Solvent effect on the enzymatic production of

Journal of Cleaner Production 185 (2018) 382e388

Contents lists available at ScienceDirect

Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro

Solvent effect on the enzymatic production of biodiesel from waste animal fat Aldricho Alpha Pollardo a, b, Hong-shik Lee a, Dohoon Lee a, c, Sangyong Kim a, c, *, Jaehoon Kim b, ** a

Green Chemistry and Materials Group, Korea Institute of Industrial Technology, 89 Yangdaegiro-gil, Ipjang-myeon, Seobuk-gu, Cheonan-si, Chungcheongnam-do, 31056, South Korea SKKU Advanced Institute of Nanotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do, 16419, South Korea c Green Process and System Engineering, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, South Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 21 February 2018

Waste animal fat is a promising feedstock to replace vegetable oil in commercial biodiesel process, however the high content of free fatty acid in waste fat makes it unfeasible to be processed with commercial base-catalytic process. Enzymatic process in supercritical fluid is a promising way to convert waste fat into biodiesel since enzyme can catalyze both esterification of free fatty acid and transesterification of triglyceride while supercritical fluid overcome mass-transfer limitation. However, the glycerol by-product needs to be separated because it might reduce the enzyme activity. Organic solvent can be used to extract the glycerol from the enzyme with destructive effect to the enzyme. Thus, the destructive effect of organic solvent on the ability of modified C.antarctica lipase B to produce biodiesel from the waste fat was investigated. And the reversibility of enzyme was tested by various ways, drowning by organic solvents, and reuse after non-solvent experiment. The activity of enzyme was considerably affected by organic solvents. The solvent-drowning test showed that the yields were similar or higher that non-solvent case. This implies that the solvent itself did not cause the permanent change in enzyme structure to decrease activity. The decrease in yield was observed in the reuse test, which is regarded to be caused by the incomplete removal of products from the first run. © 2018 Elsevier Ltd. All rights reserved.

Keywords: Biodiesel C.antarctica lipase B Solvent effect Waste animal fat Polarity

1. Introduction Lipase is a type of biocatalyst which can catalyze a broad range of reaction such as transesterification and esterification. Its wide range of activity enables lipase to be potentially used as biocatalyst in biodiesel production. Waste animal fat is a potential feedstock for the biodiesel production because the value of the feedstock is increased after its conversion. While waste animal fat contains free fatty acid and triglyceride, lipase can catalyze the conversion of both types of substrates simultaneously into biodiesel. Although the acid catalyst and heterogeneous catalyst also are applicable to

* Corresponding author. Green Chemistry and Materials Group, Korea Institute of Industrial Technology, 89 Yangdaegiro-gil, Ipjang-myeon, Seobuk-gu, Cheonan-si, Chungcheongnam-do, 31056, South Korea. ** Corresponding author. SKKU Advanced Institute of Nanotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-di, 16419, South Korea E-mail address: [email protected] (S. Kim). https://doi.org/10.1016/j.jclepro.2018.02.210 0959-6526/© 2018 Elsevier Ltd. All rights reserved.

the conversion of waste animal fat (Canakci and Van Gerpen, 2001; Chen et al., 2017; Lotero et al., 2005), relatively high reaction temperature is required compared to enzymatic process. However, the reaction involving enzyme is relatively slower than the other methods. One contributing factor is the attachment of reactant(methanol) and/or byproduct(glycerol) to the enzyme active site that might result in denaturation of the enzyme or inhibition of the enzyme (Watanabe et al., 2000). Enzyme is usually obtainable in aqueous form which makes its recyclability more difficult. It is also available in a high expense compared to any other type of catalysts. Immobilization of enzyme in polymer resin is utilized to increase its recyclability. However, in biodiesel production using the immobilized enzyme, relatively polar glycerol might get stuck inside the enzyme due to the binding of glycerol with the active site or entrapped inside the immobilization support of the enzyme. The use of supercritical CO2 as solvent can be a solution to prevent the inhibition of methanol (Lee et al., 2011, 2013, 2012). However, the accumulation of glycerol on the support also occurred because of the nonpolar nature of CO2, therefore the removal of

A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

glycerol by the solvent extraction is unavoidable. Organic solvent as reaction medium in the presence of enzymes are widely investigated. Some enzymes have a higher stability than the others in the presence of organic solvent such as phospholipase A1 which stability increased four times in the presence of 50% DMSO solution rather than no solvent condition (Song and Rhee, 2001). Enzymatic reaction in organic solvent also hold promising results in production of medicinal substances such as azole antifungal agent production which includes yeast-lipase-catalyzed acetylation of a symmetrical diol in acetonitrile(Zaks and Dodds, 1997).Some types of lipase can show various biodiesel yield depending on the variety of organic solvents (Lu et al., 2008) and water concentration (Lu et al., 2009) but can be enhanced in other type of organic solvents such as tert-butanol (Li et al., 2006), hexane (Nelson et al., 1996), or glycol ethers(Tang et al., 2013). A study in quantum mechanics and molecular dynamics simulation of CALB (PDB code 1LBS) in organic solvents demonstrated the interaction between molecules of CALB’s active sites and the molecules of organic solvents (Li et al., 2010). The study suggested that the polar organic solvent change the structure of the protein folding in the enzyme and it disrupts the hydrogen bonding between the active sites OG (Ser105) and NE2 (His224). The study also suggested that the nonpolar organic solvent also change the protein folding but it preserved the hydrogen bonding between both active sites that were previously mentioned. Furthermore, nonpolar organic solvents in their simulation was suggested to make the OG atom more negative which indicated the ease of attacks on the ester by CALB. Thus, it is hypothesized that both polar and nonpolar organic solvent will give reversible deactivation effect to the enzyme activity and nonpolar organic solvent might increase the activity of the enzyme. In this study, the activity of modified C.antarctica lipase B in organic solvents as well as the enzyme’s recyclability were investigated. 2. Materials and methods 2.1. Materials Refined waste animal fat (RWF) was obtained from Taegok Oils & Fats. The properties of RWF were provided in Table 1. Immobilized Candida Antarctica lipase B (CALB1422) was obtained from Korea Research Institute of Bioscience and Biotechnology. Acetonitrile was obtained from Fisher Brand, HPLC grade Class 1B. Tetrahydrofuran (
99.5%) was obtained from Junsei Chemical Co. Acetone (
99.5% purity) was obtained from Samchun Pure Chemical Co., Ltd. Analytical standard methyl nonadecanoate (
98.0% for GC), toluene (
99.9%), DMF (
99%), DMSO (
99.9%), heptane (
99%), DCM (
99.8%), and dioxane (
99.5%) were obtained from Sigma Aldrich, Co., 3050 Spruce Street, St.Louis, MO 63103 USA.

383

500%, 1000% in gram oil/mL solvent) of organic solvents (with methanol as exception) were applied to crude waste fat (CWF): methanol: enzyme mass in gram ratio of 50: 6: 5. The reactions are performed in 40  C and 200 rpm shaking speed in 20-mL vials. At the beginning of the reaction, the methanol was added to the reaction in a total stoichiometric amount needed to convert all the feedstock into biodiesel. The reaction in supercritical CO2 was studied using a high-pressure batch reactor at 15 MPa under the same temperature and reactant concentration. 2.2.2. Reversibility test of enzyme after organic solvent treatment (solvent-drowning test) The enzyme was prepared and put into nine 20-mL-vials with 0.1 g each and each vials was poured with 9 different types of solvent (including methanol and excluding DMSO and DMF) until the vial was full with the solvent. The mixture was kept for 6 h in room temperature and shaking speed of 200 rpm. After that, the solvent was taken from each vials until there were no more solvent can be taken using the pipette and the remaining enzyme were kept in the fume hood for 24 h to evaporate the remaining solvents. Dried immobilized enzymes were used in biodiesel experiment with the same feedstock: methanol: enzyme ratio as in previous experiment without addition of solvent. 2.2.3. Recyclability test of enzyme After the experiment of various organic solvent concentrations, reaction mixtures from previous experiment with 30% of solvent concentration was randomly selected from each type of solvents for enzyme recyclability test. For methanol case, the reaction mixture from non-solvent experiment was chosen. The waste enzymes were separated from oil mixture and drowned with acetone to take out the remaining oil mixture inside the support. Drowning with acetone was conducted several times until the brown color in the solution and immobilization support, which is an indicator of the remaining fat, vanished. After drowning, the acetone was separated from the waste enzyme and the remaining waste enzymes were kept inside a fume hood in order to evaporate the acetone left inside the support overnight. After that, the waste enzymes were used for biodiesel production without any solvent with the same condition as the previous standard experiment condition. 2.2.4. Analysis of biodiesel Biodiesel analysis of the reaction mixture was done by taking samples of every reaction mixture at sixth hours of reaction and weighed. After that, the samples were analyzed using Gas Chromatography AGILENT 6890N series. HP-Innowax with serial code 19091N-213 was used as column in the GC. The method used in the analysis followed the method provided by EN14103:2011 using methyl nonadecanoate as internal standard.

2.2. Methods

3. Result and discussion

2.2.1. Standard condition experiment and solvent concentration effect on CAL B lipase activity Standard non-solvent and various concentration (5%, 30%, 200%,

The effect of polar and non-polar solvents on enzyme activity were observed in production of biodiesel. For rationalization of the analysis, the relative polarity and the partition coefficient (log P) data of each solvent was described in Table 2 according to reference (Carnachan et al., 2006; Chakravorty et al., 2012; Reichardt, 2003). The FAME yields with the presence of variety of organic solvents are presented in Table 3. It was found that most non-polar solvents such as toluene, heptane, and hexane and some polar solvents such as DMF and DMSO inhibit the activity of modified CAL B. In lower concentration of organic solvent, polar solvents such as acetone, acetonitrile, and THF showed around 50e100% increase in FAME yield from the highest result. The FAME yield decreased when the concentrations were lower and higher than a certain number of

Table 1 Properties of refined animal fat used in this study. Density (at 15  C)

912.4 kg/m3

Kinematic viscosity (at40  C) Pour point Water content Acid value Iodine value

18.3 mm2/s 17.0  C 0.53 wt% 110.8mgKOH/g 107.4 g/100 g

384

A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

rate between the reactants which in turn increase the reaction rate in terms of mass transfer enough to compensate the lowering by dilution(Lee et al., 2011, 2013, 2012). While the use of supercritical CO2 can enhance the enzymatic reaction rate considerably, the accumulation of glycerol on the enzyme was observed due to low solubility on glycerol in scCO2(Ciftci and Temelli, 2013). Therefore, the use of co-solvent to extract the glycerol and, simultaneously, maintain high activity of enzyme is required. From previous research, it was found that the polar organic solvent interacted intensively with the active sites of the enzyme (Li et al., 2010). The organic solvents were also simulated with the CALB and it demonstrated the change of the enzyme structure in terms of protein folding. The change of structure itself is not clear to show the increase or decrease of the enzyme activity. Unless irreversible destructive effect occurred to the enzyme, the enzyme must show original activity after the organic solvent was removed from the enzyme. This issue can be checked by enzyme recyclability test. The recyclability tests were conducted by two ways. The first test was reuse of solvent-drowned enzyme and the second one was recycling of waste enzyme after biodiesel production with the presence of organic solvents. The results were shown in Table 4. The first test showed that the yields from all tested solvents except for DMSO and DMF were similar or higher that non-solvent case (14.2%). DMSO and DMF were excluded from the test due to difficulties in separation and obviously strong inhibition to immobilized enzyme activity. This implies that the solventdrowning did not cause the permanent change in enzyme structure to decrease activity. The increase of yields in several solvents was supposed to be caused by the swelling of support resin or the change of enzyme nature during the pretreatment. The second test showed that the yields from recycled enzymes were somewhat lower than those from fresh enzymes while the

Table 2 Partition coefficient and relative polarity to water of each organic solvent. Organic Solvent

Log Pb

Relative Polarity to Watera

Methanol Acetonitrile DMSO DMF Acetone Dichloromethane THF Dioxane Toluene Heptane Hexane

0.55 0.33 1.12 0.85 0.12 1.35 0.5c 0.17 2.62 3.77 3.23

0.762 0.460 0.444 0.386 0.355 0.309 0.207 0.164 0.099 0.012 0.009

a b c

Reichardt, 2003. Chakravorty et al., 2012. Carnachan et al., 2006.

solvent concentrations and the optimum yields were dependent on the types of solvent. High concentrations were tested to check the enhancement of mass transfer between the reactant and product with the enzyme’s active sites. It was found the more the concentration of solvent was increased, the stronger the inhibitory effect of the organic solvent to the reaction rate. This finding showed that higher concentration of solvent can reduce the interaction between the reactant and the enzyme. Supercritical carbon dioxide solvent was also used as comparison. In supercritical fluid reaction system, the yield was shown to be around 79% from feedstock of 10 g and around 100 g of scCO2 solvent and around 20% FAME from 50 g of feedstock in the same amount of solvent. The supercritical CO2 might also have the same effect as the organic solvent to lower the interaction between reaction components. However, it was known that the high density and high molecular mobility of scCO2 increase the mass transfer

Table 3 FAME yield in enzymatic biodiesel production at atmospheric condition in the presence of various solvents and various concentrations. The yield is stated as oil mixture mass in the sample that contain biodiesel divided by total mass of oil mixture fraction in the sample. Solvent Concentration (mL solvent/gram oil)

10

5

2

0.3

0.05

No solvent

FAME Yield in Solvent (%)

20.4 0 0 12.8 0 0 0 0 9 16.9 79.05

22.9 0 0 17.1 3.6 2.2 5.6 3.1 8.1 10.5 e

37.9 0 0 24.2 3.7 7.4 8.4 3.9 5.9 7.2 20.35

23.7 4.4 0 22.8 10.3 24.4 14.2 9.9 12.1 13.7 e

7.5 2.1 7.7 12.7 7.2 9.2 8 9. 10.5 10.8 e

14.2

Acetonitrile DMSO DMF Acetone DCM THF 1,4-Dioxane Toluene Heptane Hexane scCO2

Table 4 Enzyme productivity test. Solvents

Methanol Acetonitrile DMSO DMF Acetone Dichloromethane THF 1,4-Dioxane Toluene Heptane Hexane

Yield (%)

Enzyme loss (%)

Control

Pretreated Enzyme

Recycled Enzyme after Experiment

14.2 23.7 4.4 0 22.8 10.3 24.4 14.2 9.9 12.1 13.7

13.8 19.1 e e 21.6 18.7 18.4 12.8 18.7 20.6 23.3

8.8 12.7 3.8 0.9 12.2 6.3 7.9 14.0 10.0 10.3 10.9

2.0 0.7

2.4 5.1 2.1 0.9 0.8 0.7 1.1

A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

385

 $FHWRQLWULLOH

/CZKOWO(#/';KGNF 

  7+)

 $FHWRQH



+H[DQH



'LR[DQH



'LFKORURPHWKDQH 7ROXHQH

+HSWDQH

'0)



'062

 







NQI2









(A)  $FHWRQLWULLOH



(#/';KGNF 

 7+)



$FHWRQH

 +H[DQH



'LR[DQH +HSWDQH



7ROXHQH

'LFKORURPHWKDQH '0)



'062

 









4GNCVKXG2QNCTKV[VQ9CVGT (B)

Fig. 1. (A) Maximum yield vs logP and (B) Maximum yield vs Relative Polarity to Water.



386

A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

 

'LR[DQH $FHWRQLWULOH

(#/';KGNF 



$FHWRQH +H[DQH +HSWDQH 7ROXHQH

 0HWKDQRO



'&0 7+)

 

'062

 '0)

 















.QI2 (A)  

'LR[DQH $FHWRQH

(#/';KGNF 



$FHWRQLWULOH

+H[DQH +HSWDQH 7ROXHQH



0HWKDQRO



'&0 7+)

 

'062

 '0)

 









4GNCVKXG2QNCTKV[VQ9CVGT (B) Fig. 2. Recycled Enzyme Yield related to (A) logP and (B) Relative polarity.



A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

enzyme loss was lower than 5.1%. Based on the finding from the first test, that is, the solvent itself does not deactivate enzyme, the decrease of yields can be regarded to be caused by the incomplete removal of products from the first run. Although enzymes were washed after first run, certain amount of reactants and products could be left inside the immobilization support depending on the miscibility with each solvent. These remained products can result in disturbance of the mass transfer between the reactant and catalyst. The increase of yield in solvent-drowning test might give a clue about how the organic solvents interact with enzyme. Although previous study claimed that the presence of nonpolar solvent might increase the ease of CALB to attack the ester bond, the polar solvent might also increase the activity of the enzyme. Pretreatment of enzyme with polar solvent might stripped off the water molecules(Gorman and Dordick, 1992) in the enzyme and the polar solvent exchange the position of the water molecules inside the enzyme. As initially stated, the water has relative polarity of 1, making it the most polar solvent than any other solvents tested in this experiment. The water has the function to preserve the natural conformity of the enzyme (Rezaei et al., 2007). However, in modified CAL B, some of its structure was altered with additional functional groups which make its natural structure also altered. It is proposed that the presence of organic solvent altered the natural structure. Such alteration made the enzyme in more activated state after the organic solvent was removed. Further study and more evidence needs to be conducted to prove this hypothesis. The correlation between the physicochemical properties of solvents with the biodiesel yield from those solvents was described in Figs. 1 and 2. The relation between the maximum yield with log P and relative polarity as a scattered point graph and the relation of yield with relative polarity was shown in Fig. 1(A) and (B) respectively. As can be seen in Fig. 1 (A), the enzyme seems to work well in logP around 0. Fig. 1 (B) showed that there is no obvious relationship between the maximum yield and the relative polarity. Effect of solvent related to the enzyme activity after being recycled was also investigated in relation with logP and relative polarity as shown in Fig. 2. From Fig. 2 (A), it was found that most solvent that yield high biodiesel content solution tend to gather around log P of 0. From Fig. 2 (B), clear conclusion was not found from the random scattering graph. DMSO and DMF was found in other enzyme to be enhancing its activity (Song and Rhee, 2001). However, in this study, it was confirmed that DMSO and DMF can potentially damage the polymer support of the enzyme by UV Spectrophotometer measurement. Therefore, DMSO and DMF effect can be excluded from the overall activity evaluation. From Figs. 1 and 2, the logP near 0 means that the solvent has the intermediate polarity. High reactivity in the solvents with the near-zero log P is considered to be caused by their miscibility with both fat and methanol. These solvents can make the biphasic mixture of fat and methanol homogeneous to enhance the mass transfer between reactants. Consequently, the mass transfer enhancement from the bipolar property of solvent is supposed to be an important mechanism for the increase of FAME yield. Glycerol extraction from the immobilized enzyme is the main reason of the usage of organic solvents. From all types of solvent, methanol would be the most promising solvent for washing the enzymes after enzymatic biodiesel production in supercritical CO2. This is because methanol is relatively polar like the glycerol. Furthermore, according to the rules “like dissolves like”, the interaction between methanol and glycerol will be adequate for dissolution of each other. Methanol is also easily separated from the reaction mixture due to its low boiling point, easily obtainable, and no need for additional equipment in supercritical fluid reaction apparatus because it is already a requirement of biodiesel

387

production in the apparatus. The previous experiment also showed the same yield with non-solvent experiment when the enzyme was pretreated with methanol for 6 h, showing the potential of methanol to recycle the cost intensive biocatalyst. As for glycerol þ oil mixture extraction, methanol dissolving power can be predicted with the ternary diagram in reference (Zhou et al., 2006). 4. Conclusion The organic solvent was confirmed to be indestructible to the activity of the enzyme. It was also found that pre-treatment of enzyme using organic solvent might increase the yield of biodiesel to around 50% more compared to the atmospheric condition without any solvent addition. Recycling of enzyme using the organic solvent as washing agent after the reaction showed reduced biodiesel yield. This decrease might be attributed to the presence of glycerol and oil mixture in the enzyme immobilization support that was remained even after extraction. The enzyme was intentionally destroyed by using organic solvent because the previous results showed deactivation of enzyme in organic solvent. This study suggests that the organic solvents with log P value near 0 might have some contribution in enhancing the mass transfer and increasing the solubility between methanol and oil. In glycerol extraction issue, the most promising organic solvent for extraction is methanol because it can easily be found, common solvent, relatively non-destructive to enzyme activity, and no additional apparatus should be prepared. Acknowledgement This work was supported by the New & Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (No. 20153010092130). References Canakci, M., Van Gerpen, J., 2001. Biodiesel production from oils and fats with high free fatty acids. Trans. ASAE 44 (6), 1429e1436. Carnachan, R.J., Bokhari, M., Przyborski, S.A., Cameron, N.R., 2006. Tailoring the morphology of emulsion-templated porous polymers. Soft. Matter. 2 (7), 608e616. Chakravorty, D., Parameswaran, S., Dubey, V.K., Patra, S., 2012. Unraveling the rationale behind organic solvent stability of lipases. Appl. Biochem. Biotechnol. 167 (3), 439e461. Chen, S.S., Maneerung, T., Tsang, D.C.W., Ok, Y.S., Wang, C.-H., 2017. Valorization of biomass to hydroxymethylfurfural, levulinic acid, and fatty acid methyl ester by heterogeneous catalysts. Chem. Eng. J. 328, 246e273. Ciftci, O.N., Temelli, F., 2013. Continuous biocatalytic conversion of the oil of corn distiller’s dried grains with solubles to fatty acid methyl esters in supercritical carbon dioxide. Biomass Bioenergy 54, 140e146. Gorman, L.A.S., Dordick, J.S., 1992. Organic solvents strip water off enzymes. Biotechnol. Bioeng. 39 (4), 392e397. Lee, J.H., Kim, S.B., Kang, S.W., Song, Y.S., Park, C., Han, S.O., Kim, S.W., 2011. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process. Bioresour. Technol. 102 (2), 2105e2108. Lee, M., Lee, D., Cho, J., Kim, S., Park, C., 2013. Enzymatic biodiesel synthesis in semipilot continuous process in near-critical carbon dioxide. Appl. Biochem. Biotechnol. 171 (5), 1118e1127. Lee, M., Lee, D., Cho, J.K., Cho, J., Han, J., Park, C., Kim, S., 2012. Improved highpressure enzymatic biodiesel batch synthesis in near-critical carbon dioxide. Bioproc. Biosyst. Eng. 35 (1), 105e113. Li, C., Tan, T., Zhang, H., Feng, W., 2010. Analysis of the conformational stability and activity of Candida Antarctica lipase B in organic solvents insight from molecular dynamics and quantum mechanics/simulations. J. Biol. Chem. 285 (37), 28434e28441. Li, L., Du, W., Liu, D., Wang, L., Li, Z., 2006. Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium. J. Mol. Catal. B Enzym. 43 (1), 58e62. Lotero, E., Liu, Y., Lopez, D.E., Suwannakarn, K., Bruce, D.A., Goodwin, J.G., 2005. Synthesis of biodiesel via acid catalysis. Ind. Eng. Chem. Res. 44 (14), 5353e5363.

388

A.A. Pollardo et al. / Journal of Cleaner Production 185 (2018) 382e388

Lu, J., Chen, Y., Wang, F., Tan, T., 2009. Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida sp. 99e125 in organic solvent system. J. Mol. Catal. B Enzym. 56 (2), 122e125. Lu, J., Nie, K., Wang, F., Tan, T., 2008. Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect. Bioresour. Technol. 99 (14), 6070e6074. Nelson, L.A., Foglia, T.A., Marmer, W.N., 1996. Lipase-catalyzed production of biodiesel. J J. Am. Oil Chem. Soc. 73 (9), 1191e1195. Reichardt, C., 2003. Solvents and Solvent Effects in Organic Chemistry, third ed. Wiley-VCH Publishers. Rezaei, K., Jenab, E., Temelli, F., 2007. Effects of water on enzyme performance with an emphasis on the reactions in supercritical fluids. Crit. Rev. Biotechnol. 27 (4), 183e195.

Song, J.K., Rhee, J.S., 2001. Enhancement of stability and activity of phospholipase A1 in organic solvents by directed evolution. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1547 (2), 370e378. Tang, S., Jones, C.L., Zhao, H., 2013. Glymes as new solvents for lipase activation and biodiesel preparation. Bioresour. Technol. 129, 667e671. Watanabe, Y., Shimada, Y., Sugihara, A., Noda, H., Fukuda, H., Tominaga, Y., 2000. Continuous production of biodiesel fuel from vegetable oil using immobilized Candida Antarctica lipase. J. Am. Oil Chemist Soc. 77 (4), 355e360. Zaks, A., Dodds, D.R., 1997. Application of biocatalysis and biotransformations to the synthesis of pharmaceuticals. Drug Discov. Today 2 (12), 513e531. Zhou, H., Lu, H., Liang, B., 2006. Solubility of multicomponent systems in the biodiesel production by transesterification of jatropha curcas L. Oil with methanol. J. Chem. Eng. Data 51 (3), 1130e1135.