. Reaction conditions were 1 g Jatropha oil, 160 U CRL2 solution, 50% water, 250 rpm, and 37 °C for 72 h.
After the feed-batch transesterification reaction occurred, three phases were formed by centrifugation in the reaction mixture. The upper layer was the FAME phase, which contained the FAME product and the remaining glycerides. The combination of the middle and lower layers is called the glycerol-water phase, which contained water, produced glycerol, used CRL2, and the residual methanol. The glycerol-water phase can be recovered for use in the subsequent batch reaction. When the glycerol-water phase was repeated, the residual methanol (approximately 0.5 eq.) was considered when adding methanol for the subsequent batch. In batch numbers 2–4, the feeding amount of the initial methanol did not exceed 0.5 eq. so that it could limit the total methanol
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not to more than 1 eq.; an additional 0.5 eq. feeding of methanol occurred at 24 h, and the reaction was performed for 48 h. The FAME yields of the first, second and third reuse cycles were 94.9%, 81.1%, and 53%, respectively. Furthermore, the liquid CRL2 retained 56% of its relative activity after 6 days and three reuse cycles, and 37.5% of its relative activity after 8 days and four reuse cycles. 4. Conclusion The recombinant CRL2 and CRL4 were the superior types of isozymes for biodiesel production from Jatropha oil. Optimal reaction parameters of the liquid CRL2-based process were determined as follows: 160 U CRL2 per gram of oil, 50 wt% water, 1 eq. of methanol, an additional 0.5 eq. of methanol feeds at 24 h, and a total reaction time of 48 h at 37 °C. The maximum 95.3% of the FAME yield could be achieved. These results implied that this enzymatic process may provide a promising solution on an industrial scale for biodiesel production from low-cost crude Jatropha oil. Acknowledgements Our gratitude is extended to the Academic Paper Editing Clinic, NTNU. References Abdulla, R., Chan, E.S., Ravindra, P., 2011. Biodiesel production from Jatropha curcas: a critical review. Crit. Rev. Biotechnol. 31, 53–64. Akoh, C.C., Lee, G.C., Shaw, J.F., 2004. Protein engineering and applications of Candida rugosa lipase isoforms. Lipids 39, 513–526. Cesarini, S., Diaz, P., Nielsen, P.M., 2013. Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock. Process Biochem. 48, 484–487. Chang, S.W., Huang, M., Hsieh, Y.H., Luo, Y.T., Wu, T.T., Tsai, C.W., Chen, C.S., Shaw, J.F., 2014. Simultaneous production of fatty acid methyl esters and diglycerides by four recombinant Candida rugosa lipase’s isozymes. Food Chem. 155, 140– 145. Chang, S.W., Lee, G.C., Shaw, J.F., 2006a. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J. Agric. Food Chem. 54, 815–822. Chang, S.W., Lee, G.C., Shaw, J.F., 2006b. Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J. Agric. Food Chem. 54, 5831–5838. Chang, S.W., Li, C.F., Lee, G.C., Yeh, T., Shaw, J.F., 2011. Engineering the expression and biochemical characteristics of recombinant Candida rugosa LIP2 lipase by removing the additional N-terminal peptide and regional codon optimization. J. Agric. Food Chem. 59, 6710–6719. Dalmau, E., Montesinos, J.L., Lotti, M., Casas, C., 2000. Effect of different carbon sources on lipase production by Candida rugosa. Enzyme Microb. Technol. 26, 657–663. de Oliveira, J.S., Leite, P.M., de Souza, L.B., Mello, V.M., Silva, E.C., Rubim, J.C., Meneghetti, S.M.P., Suarez, P.A.Z., 2009. Characteristics and composition of Jatropha gossypiifolia and Jatropha curcas L. oils and application for biodiesel production. Biomass Bioenergy 33, 449–453.
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Dominguez de Maria, P., Sanchez-Montero, J.M., Sinisterra, J.V., Alcantara, A.R., 2006. Understanding Candida rugosa lipases: an overview. Biotechnol. Adv. 24, 180–196. Hama, S., Kondo, A., 2013. Enzymatic biodiesel production: an overview of potential feedstocks and process development. Bioresour. Technol. 135, 386–395. Huang, J., Xia, J., Jiang, W., Li, Y., Li, J., 2015. Biodiesel production from microalgae oil catalyzed by a recombinant lipase. Bioresour. Technol. 180, 47–53. Kawakami, K., Oda, Y., Takahashi, R., 2011. Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil. Biotechnol. Biofuels 4, 42. Kuo, C.H., Peng, L.T., Kan, S.C., Liu, Y.C., Shieh, C.J., 2013. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. 145, 229– 232. Lee, G.C., Lee, L.C., Sava, V., Shaw, J.F., 2002. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. Biochem. J. 366, 603–611. Lee, L.C., Yen, C.C., Malmis, C.C., Chen, L.F., Chen, J.C., Lee, G.C., Shaw, J.F., 2011. Characterization of codon-optimized recombinant candida rugosa lipase 5 (LIP5). J. Agric. Food Chem. 59, 10693–10698. Li, X., Qian, P., Wu, S.G., Yu, H.Y., 2014. Characterization of an organic solventtolerant lipase from Idiomarina sp. W33 and its application for biodiesel production using Jatropha oil. Extremophiles 18, 171–178. Linko, Y.Y., Lamsa, M., Wu, X., Uosukainen, E., Seppala, J., Linko, P., 1998. Biodegradable products by lipase biocatalysis. J. Biotechnol. 66, 41–50. Lotti, M., Grandori, R., Fusetti, F., Longhi, S., Brocca, S., Tramontano, A., Alberghina, L., 1993. Cloning and analysis of Candida cylindracea lipase sequences. Gene 124, 45–55. Moser, B.R., 2008. Influence of blending canola, palm, soybean, and sunflower oil methyl esters on fuel properties of biodiesel. Energy Fuels 22, 4301–4306. Nordblad, M., Silva, V.T., Nielsen, P.M., Woodley, J.M., 2014. Identification of critical parameters in liquid enzyme-catalyzed biodiesel production. Biotechnol. Bioeng. 111, 2446–2453. Noureddini, H., Gao, X., Philkana, R.S., 2005. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour. Technol. 96, 769–777. Park, E.Y., Sato, M., Kojima, S., 2008. Lipase-catalyzed biodiesel production from waste activated bleaching earth as raw material in a pilot plant. Bioresour. Technol. 99, 3130–3135. Ranganathan, S.V., Narasimhan, S.L., Muthukumar, K., 2008. An overview of enzymatic production of biodiesel. Bioresour. Technol. 99, 3975–3981. Sagiroglu, A., 2008. Conversion of sunflower oil to biodiesel by alcoholysis using immobilized lipase. Artif. Cells Blood Substit. Immobil. Biotechnol. 36, 138–149. Shah, S., Gupta, M.N., 2007. Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system. Process Biochem. 42, 409–414. Shah, S., Sharma, S., Gupta, M.N., 2004. Biodiesel preparation by lipase-catalyzed tranesterification of Jatropha oil. Energy Fuels 18, 154–159. Su, E., You, P., Wei, D., 2009. In situ lipase-catalyzed reactive extraction of oilseeds with short-chained dialkyl carbonates for biodiesel production. Bioresour. Technol. 100, 5813–5817. Tang, S.J., Shaw, J.F., Sun, K.H., Sun, G.H., Chang, T.Y., Lin, C.K., Lo, Y.C., Lee, G.C., 2001. Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability. Arch. Biochem. Biophys. 387, 93–98. Toftgaard Pedersen, A., Nordblad, M., Nielsen, P.M., Woodley, J.M., 2014. Batch production of FAEE-biodiesel using a liquid lipase formulation. J. Mol. Catal. B: Enzym. 105, 89–94. Wang, Y., Liu, J., Gerken, H., Zhang, C., Hu, Q., Li, Y., 2014. Highly-efficient enzymatic conversion of crude algal oils into biodiesel. Bioresour. Technol. 172, 143–149. You, Q., Yin, X., Zhao, Y., Zhang, Y., 2013. Biodiesel production from Jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite. Bioresour. Technol. 148, 202–207.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes Ting-Chun Kuo a, Jei-Fu Shaw b, Guan-Chiun Lee a,⇑ a b
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan
h i g h l i g h t s Recombinant CRL2 isozyme efficiently converted crude Jatropha oil into biodiesel. Optimum reaction parameters of the liquid CRL2-based process were determined. CRL2 isozyme achieved 95.3% of FAME yield in a methanol feed-batch process. Liquid CRL2 retained 53.0% of FAME yield after 3 reuse cycles for 6 days.
a r t i c l e
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Article history: Received 3 April 2015 Received in revised form 3 May 2015 Accepted 4 May 2015 Available online 19 May 2015 Keywords: Biodiesel Jatropha curcas Crude seed oil Candida rugosa lipase (CRL) isozyme Soluble lipase
a b s t r a c t The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1–CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24 h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, commonly called fatty acid methyl ester (FAME) and synthesized using the transesterification of edible, non-edible, and waste oils, is a renewable alternative to diesel fuel (Park et al., 2008). Commercially, biodiesel is typically produced using efficient chemical processes that exhibit rapid reaction times and high yields; however, this process also presents various drawbacks, such as soap formation, difficult recovery of glycerol, the need to remove salt residues, the production of a large amount of wastewater, and high energy costs. Therefore, lipase-catalyzed biodiesel production under mild conditions is considered a more environmental friendly process than the use of other chemical processes to produce biodiesel (Wang et al., 2014).
⇑ Corresponding author at: Department of Life Science, National Taiwan Normal University, Taipei 11677, Taiwan. Tel.: +886 2 7734 6351; fax: +886 2 2931 2904. E-mail address: [email protected] (G.-C. Lee). http://dx.doi.org/10.1016/j.biortech.2015.05.008 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
The various types of vegetable oils used for biodiesel synthesis are high-cost refined edible oils, such as soybean oil, rapeseed oil, cotton seed oil, and sunflower oil (Ranganathan et al., 2008). However, the primary raw materials used to produce biodiesel account for over 85% of biodiesel production costs (Moser, 2008). Moreover, biodiesel production is expensive in numerous developing countries because of the lack of edible oils. Therefore, the use of non-edible plant feedstocks, such as Jatropha (Jatropha curcas), microalgae, Neem (Azadirachta indica), Karanja (Pongamia pinnata), and rubber seed, for biodiesel synthesis is a more economical process than the use of edible oils (Abdulla et al., 2011). Unlike the chemical process, the enzymatic process for biodiesel production allows the use of less expensive and more varied types of feedstock with a free fatty acid content as high as 100% including non-edible oils (Hama and Kondo, 2013). Many studies used non-edible oils as feedstocks for enzymatic biodiesel productions. For example, nowadays at least eight sources of lipase were identified to convert Jatropha oil into biodiesel with yields ranging from 70% to 98% (Abdulla et al., 2011; Li et al., 2014; You et al.,
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2013). Immobilized lipases have been extensively used in previous studies. However, the use of immobilized lipases is often affected by their high costs; the price of biocatalysts used in the industrial enzymatic biodiesel production process should be reduced. Therefore, liquid enzymatic biodiesel processing has proven technically and economically viable recently. An increasing interest has been shown in the application of low-cost liquid lipase formulations (e.g., Callera™ Trans L) for biodiesel production (Cesarini et al., 2013; Toftgaard Pedersen et al., 2014). Liquid lipase can be reused multiple times, making the production cost per gallon significantly lower compared to that of traditional biodiesel (Huang et al., 2015). Candida rugosa lipase (CRL) is a well-known and widely used enzyme in biotransformation reactions. It has been used to produce valuable materials for foods, flavors, fragrances, cosmetics, pharmaceuticals, and other industrial applications (Akoh et al., 2004). Previous studies have shown that commercial CRL displayed particularly high catalytic activity toward alcoholysis of soybean oil (Kuo et al., 2013), rapeseed oil (Linko et al., 1998), and sunflower oil (Sagiroglu, 2008). However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL. Data have shown that commercial CRL has not been able to efficiently catalyze FAME production from Jatropha oil (Shah and Gupta, 2007). Several studies have indicated that commercial CRL samples are crude enzyme preparations which contain various types of isozymes. The isozyme profiles vary among suppliers and may be affected by fermentation conditions (Dalmau et al., 2000). Therefore, commercial CRL used as a biocatalyst typically exhibits remarkable variation in catalytic efficiency, regioselectivity, and stereospecificity (Dominguez de Maria et al., 2006). We successfully expressed individual recombinant CRL isozymes (CRL1 to CRL5) in Pichia pastoris and demonstrated that they exhibited distinct substrate preferences and catalytic activities (Chang et al., 2006a,b; Lee et al., 2002, 2011; Tang et al., 2001), which suggests a new opportunity for biodiesel production using specific recombinant CRL isozymes. In our previous study, we explored the transesterification of soybean oil with various recombinant CRL isozymes (CRL1–CRL4), and demonstrated that CRL1 exhibits a superior performance with a FAME conversion rate of 61.5% (Chang et al., 2014). However, individual recombinant CRL isozymes have not been used in biodiesel synthesis from non-edible plant oils. In this study, the catalytic efficiencies of the four recombinant CRL isozymes for biodiesel production using three non-edible oils (i.e., Jatropha, Karanja, and castor oils) as feedstocks were studied. The optimal combination of recombinant CRL isozyme and feedstock with the highest catalytic efficiency was identified and investigated to identify optimal biodiesel production conditions, such as water content, reaction temperature, molar ratio of substrates, method of methanol addition, and time course of FAME production. Moreover, the reusability of liquid lipase solution was also investigated.
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2.2. Strains and lipase production Recombinant CRL isozymes from P. pastoris were used in this study. Four recombinant P. pastoris strains harboring recombinant CRL1, CRL2, CRL3, and CRL4 expression vectors were established in our previous studies (Chang et al., 2006a,b; Lee et al., 2002; Tang et al., 2001). An extracellular CRL isozyme was produced by culturing P. pastoris in a shaking flask containing a 50-mL glycerol medium (2% glycerol, 1% yeast extract, and 0.5% ammonium sulfate), comprising 100 lg/mL Zeocin at 20 °C and at 200 rpm for 5 days. At the end of the incubation period, the fermentation supernatant containing CRL was collected using centrifugation at 7000g for 10 min, and was concentrated using ultrafiltration with an Amicon Ultra-4 10 kDa cut-off centrifugal filter (Merk KGaA, Darmstadt, Germany). The concentrated enzyme samples were used in the synthesis of FAME. 2.3. Enzyme assay Lipase activity was determined using p-nitrophenyl butyrate as a substrate in a microplate spectrophotometer (Multiskan FC Microplate Photometer, Thermo Scientific). The reaction contained a 10-lL concentrated fermentation supernatant, 100-lL 20 mM phosphate buffer (pH 7.0), 0.25% Triton X-100, and 0.5 mM substrate. The reaction was performed at 37 °C. The hydrolysis product p-nitrophenol was measured using absorbance at 405 nm. The increase in absorbance was recorded for 10 min to calculate the initial rate of lipase. One unit of activity was defined as the quantity of enzyme necessary to release 1 lmol p-nitrophenol per minute under the stated conditions. 2.4. Enzymatic synthesis of fatty acid methyl ester To identify specific CRL isozymes for converting crude non-edible oils into biodiesel, a enzymatic transesterification reaction was performed in 20-mL screw-cap glass vials on an incubator shaker at 250 rpm. During the reaction, 0.5 g of crude Jatropha, Karanja, or castor oil was mixed with 1 equivalent (eq.) amount of methanol (1 eq. = 3 mol of alcohol per mole of triglycerides), lipase solution, and demineralized water. Water and lipase solution were first mixed and then added to each reaction in weight percent relative to the mass of oil used. The lipase solution contributed to the water content (e.g., 50 lL of lipase solution equivalently contributed 50 mg of water). The reaction was incubated at 37 °C for 24 h. To optimize the biodiesel production condition with the identified CRL isozyme, the effects of water content, enzyme amount, reaction temperature, various substrate molar ratios, and stepwise addition of methanol were studied using single-factor experimental design (i.e., when one variable range was studied, other variables were kept constant). 2.5. Sampling and analysis
2. Methods 2.1. Materials Jatropha seeds were used in this study that were gifts from Bioptik Technology Inc. (Miaoli County, Taiwan) and Shan-Hai-Guan riding club (New Taipei City, Taiwan). Karanja seeds and castor seeds were collected from wild species in the north and south of Taiwan, respectively. Crude oils were extracted using n-hexane in a Soxhlet apparatus (de Oliveira et al., 2009). Standard fatty acid esters were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other types of reagents and solvents used in this study were of analytical reagent grade.
Samples were collected from the reaction mixture at specified time points for the FAME assay using gas chromatography. The reaction mixture was immediately centrifuged at 8000g for 1 min. The upper layer containing FAME was then transferred to clean bottles for further analysis. Ten-milligram samples were added to 600 lL of methyl heptadecanoate (1 mg/mL in n-hexane) which is an internal standard for quantitative analysis. The quantification of FAME contents was performed according to the European Standard Method, EN 14103. Analysis of the FAME was performed by a Thermo TRACE™ 1300 gas chromatograph equipped with a flame-ionization detector, programmable temperature vaporizing injector, and a TR-BioDiesel (F) column
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3. Results and discussion 3.1. Screening of CRL isozymes for the conversion of non-edible oils Four recombinant enzymes (CRL1–CRL4) were expressed in P. pastoris and collected from the fermentation supernatant. Forty units (U) of each concentrated CRL were used to determine the biocatalytic efficiency on FAME production by using Jatropha, Karanja, or castor oils. As shown in Table 1, the catalytic efficiencies of CRLs on Jatropha oil were superior to Karanja and castor oils. The FAME yield from Jatropha oil catalyzed by CRL2 (36.0 ± 2.5%) was similar to that catalyzed by CRL4 (36.9 ± 0.3%), and was higher than that catalyzed by CRL1 (17.3 ± 1.7%) or CRL3 (24.3 ± 0.9%). Based on these results, CRL2 and CRL4 were the superior types of isozymes for biodiesel production using Jatropha oil, and CRL2 was then used as a biocatalyst to study the optimal conditions for transesterification. Phylogenetic analysis based on the amino acid sequence alignment of the four CRL isozymes revealed that CRL2 and CRL4 shared a high homology and CRL1, CRL3, and CRL5 displayed a high degree of homology (Lotti et al., 1993). Sequence similarity may reflect similar protein structure and function. In our previous studies, a comparison of the modeled structures of CRL2 and CRL4 revealed high degrees of similarity, and both showed higher amounts of activity toward medium- and long-chain fatty acid esters (C12– C18) (Lee et al., 2002). In addition, the favorable substrates of CRL1 and CRL3 were medium-chain fatty acid esters, C8–C12 and C8–C14, respectively (Chang et al., 2006a,b). The fatty acid composition of Jatropha oil is 14.6% palmitic acid (16:0), 6.9% stearic acid (18:0), 46.2% oleic acid (18:1), and 30.8% linoleic acid (18:2) (Kawakami et al., 2011), which indicates that long-chain fatty acids are predominant in the Jatropha oil and may account for the preferable conversion of this oil by CRL2 and CRL4. Previous studies have shown that commercial crude CRLs displayed no transesterification activity for Jatropha oil (Shah and Gupta, 2007; Shah et al., 2004). The CRL isozyme composition of commercial CRL samples depended on the suppliers, and varied among lots within the same supplier. It has been reported that CRL1 is the major component in most commercial preparations, and that CRL3 and CRL2 range only from approximately 0–25% (Dominguez de Maria et al., 2006). Our results confirmed that individual CRL isozymes possess distinctive substrate specificities, and that no activity on the transesterification of Jatropha oil by commercial CRL preparations may be due to the lack of CRL2 and CRL4 isozymes. Our research revealed an important new finding
Table 1 Comparison of CRL isozyme transesterification activities for three non-edible oils. Reaction conditions were 0.5 g oil, 1 eq. methanol, 30% water, 40 U of each isozyme, 250 rpm, and 37 °C for 24 h. Lipase activities of each recombinant isozyme were calculated as 2857 U/mL for CRL1, 674 U/mL for CRL2, 307 U/mL for CRL3, and 586 U/ mL for CRL4. FAME yield (%)
CRL1 CRL2 CRL3 CRL4
that instead of commercial crude CRL, the specific recombinant CRL isozymes could provide a promising solution for the biodiesel synthesis from Jatropha oil.
3.2. Optimization of reaction conditions 3.2.1. Effects of water content and enzyme dosage During the transesterification reaction, water is necessary to maintain enzyme configuration and to increase the available interfacial area between water and oil. However, excess water may dilute the amount of methanol available and reverse the esterification reaction into the hydrolysis reaction. The optimal water content depends on the lipase, feedstock oil, and enzyme-immobilized support (You et al., 2013). Therefore, appropriate water content is an essential parameter in enzymatic biodiesel synthesis. The influence of water content on the initial rates and FAME yields that were catalyzed by various CRL2 concentrations (20 U, 40 U, and 80 U per 0.5 g of oil) was investigated (Figs. 1 and 2), which showed that 20% water content was not sufficient for the reactions catalyzed by any CRL2 loads. Sufficient interfacial area was provided by 30% water content for 20 U CRL2, and further increases in water content did not increase or decrease the initial rate due to the dilution of enzymes. A similar situation was observed in 40 U CRL2 loading with 40% water content. In this study, 80 U CRL2 loading with 50% water content achieved the highest initial rate (3.25% h1) (Fig. 1). Moreover, after a reaction lasting 24 h, the FAME yield of 20 U CRL2 loading with 30% water content was 34%, which was higher than 20 U CRL2 loading with 40% and 50% water content (Fig. 2B–D). The 24-h FAME yield of 40 U CRL2 loading with 40% water content reached 49.1%, which was higher than 40 U CRL2 loading with 50% water content (Fig. 2C and D). The 24-h FAME yield of 80 U CRL2 loading was particularly enhanced by increasing water content, and reached 62.9% when water content was 50% (Fig. 2D). These results suggested that the FAME production of each enzyme loading was strongly affected by various percentages of water content. As previous studies have shown, the biodiesel production which was catalyzed using a higher enzyme concentration benefited more from the larger interfacial area provided by the higher level of water content (Nordblad et al., 2014). Although 50% water content provided the maximum interfacial area in this study and achieved the highest 24-h FAME yield when 80 U CRL2 was used, excessive volume of the polar phase occupied part of the reactor capacity and reduced
4 -1
(30 m 0.25 mm; 0.25-lm film thickness). A sample volume of 1 lL was injected into the column using the split mode (1:100 split ratio). The type of carrier gas used was high purity nitrogen at a flow rate 1 mL/min. The oven temperature was increased by 2 °C/min from 200 °C to 220 °C, and maintained at 260 °C for 10 min. The injector and the detector temperature were set at 260 °C and 270 °C, respectively.
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1.51% ± 0.11 1.47% ± 0.04 19.08% ± 0.29 0.22% ± 0.10
Fig. 1. Effects of water content and enzyme dosage on the initial rate of FAME production. Lipase activity of the concentrated CRL2 was calculated to be 674 U/mL, and the CRL2 solution also contributed to the water content (wt% based on oil weight). Reaction conditions were 0.5 g Jatropha oil, 1 eq. methanol, 250 rpm, and 37 °C for 4 h.
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Fig. 2. Time courses of FAME yield for the effects of water content and enzyme dosage. (A–D) indicate the effects of 20%, 30%, 40%, and 50% water contents on 24-h FAME yield, respectively. Reaction conditions were 0.5 g Jatropha oil, 1 eq. methanol, and 250 rpm, and 20 U (d), 40 U (s), or 80 U (.) of CRL2 solution was loaded to start the reaction at 37 °C for 24 h.
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volumetric efficiency (i.e., the ratio of oil to polar phase) of the process. Therefore, the FAME yield should be a compromise between the water content and the enzyme loading amount. 3.2.2. Effect of temperature To examine the effect of temperature on the biodiesel production catalyzed by CRL2, the experiments in this study were performed between 10 °C and 50 °C (Fig. 3). The reaction mixture
included 0.5 g Jatropha oil, 66 mg methanol (molar ratio of oil to methanol was 1:3), 50% water, and 80 U CRL2; the reaction lasted for 24 h. FAME yield increased with the increasing temperatures from 10 °C to 37 °C and reached a maximum at 37 °C (56.9% FAME yield); however, they rapidly decreased at 50 °C. When the reaction temperature was at 10 °C, 20 °C, and 30 °C, CRL2 remained around 57.7%, 84.8% and 88.4% of the activity, respectively it had at 37 °C. In our previous study, the CRL2-catalyzed conversion of soybean oil reached a maximum at 40 °C (40.2% FAME yield) and remained above 80% of the activity it had at 40 °C when the temperature was between 10 °C and 40 °C (Chang et al., 2014). Our results implied that CRL2 can be applied in biodiesel production by using refined oil or crude non-edible oil. Notably, the catalytic efficiency on Jatropha oil was even higher than that on soybean oil. In addition, the mild and broad-range optimal production temperatures may reduce the energy cost for the industrial process, and thus reduce the production cost of biodiesel.
3.2.3. Effect of substrate molar ratio A stoichiometric amount (1 eq.) of methanol is typically required for the complete conversion of triacylglycerols into FAMEs. Usually, adding more alcohol could increase the transesterification yield. However, excessive amount of alcohol can also inhibit the enzyme activity and thus decrease biodiesel production (Noureddini et al., 2005). In this study, the optimal substrate molar ratio (oil: methanol) was investigated in a range from 1:3 to 1:6 (i.e., 1–2 eq.) (Fig. 4). The reactions containing 50% water content, 80 U CRL2 per 0.5 g of oil, and various substrate ratios were
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performed at 37 °C for 72 h. As shown in Fig. 4, the oil to methanol molar ratios of 1:3 and 1:4 resulted in higher FAME yields (93.5% for 1:3 and 88.8% for 1:4); however, methanol amounts of 1:5 or 1:6 decrease the FAME production. The methanol amounts of 1:5 and 1:6 are equivalent to 23.0% and 26.4% (w/w) in the water phase, respectively. The effect of methanol on the activity of recombinant CRL2 was investigated previously (Chang et al., 2011). After incubation at 37 °C in 0.1 M Tris/HCl buffer containing 30% methanol for 1 h, the activity of CRL2 decreased significantly. Therefore, the higher amounts of methanol in the water phase may inactivate CRL2, and thus decrease the FAME production.
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To prevent the inactivation of enzymes by methanol, previous studies have attempted to add methanol stepwise into the reaction system or to maintain the oil to methanol ratio below 1:5 (Wang et al., 2014). Based on the optimal oil to methanol molar ratio of 1:3 (1 eq. of methanol), we investigated the time courses of FAME yields for various stepwise methanol feeding profiles; 1 eq. of methanol was initially added to the reaction, and then 1 eq. or 0.5 eq. of methanol was additionally added at various reaction times (8 h, 16 h, or 24 h). As shown in Fig. 5A, an additional 1 eq. feeding of methanol at 8 h would inactivate CRL2, and thus reduce the FAME yield; additional feeding at 16 h or 24 h resulted in a similar 72-h FAME yield when compared with a reaction without additional methanol feeding. However, 91.6% of a 48-h FAME yield could be achieved for the additional 1 eq. feeding at 24 h. The amount of methanol does not exceed 1.4 eq. (i.e., 3 3 60% + 3 = 4.2 mol = 1.4 eq. for about 60% conversion at 24 h) when an additional 1 eq. feeds at 24 h, and this substrate molar ratio would not inactivate CRL2, as described in the results and discussion section. As shown in Fig. 5B, an additional 0.5 eq. feeding of methanol at 8 h, 16 h, or 24 h achieved 94.5%, 94.8%, or 95.3%, respectively, of 48-h FAME yields, which were all higher than the yield from no additional methanol feeding. These results suggested that an initial 1 eq. of methanol feeding followed by an additional 0.5 eq. feeding of methanol at 24 h was the optimal methanol feeding profile because of no inhibition of CRL2. In the previous published reports about enzymatic biodiesel production from Jatropha oil, they all used high-cost immobilized biocatalysts. In the present study, we showed that the low-cost liquid enzyme-based process can achieve a FAME yield which is comparable to those best yields of the immobilized enzyme-based studies (Shah and Gupta, 2007; Su et al., 2009; You et al., 2013).
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Reaction time (hr) Fig. 5. Time courses of FAME yield for various stepwise methanol feeding profiles. (A) 1 eq. methanol was initially added to the reaction and an additional 1 eq. methanol was added at 8 h (d), 16 h (s), or 24 h (.) during biodiesel production. (B) 1 eq. methanol was initially added to the reaction and an additional 0.5 eq. methanol was added at 8 h (d), 16 h (s), or 24 h (.) during biodiesel production. Symbol (4) indicates the reaction without additional methanol feeding.
We used the liquid recombinant CRL2 directly from fermentation medium for the transesterification of Jatropha oil without enzyme preparation process. P. pastoris is a well-established system for recombinant protein expression, which can produce a large amount of recombinant protein through high-density fermentation, and thus may be used to reduce the cost of enzymes in industrial applications. Using the P. pastoris expression system, a large amount of recombinant CRL2 and CRL4 isozymes could be obtained, implying that this enzymatic process may provide a solution for the biodiesel synthesis of Jatropha oil on an industrial scale.
3.4. Reusability of liquid lipase
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Reaction time (hr) Fig. 4. Time courses of FAME yield for the effect of substrate molar ratio. Reaction conditions were 1 g Jatropha oil, 160 U CRL2 solution, 50% water, 250 rpm, and 37 °C for 72 h.
After the feed-batch transesterification reaction occurred, three phases were formed by centrifugation in the reaction mixture. The upper layer was the FAME phase, which contained the FAME product and the remaining glycerides. The combination of the middle and lower layers is called the glycerol-water phase, which contained water, produced glycerol, used CRL2, and the residual methanol. The glycerol-water phase can be recovered for use in the subsequent batch reaction. When the glycerol-water phase was repeated, the residual methanol (approximately 0.5 eq.) was considered when adding methanol for the subsequent batch. In batch numbers 2–4, the feeding amount of the initial methanol did not exceed 0.5 eq. so that it could limit the total methanol
T.-C. Kuo et al. / Bioresource Technology 192 (2015) 54–59
not to more than 1 eq.; an additional 0.5 eq. feeding of methanol occurred at 24 h, and the reaction was performed for 48 h. The FAME yields of the first, second and third reuse cycles were 94.9%, 81.1%, and 53%, respectively. Furthermore, the liquid CRL2 retained 56% of its relative activity after 6 days and three reuse cycles, and 37.5% of its relative activity after 8 days and four reuse cycles. 4. Conclusion The recombinant CRL2 and CRL4 were the superior types of isozymes for biodiesel production from Jatropha oil. Optimal reaction parameters of the liquid CRL2-based process were determined as follows: 160 U CRL2 per gram of oil, 50 wt% water, 1 eq. of methanol, an additional 0.5 eq. of methanol feeds at 24 h, and a total reaction time of 48 h at 37 °C. The maximum 95.3% of the FAME yield could be achieved. These results implied that this enzymatic process may provide a promising solution on an industrial scale for biodiesel production from low-cost crude Jatropha oil. Acknowledgements Our gratitude is extended to the Academic Paper Editing Clinic, NTNU. References Abdulla, R., Chan, E.S., Ravindra, P., 2011. Biodiesel production from Jatropha curcas: a critical review. Crit. Rev. Biotechnol. 31, 53–64. Akoh, C.C., Lee, G.C., Shaw, J.F., 2004. Protein engineering and applications of Candida rugosa lipase isoforms. Lipids 39, 513–526. Cesarini, S., Diaz, P., Nielsen, P.M., 2013. Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock. Process Biochem. 48, 484–487. Chang, S.W., Huang, M., Hsieh, Y.H., Luo, Y.T., Wu, T.T., Tsai, C.W., Chen, C.S., Shaw, J.F., 2014. Simultaneous production of fatty acid methyl esters and diglycerides by four recombinant Candida rugosa lipase’s isozymes. Food Chem. 155, 140– 145. Chang, S.W., Lee, G.C., Shaw, J.F., 2006a. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J. Agric. Food Chem. 54, 815–822. Chang, S.W., Lee, G.C., Shaw, J.F., 2006b. Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J. Agric. Food Chem. 54, 5831–5838. Chang, S.W., Li, C.F., Lee, G.C., Yeh, T., Shaw, J.F., 2011. Engineering the expression and biochemical characteristics of recombinant Candida rugosa LIP2 lipase by removing the additional N-terminal peptide and regional codon optimization. J. Agric. Food Chem. 59, 6710–6719. Dalmau, E., Montesinos, J.L., Lotti, M., Casas, C., 2000. Effect of different carbon sources on lipase production by Candida rugosa. Enzyme Microb. Technol. 26, 657–663. de Oliveira, J.S., Leite, P.M., de Souza, L.B., Mello, V.M., Silva, E.C., Rubim, J.C., Meneghetti, S.M.P., Suarez, P.A.Z., 2009. Characteristics and composition of Jatropha gossypiifolia and Jatropha curcas L. oils and application for biodiesel production. Biomass Bioenergy 33, 449–453.
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