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Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst
Biochemical Engineering Journal 39 (2008) 185–189
Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst Sriappareddy Tamalampudi a , Mahabubur Rahman Talukder e , Shinji Hama d , Takao Numata b , Akihiko Kondo b , Hideki Fukuda a,c,∗ a
Department of Molecular Science and Material Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan b Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan c Organization of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan d Bio-energy Corporation, 9-7-2 Minaminanamatsu, Amgasaki 660-0053, Japan e Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore Received 27 March 2007; received in revised form 17 July 2007; accepted 15 September 2007
Abstract The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80 wt.% after 60 h using ROL, whereas it is 76% after 90 h using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from oil. © 2007 Elsevier B.V. All rights reserved. Keywords: Lipase; Transesterification; Filamentous fungi; Immobilized cells
1. Introduction Biodiesel (BDF) produced by alcoholysis of vegetables oils or animal fats is viewed as promising renewable resources of fuel. The use of BDF is becoming increasingly important due to diminishing petroleum reserves and environmental regulations. BDF is expensive in comparison with petroleum-based fuel and 60–75% of the cost is associated with feedstock oil [1]. Therefore, the exploring ways to reduce the cost of BDF with respect
∗ Corresponding author at: Department of Molecular Science and Material Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan. Tel.: +81 78 803 6192; fax: +81 78 803 6192. E-mail address: [email protected] (H. Fukuda).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.09.002
to enzyme and substrate oils are of prime interest in the recent BDF research. Jatropha curcas, an agro-forestry crop is a genus comprising 70 species growing in topical and sub-tropical countries. Jatropha grows as a natural habitat across sub-Sahara Africa, India, South East Asia and China. It grows rapidly, takes approximately 2–3 years to reach maturity and generate economic yields. It has a productive lifespan in excess of 30 years. The fatty acid composition of Jatropha oil is similar to other edible oils but the presence of some anti-nutritional factors such as toxic phorbol esters renders this oil unsuitable for cooking purposes [2]. Jatropha oil is thus a promising candidate for BDF production in terms of availability and cost. BDF is industrially produced via chemical catalysis using strong bases as a catalyst. The strong base process suffers
186
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from several drawbacks such as difficulty in recovery of glycerol, removal of base catalyst from product and the treatment of alkaline wastewater. The utilization of lipases for the production of BDF has been reported as an effective means of circumventing the aforementioned problems [3,4]. The first difficulty of using lipase is that it is more expensive than the base catalyst like NaOH. Immobilized lipase is distinguished from free lipase because of its easy recovery from the reaction mixture facilitating its repeated use. Several researchers [5–7] have reported that the commercially available Novozym 435 (Candida antarctica lipase B immobilized on acrylic resin) was the most effective catalyst among tested lipases for the production of BDF. However, laborious and expensive purification processes of this lipase from culture broth are restricting its application for BDF production in industrial scale. Here, we report that whole cells of lipase producing Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) made of reticulated polyurethane foam can catalyze the alcoholysis of Jatropha oil more effectively than Novozym 435. The advantage of using whole cells of R. oryzae immobilized onto BSP over Novozym 435 is that no labor intensive and cost associated lipase purification steps prior to the immobilization is required because the whole cells of R. oryzae can spontaneously immobilized onto BSPs during cultivation. Moreover, Jatropha oil makes the biodiesel fuel production more feasible for industrial applications than the other edible vegetable oils. 2. Materials and methods 2.1. Materials Jatropha oil was obtained as a gift from Dr. Jayaveera, Jawaharlal Technological University (JNTU), Oil Technological Research Institute, Anantapur, India. The saponification value of Jatropha oil is 210. The water content in Jatropha oil is 1.5 wt.%. Candida antarctica lipase B immobilized on macro-porous acrylic resin (Novozym 435) was purchased from Sigma–Aldrich Japan K.K, Tokyo, Japan. According to the manufacturer, the enzyme belongs to the class of triacylglycerol hydrolases (EC 3.1.3.3), with a declared activity of ≥10,000 U/g (propyl laurate units per gram). All other chemicals are of analytical grade. 2.2. Microorganism and culture medium R. oryzae IFO 4697 which has 1,3-positional specificity lipase was used as the whole-cell biocatalyst. The organism was maintained on 4% potato dextrose agar (Difco, Sparks, MD, USA) slants. R. oryzae was grown in basal medium containing 1% glucose/olive oil, polypepton 70 g, NaNO3 1.0 g, KH2 PO4 1.0 g and MgSO4 ·7H2 O 0.5 g in 1 l distilled water. Reticulated polyurethane foam particles (Bridge Stone Co. Ltd., Osaka, Japan) with a particle voidage of more than 97% and a pore size of 50 pores per linear inch were used for the immobilization of R. oryzae. In all the methanolysis experiments, 0.2 g of
BSPs containing immobilized R. oryzae and Novozym 435 were used. 2.3. Air-lift bioreactor cultivation Seed culture of R. oryzae was grown in 500 ml Sakaguchi flask containing 100 ml basal medium with 1% glucose. After cultivation for 24 h, the seed culture was transferred to air-lift bioreactor containing 10 l basal medium with 30 g/l olive oil and 12,000 BSPs. The bioreactor was maintained at 2.5 vvm at 30 ◦ C. During the growth, R. oryzae cells were naturally immobilized in BSPs during the cultivation in air-lift bioreactor. After cultivation, the BSP-immobilized cells were separated from the culture medium by filtration, washed with tap water and dried at room temperature for around 24 h followed by cross linking with glutaraldehyde. 2.4. Glutaraldehyde treatment of immobilized cells To stabilize the lipase activity, separated whole-cell biocatalysts were incubated with 0.1 vol.% glutaraldehyde (GA) solution at 25 ◦ C for 1 h. BSPs were separated from the GA solution and were shaken in phosphate buffer at 4 ◦ C for few minutes, washed with tap water for 1 min followed by drying at room temperature for 1 day. 2.5. Alcoholysis Alcoholysis was carried out at 30 ◦ C in 50 ml screw-capped vessel with reciprocal shaking at 150 rpm. A typical reaction mixture consisted of Jatropha oil (5 g), alcohol–oil molar ratio (3:1) and lipases (0.2 g) for the complete conversion of triglycerides to methyl esters. Reaction was started by adding lipase into pre-incubated reaction mixture. The alkyl ester contents were analyzed by capillary gas chromatography (GC) as described below [8]. The activity of lipases is expressed as amount of methyl esters (ME) produced per hour per gram lipase. 2.6. Analysis Samples (150 l) were taken from the reaction mixture at specified time and centrifuged at 12,000 rpm for 5 min to obtain the upper layer. The upper layer (80 l) and tricaprylin (20 l) were precisely weighed into a 10 ml bottle, to which a small amount of anhydrous sodium sulfate and hexane (3 ml) were added. Tricaprylin and sodium sulfate served as the internal standard and dehydrating agent, respectively. A 1.0 l aliquot of the treated sample was injected into GC-18A gas chromatograph (Shimadzu Corp., Kyoto, Japan) connected to a DB-5 capillary column (0.25 mm × 10 m, J&W Scientific, Folsom, CA, USA). The column temperature was held at 150 ◦ C for 0.5 min, raised to 300 ◦ C at 10 ◦ C/min, and maintained at this temperature for 3 min. The temperature for injector and flame ionization detector (FID) were set at 245 and 250 ◦ C, respectively.
S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189
Fig. 1. Effect of different alcohols on lipase activities. Reaction conditions: Jatropha oil 5 g; alcohol–oil molar ratio 1:1; lipases 0.2 g; reaction temperature 30 ◦ C; reaction time 60 min.
3. Results and discussion
187
Fig. 2. Effect of methanol–oil molar ratios on lipases activities. Reaction conditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C; reaction time 60 min.
foam may adsorb more methanol than acrylic resin particles so that activity of ROL decreased more rapidly than that of Novozym 435.
3.1. Effect of alcohol type on biodiesel production 3.3. Effect of lipases loaded More commonly used alcohols for biodiesel production are methanol, ethanol, propanol and butanol. The short chain normal alcohols were employed in the transesterification reaction in order to know their effect on whole-cell R. oryzae and Novozym 435 (Fig. 1). Although, both the lipases can able to catalyze the reaction, ROL is more efficient than Novozym 435 regardless of alcohol type. Among alcohols tested, methanol is most active for production of biodiesel from Jatropha oil and the activities of lipases decrease with the increase in alcohol chain length. Due to low molecular weight and higher polarity, methanol may easily diffuse and access the lipase enzyme localized in the cell membrane of R. oryzae [9] resulting in higher reaction rate.
In order to investigate the effect of the weight of lipase enzyme on methanolysis of Jatropha oil, the amount of lipase is varied while keeping the amount of oil constant. Fig. 3 shows that the specific activity of ROL remains constant up to lipase amount equivalent to 6 wt.% of Jatropha oil, while it is only 2 wt.% in case of Novozym 435. This result suggested that the amount of Novozym 435 added was much greater than the required and external mass transfer resistance had limited the rate of transesterification reaction. 3.4. Time course methanolysis of Jatropha oil
3.2. Comparison of ROL and Novozym 435 resistance to methanol
Since the concentration of methanol more than 1/3 Mequiv. has adverse effects on Novozym 435 and ROL, the time course
For the complete conversion of palm oil to methyl esters, at least 3 Mequiv. of methanol (i.e. methanol–oil ratio 3:1) need to be added in the reaction mixture. However, maximum 1/2 Mequiv. of methanol is generally miscible with vegetable oils and the insoluble methanol in reaction mixture inactivates lipases [10,11]. The activities of ROL and Novozym 435 at different methanol–oil ratios are thus investigated to compare their tolerance to methanol (Fig. 2). Activities of lipases increase with the increase in methanol–oil ratio up to 1 corresponding to 1/3 Mequiv. of methanol. Both lipases activities are decreased after methanol–oil ratio 1 and compared with Novozym 435, ROL is more susceptible to be poisoned by methanol. The porous materials used for immobilizing ROL and Novozym 435 are polyurethane foam and acrylic resin particles, respectively. These materials could adsorb polar compounds such as methanol. When methanol–oil ratio is high, the immiscible methanol droplets attached to the materials reducing or blocking the entry of Jatropha oil (apolar) to lipases [10]. Polyurethane
Fig. 3. Effect of lipase loaded on methanolysis Jatropha oil. Reaction conditions: Jatropha oil 5 g; alcohol–oil molar ratio 1:1; reaction temperature 30 ◦ C; reaction time 60 min.
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Fig. 4. Time course methanolysis of Jatropha oil at different water content. The molar equivalent of methanol is added by three successive addition of 1/3 Mequiv. of methanol at 0, 4 and 17 h for ROL (a) and at 0, 20 and 63 h for Novozym 435 (b). Reaction conditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C. Table 1 Recyclability of the whole-cell biocatalyst (ROL) and Novozyme 435 Cycle number
1 2 3 4 5
ROL
Novozym 435
ME content (%)
Residual activity (%)
ME content (%)
Residual activity (%)
80.2 78.3 78.2 75.5 73.1
100 97.6 97.5 94.1 91.1
75.1 73.2 71.6 70.5 70.5
100 97.4 95.3 93.8 93.8
of methyl esters production was carried out by three successive additions of 1/3 Mequiv. of methanol. The progress of methanolysis reaction catalyzed either by ROL or Novozym 435 is shown in Fig. 4a and b, respectively. In case of ROL, ME content reaches 80% after 60 h at added water 5% (v/v), above which (e.g. at 10% water) methyl ester yield decreased. Since acyl migration occurs with the intracellular lipase of immobilized cell and water may improve the cell permeability, the rate of methanolysis catalyzed by ROL increases in presence of added water. However, excess water reduces methanolysis as it also acts as a competitive inhibitor for lipase-catalyzed esterification or transesterification [12]. In case of Novozym 435, the rate of methanolysis decreases with increase in water content and ME content reaches 75% after 90 h at 0% added water. The results suggest that ROL catalyzes methanolysis of Jatropha oil more efficiently than Novozym 435. In addition, ROL is active even at 5–10% water content in Jatropha oil, while Novozym 435 needs nearly anhydrous reaction medium. Shimada et al. [11] reported that water (>500 ppm) in soybean oil decreased the rate of methanolysis catalyzed by Novozym 435. It should be mentioned that crude vegetable oil usually contains 3–5% water. Therefore, the step for removing water from crude Jatropha oil can be avoided when ROL is used for production of biodiesel.
directly used for the next batch. The time of methanolysis using ROL and Novozym 435 are kept constant at 60 and 90 h, respectively for each reaction cycle. Table 1 shows that no significant decrease in BDF content is observed after five repeated cycles and both lipases exhibit more than 90% of their initial activities. It is observed that ROL is easier to separate from reaction mixture because of its larger size (4 mm × 4 mm) compared with Novozym 435 (<1 mm in diameter).
3.5. Repeated use of ROL and Novozym 435
References
In order to test the reusability of ROL and Novozym 435, both the biocatalysts were filtered from the reaction mixture and
4. Conclusions Whole-cell R. oryzae immobilized onto BSP catalyzes the methanolysis of Jatropha oil more efficiently than Novozym 435. The presence of water in Jatropha oil has significant effect on the rate of methanolysis and ROL exhibits highest activity in presence of 5% (v/v) added water. In contrast to ROL, Novozym 435 activity is inhibited by the presence of added water and it needs nearly anhydrous media for efficient catalyzing. The results obtained here suggest that whole-cell ROL immobilized onto BSP can be used as low cost biocatalyst for production of biodiesel from crude Jatropha oil. Furthermore, expensive down stream processing steps for lipase enzyme and refining methods for the Jatropha oil can be avoided.
[1] T. Krawczyk, Biodiesel-alternative fuel makes inroads but hurdles remain, Informatics 7 (1996) 801–829.
S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189 [2] S. Shah, S. Sharma, M.N. Gupta, Biodiesel preparation by lipase-catalyzed transesterification of Jatropha oil, Energy Fuel 18 (2004) 154–159. [3] H. Fukuda, A. Kondo, H. Noda, Biodiesel fuel production by transesterification of oils—a review, J. Biosci. Bioeng. 92 (2001) 405–416. [4] L.A. Nelson, T.A. Foglia, W.N. Marmer, Lipase-catalyzed production of biodiesel, J. Am. Oil Chem. Soc. 73 (1996) 1191–1195. [5] W. Du, Y. Xu, D. Liu, J. Zeng, Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors, J. Mol. Catal. B: Enzym. 30 (2004) 125–129. [6] Y. Shimada, Y. Watanabe, T. Samukawa, A. Sugihara, H. Noda, H. Fukuda, Y. Tominaga, Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase, J. Am. Oil Chem. Soc. 76 (1999) 789–793. [7] Y. Xu, W. Du, D. Liu, J. Zeng, A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium, Biotechnol. Lett. 25 (2003) 1239–1241. [8] M. Kaieda, T. Samukawa, T. Matsumoto, K. Ban, A. Kondo, Y. Shimada, H. Noda, F. Nomoto, K. Ohtsuka, E. Izumoto, H. Fukuda, Biodiesel fuel
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production from plant oil catalyzed by Rhizopus oryzae lipase in a watercontaining system without an organic solvent, J. Biosci. Bioeng. 88 (1999) 627–631. S. Hama, S. Tamalampudi, T. Fukumizu, K. Miura, H. Yamaji, A. Kondo, H. Fukuda, Lipase localization in Rhizopus oryzae cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel fuel production, J. Biosci. Bioeng. 101 (2006) 328–333. J.W. Chen, W.T. Wu, Regeneration of immobilized Candida ntarctica lipase for transesterification, J. Biosci. Bioeng. 95 (2003) 466–469. Y. Shimada, Y. Watanabe, A. Sugihara, Y. Tominaga, Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing, J. Mol. Catal. B: Enzym. 76 (2002) 133–142. R.H. Valivety, G.A. Johnston, C.J. Suckling, P.J. Halling, Solvent effects on biocatalysis in organic systems: equilibrium position and rates of lipase-catalysed esterification, Biotechnol. Bioeng. 38 (1991) 1137–1143.
Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst Sriappareddy Tamalampudi a , Mahabubur Rahman Talukder e , Shinji Hama d , Takao Numata b , Akihiko Kondo b , Hideki Fukuda a,c,∗ a
Department of Molecular Science and Material Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan b Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan c Organization of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan d Bio-energy Corporation, 9-7-2 Minaminanamatsu, Amgasaki 660-0053, Japan e Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore Received 27 March 2007; received in revised form 17 July 2007; accepted 15 September 2007
Abstract The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80 wt.% after 60 h using ROL, whereas it is 76% after 90 h using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from oil. © 2007 Elsevier B.V. All rights reserved. Keywords: Lipase; Transesterification; Filamentous fungi; Immobilized cells
1. Introduction Biodiesel (BDF) produced by alcoholysis of vegetables oils or animal fats is viewed as promising renewable resources of fuel. The use of BDF is becoming increasingly important due to diminishing petroleum reserves and environmental regulations. BDF is expensive in comparison with petroleum-based fuel and 60–75% of the cost is associated with feedstock oil [1]. Therefore, the exploring ways to reduce the cost of BDF with respect
∗ Corresponding author at: Department of Molecular Science and Material Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan. Tel.: +81 78 803 6192; fax: +81 78 803 6192. E-mail address: [email protected] (H. Fukuda).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.09.002
to enzyme and substrate oils are of prime interest in the recent BDF research. Jatropha curcas, an agro-forestry crop is a genus comprising 70 species growing in topical and sub-tropical countries. Jatropha grows as a natural habitat across sub-Sahara Africa, India, South East Asia and China. It grows rapidly, takes approximately 2–3 years to reach maturity and generate economic yields. It has a productive lifespan in excess of 30 years. The fatty acid composition of Jatropha oil is similar to other edible oils but the presence of some anti-nutritional factors such as toxic phorbol esters renders this oil unsuitable for cooking purposes [2]. Jatropha oil is thus a promising candidate for BDF production in terms of availability and cost. BDF is industrially produced via chemical catalysis using strong bases as a catalyst. The strong base process suffers
186
S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189
from several drawbacks such as difficulty in recovery of glycerol, removal of base catalyst from product and the treatment of alkaline wastewater. The utilization of lipases for the production of BDF has been reported as an effective means of circumventing the aforementioned problems [3,4]. The first difficulty of using lipase is that it is more expensive than the base catalyst like NaOH. Immobilized lipase is distinguished from free lipase because of its easy recovery from the reaction mixture facilitating its repeated use. Several researchers [5–7] have reported that the commercially available Novozym 435 (Candida antarctica lipase B immobilized on acrylic resin) was the most effective catalyst among tested lipases for the production of BDF. However, laborious and expensive purification processes of this lipase from culture broth are restricting its application for BDF production in industrial scale. Here, we report that whole cells of lipase producing Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) made of reticulated polyurethane foam can catalyze the alcoholysis of Jatropha oil more effectively than Novozym 435. The advantage of using whole cells of R. oryzae immobilized onto BSP over Novozym 435 is that no labor intensive and cost associated lipase purification steps prior to the immobilization is required because the whole cells of R. oryzae can spontaneously immobilized onto BSPs during cultivation. Moreover, Jatropha oil makes the biodiesel fuel production more feasible for industrial applications than the other edible vegetable oils. 2. Materials and methods 2.1. Materials Jatropha oil was obtained as a gift from Dr. Jayaveera, Jawaharlal Technological University (JNTU), Oil Technological Research Institute, Anantapur, India. The saponification value of Jatropha oil is 210. The water content in Jatropha oil is 1.5 wt.%. Candida antarctica lipase B immobilized on macro-porous acrylic resin (Novozym 435) was purchased from Sigma–Aldrich Japan K.K, Tokyo, Japan. According to the manufacturer, the enzyme belongs to the class of triacylglycerol hydrolases (EC 3.1.3.3), with a declared activity of ≥10,000 U/g (propyl laurate units per gram). All other chemicals are of analytical grade. 2.2. Microorganism and culture medium R. oryzae IFO 4697 which has 1,3-positional specificity lipase was used as the whole-cell biocatalyst. The organism was maintained on 4% potato dextrose agar (Difco, Sparks, MD, USA) slants. R. oryzae was grown in basal medium containing 1% glucose/olive oil, polypepton 70 g, NaNO3 1.0 g, KH2 PO4 1.0 g and MgSO4 ·7H2 O 0.5 g in 1 l distilled water. Reticulated polyurethane foam particles (Bridge Stone Co. Ltd., Osaka, Japan) with a particle voidage of more than 97% and a pore size of 50 pores per linear inch were used for the immobilization of R. oryzae. In all the methanolysis experiments, 0.2 g of
BSPs containing immobilized R. oryzae and Novozym 435 were used. 2.3. Air-lift bioreactor cultivation Seed culture of R. oryzae was grown in 500 ml Sakaguchi flask containing 100 ml basal medium with 1% glucose. After cultivation for 24 h, the seed culture was transferred to air-lift bioreactor containing 10 l basal medium with 30 g/l olive oil and 12,000 BSPs. The bioreactor was maintained at 2.5 vvm at 30 ◦ C. During the growth, R. oryzae cells were naturally immobilized in BSPs during the cultivation in air-lift bioreactor. After cultivation, the BSP-immobilized cells were separated from the culture medium by filtration, washed with tap water and dried at room temperature for around 24 h followed by cross linking with glutaraldehyde. 2.4. Glutaraldehyde treatment of immobilized cells To stabilize the lipase activity, separated whole-cell biocatalysts were incubated with 0.1 vol.% glutaraldehyde (GA) solution at 25 ◦ C for 1 h. BSPs were separated from the GA solution and were shaken in phosphate buffer at 4 ◦ C for few minutes, washed with tap water for 1 min followed by drying at room temperature for 1 day. 2.5. Alcoholysis Alcoholysis was carried out at 30 ◦ C in 50 ml screw-capped vessel with reciprocal shaking at 150 rpm. A typical reaction mixture consisted of Jatropha oil (5 g), alcohol–oil molar ratio (3:1) and lipases (0.2 g) for the complete conversion of triglycerides to methyl esters. Reaction was started by adding lipase into pre-incubated reaction mixture. The alkyl ester contents were analyzed by capillary gas chromatography (GC) as described below [8]. The activity of lipases is expressed as amount of methyl esters (ME) produced per hour per gram lipase. 2.6. Analysis Samples (150 l) were taken from the reaction mixture at specified time and centrifuged at 12,000 rpm for 5 min to obtain the upper layer. The upper layer (80 l) and tricaprylin (20 l) were precisely weighed into a 10 ml bottle, to which a small amount of anhydrous sodium sulfate and hexane (3 ml) were added. Tricaprylin and sodium sulfate served as the internal standard and dehydrating agent, respectively. A 1.0 l aliquot of the treated sample was injected into GC-18A gas chromatograph (Shimadzu Corp., Kyoto, Japan) connected to a DB-5 capillary column (0.25 mm × 10 m, J&W Scientific, Folsom, CA, USA). The column temperature was held at 150 ◦ C for 0.5 min, raised to 300 ◦ C at 10 ◦ C/min, and maintained at this temperature for 3 min. The temperature for injector and flame ionization detector (FID) were set at 245 and 250 ◦ C, respectively.
S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189
Fig. 1. Effect of different alcohols on lipase activities. Reaction conditions: Jatropha oil 5 g; alcohol–oil molar ratio 1:1; lipases 0.2 g; reaction temperature 30 ◦ C; reaction time 60 min.
3. Results and discussion
187
Fig. 2. Effect of methanol–oil molar ratios on lipases activities. Reaction conditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C; reaction time 60 min.
foam may adsorb more methanol than acrylic resin particles so that activity of ROL decreased more rapidly than that of Novozym 435.
3.1. Effect of alcohol type on biodiesel production 3.3. Effect of lipases loaded More commonly used alcohols for biodiesel production are methanol, ethanol, propanol and butanol. The short chain normal alcohols were employed in the transesterification reaction in order to know their effect on whole-cell R. oryzae and Novozym 435 (Fig. 1). Although, both the lipases can able to catalyze the reaction, ROL is more efficient than Novozym 435 regardless of alcohol type. Among alcohols tested, methanol is most active for production of biodiesel from Jatropha oil and the activities of lipases decrease with the increase in alcohol chain length. Due to low molecular weight and higher polarity, methanol may easily diffuse and access the lipase enzyme localized in the cell membrane of R. oryzae [9] resulting in higher reaction rate.
In order to investigate the effect of the weight of lipase enzyme on methanolysis of Jatropha oil, the amount of lipase is varied while keeping the amount of oil constant. Fig. 3 shows that the specific activity of ROL remains constant up to lipase amount equivalent to 6 wt.% of Jatropha oil, while it is only 2 wt.% in case of Novozym 435. This result suggested that the amount of Novozym 435 added was much greater than the required and external mass transfer resistance had limited the rate of transesterification reaction. 3.4. Time course methanolysis of Jatropha oil
3.2. Comparison of ROL and Novozym 435 resistance to methanol
Since the concentration of methanol more than 1/3 Mequiv. has adverse effects on Novozym 435 and ROL, the time course
For the complete conversion of palm oil to methyl esters, at least 3 Mequiv. of methanol (i.e. methanol–oil ratio 3:1) need to be added in the reaction mixture. However, maximum 1/2 Mequiv. of methanol is generally miscible with vegetable oils and the insoluble methanol in reaction mixture inactivates lipases [10,11]. The activities of ROL and Novozym 435 at different methanol–oil ratios are thus investigated to compare their tolerance to methanol (Fig. 2). Activities of lipases increase with the increase in methanol–oil ratio up to 1 corresponding to 1/3 Mequiv. of methanol. Both lipases activities are decreased after methanol–oil ratio 1 and compared with Novozym 435, ROL is more susceptible to be poisoned by methanol. The porous materials used for immobilizing ROL and Novozym 435 are polyurethane foam and acrylic resin particles, respectively. These materials could adsorb polar compounds such as methanol. When methanol–oil ratio is high, the immiscible methanol droplets attached to the materials reducing or blocking the entry of Jatropha oil (apolar) to lipases [10]. Polyurethane
Fig. 3. Effect of lipase loaded on methanolysis Jatropha oil. Reaction conditions: Jatropha oil 5 g; alcohol–oil molar ratio 1:1; reaction temperature 30 ◦ C; reaction time 60 min.
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Fig. 4. Time course methanolysis of Jatropha oil at different water content. The molar equivalent of methanol is added by three successive addition of 1/3 Mequiv. of methanol at 0, 4 and 17 h for ROL (a) and at 0, 20 and 63 h for Novozym 435 (b). Reaction conditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C. Table 1 Recyclability of the whole-cell biocatalyst (ROL) and Novozyme 435 Cycle number
1 2 3 4 5
ROL
Novozym 435
ME content (%)
Residual activity (%)
ME content (%)
Residual activity (%)
80.2 78.3 78.2 75.5 73.1
100 97.6 97.5 94.1 91.1
75.1 73.2 71.6 70.5 70.5
100 97.4 95.3 93.8 93.8
of methyl esters production was carried out by three successive additions of 1/3 Mequiv. of methanol. The progress of methanolysis reaction catalyzed either by ROL or Novozym 435 is shown in Fig. 4a and b, respectively. In case of ROL, ME content reaches 80% after 60 h at added water 5% (v/v), above which (e.g. at 10% water) methyl ester yield decreased. Since acyl migration occurs with the intracellular lipase of immobilized cell and water may improve the cell permeability, the rate of methanolysis catalyzed by ROL increases in presence of added water. However, excess water reduces methanolysis as it also acts as a competitive inhibitor for lipase-catalyzed esterification or transesterification [12]. In case of Novozym 435, the rate of methanolysis decreases with increase in water content and ME content reaches 75% after 90 h at 0% added water. The results suggest that ROL catalyzes methanolysis of Jatropha oil more efficiently than Novozym 435. In addition, ROL is active even at 5–10% water content in Jatropha oil, while Novozym 435 needs nearly anhydrous reaction medium. Shimada et al. [11] reported that water (>500 ppm) in soybean oil decreased the rate of methanolysis catalyzed by Novozym 435. It should be mentioned that crude vegetable oil usually contains 3–5% water. Therefore, the step for removing water from crude Jatropha oil can be avoided when ROL is used for production of biodiesel.
directly used for the next batch. The time of methanolysis using ROL and Novozym 435 are kept constant at 60 and 90 h, respectively for each reaction cycle. Table 1 shows that no significant decrease in BDF content is observed after five repeated cycles and both lipases exhibit more than 90% of their initial activities. It is observed that ROL is easier to separate from reaction mixture because of its larger size (4 mm × 4 mm) compared with Novozym 435 (<1 mm in diameter).
3.5. Repeated use of ROL and Novozym 435
References
In order to test the reusability of ROL and Novozym 435, both the biocatalysts were filtered from the reaction mixture and
4. Conclusions Whole-cell R. oryzae immobilized onto BSP catalyzes the methanolysis of Jatropha oil more efficiently than Novozym 435. The presence of water in Jatropha oil has significant effect on the rate of methanolysis and ROL exhibits highest activity in presence of 5% (v/v) added water. In contrast to ROL, Novozym 435 activity is inhibited by the presence of added water and it needs nearly anhydrous media for efficient catalyzing. The results obtained here suggest that whole-cell ROL immobilized onto BSP can be used as low cost biocatalyst for production of biodiesel from crude Jatropha oil. Furthermore, expensive down stream processing steps for lipase enzyme and refining methods for the Jatropha oil can be avoided.
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