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Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production
Biochemical Engineering Journal 23 (2005) 45–51
Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production Mitsuhiro Odaa , Masaru Kaiedab , Shinji Hamab , Hideki Yamajia , Akihiko Kondoa , Eiji Izumotoc , Hideki Fukudab,∗ a
Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkoudai, Nada, Kobe 657-8501, Japan b Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkoudai, Nada, Kobe 657-8501, Japan c Process Technology and Research Group, Production Technology RD Center, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan Received 25 June 2004; accepted 5 October 2004
Abstract For biodiesel-fuel production by methanolysis of plant oils, Rhizopus oryzae cells producing a 1,3-positional specificity lipase were cultured with polyurethane foam biomass support particles (BSPs) in a 20 l air-lift bioreactor, and the cells immobilized within BSPs were used as whole-cell biocatalyst in repeated batch-cycle methanolysis reaction of soybean oil. The whole-cell biocatalyst had a higher durability in the methanolysis reaction when obtained from air-lift bioreactor cultivation than from shake-flask cultivation. Following repeated methanolysis reaction using the whole-cell biocatalyst, analysis of the reaction mixture composition indicated that monoglycerides (MGs) decreased and free fatty acids (FFAs) increased with increasing water content in the reaction mixture, and that MGs, diglycerides (DGs), and triglycerides (TGs) increased with increasing number of reaction cycles. The isomers of MGs and DGs generated during the 20th methanolysis reaction cycle consisted of 2-MGs and 1,2(2,3)-DGs, respectively. The hydrolytic activity of the whole-cell biocatalyst, on the other hand, was stable regardless of the number of reaction cycles. It was demonstrated thus that the whole cell biocatalyst promotes acyl migration of partial glycerides, and that the facilitatory effect is increased by increase in the water content of the reaction mixture but it is lost gradually with increasing number of reaction cycles. © 2004 Elsevier B.V. All rights reserved. Keywords: Biodiesel fuel; Methanolysis; Rhizopus oryzae; Lipase; Whole-cell biocatalyst; Air-lift bioreactor; Acyl migration
1. Introduction Biodiesel fuel consists of methyl ester (ME) produced by transesterification of triglycerides (TGs) with methanol (i.e. methanolysis). As a fuel with the environmental advantages of renewability, biodegradability, and non-toxicity, it has attracted considerable attention, and is in current production using plant oil in Europe and the USA and waste oil in Japan. A number of processes have been developed for biodieselfuel production involving chemical or enzyme catalysis or supercritical alcohol treatment [1–4]. Although each process ∗
Corresponding author. Tel.: +81 78 803 6192; fax: +81 78 803 6192. E-mail address: [email protected] (H. Fukuda).
1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2004.10.009
has drawbacks and advantages [1], increasing environmental concerns have led to growing interest in the use of enzyme catalysis. There are many reports on biodiesel-fuel production using enzyme catalysis by free or immobilized lipase [5–12]. Immobilized lipase in particular is suitable for continuous biodiesel-fuel production because of its ease of recovery from the reaction mixture. However, the various processes involved result in high cost and can be a barrier to widespread use of enzymatic processing. The use of lipase-producing microbial cells immobilized within porous biomass support particles (BSPs) as whole-cell biocatalyst is effective in improving cost efficiency since the immobilization can be achieved spontaneously during batch cultivation and no purification of lipase is necessary. In a previous study [13], we found that
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glutaraldehyde (GA)-treated Rhizopus oryzae cells immobilized within polyurethane foam BSPs had a high durability in comparison with non-treated cells and were suitable for repeated use. For industrial application of whole-cell biocatalyst to biodiesel-fuel production, a technique for preparing whole-cell biocatalyst in large quantity is needed. In the present study, R. oryzae was cultured in an air-lift bioreactor (20 l), one of whose advantages is the low level of shear stress during cell growth compared to the stirredtank bioreactor. Immobilized cells from shake-flask and airlift cultures were compared for their durability in repeated methanolysis reaction. By analyzing the composition of the resultant reaction mixture, the effect of the number of reaction cycles on the methanolysis activity of the immobilized cells was also investigated.
2. Materials and methods 2.1. Microorganism, culture media, and BSPs All experiments were carried out using the filamentous fungus R. oryzae IFO 4697, which has a 1,3-positional specificity lipase [14–16]. The agar slant was made from 4% potato dextrose agar and 2% agar powder. R. oryzae was grown in basal medium (polypepton 70 g, NaNO3 1.0 g, KH2 PO4 1.0 g, and MgSO4 ·7H2 O 0.5 g in 1 l of distilled water) with olive oil or glucose as carbon source. The pH of the medium was initially adjusted to 5.6 and then allowed to follow its natural course. Six millimeters cubes of reticulated polyurethane foam (Bridgestone Co. Ltd., Osaka, Japan) with a particle voidage of more than 97% and a pore size of 50 pores per linear inch were used as BSPs. 2.2. Air-lift bioreactor Fig. 1 is a diagrammatic illustration of the air-lift bioreactor (20 l) used. The bioreactor is divided into an upper part (200 mm in internal diameter and 350 mm in height) and
a lower part (150 mm in internal diameter and 415 mm in height), and has an inner draft tube of 80 mm diameter and 350 mm height. 2.3. Shake-flask and air-lift bioreactor cultivation For shake-flask cultivation, R. oryzae hyphae and spores grown for 72 h on an agar slant were aseptically inoculated into a Sakaguchi flask containing 100 ml of basal medium with 30 g/l olive oil and 150 BSPs, and cultivated for 90 h at 35 ◦ C on a reciprocal shaker (150 oscillations/min, amplitude 70 mm). For air-lift cultivation, meanwhile, the seed culture was processed for 24 h in a Sakaguchi flask containing 100 ml basal medium with 10 g/l glucose. The resultant culture medium and the R. oryzae cells were then transferred to the air-lift bioreactor containing 10 l basal medium with 30 g/l olive oil and 12,000 BSPs. The bioreactor was aerated at 2.5 vvm at 35 ◦ C. The R. oryzae cells became well immobilized within the BSPs as a natural consequence of their growth during shake-flask and air-lift cultivation. After cultivation, the BSP-immobilized cells were separated from the culture broth by filtration, washed with tap water, dried at room temperature for around 24 h, and crosslinked with GA. 2.4. GA treatment of BSP-immobilized cells The GA treatment was carried out by adding 0.1 vol.% GA solution to BSP-immobilized cells, which were separated from the culture broth, and incubating them at 25 ◦ C for 1 h [13]. In the case of BSP-immobilized cells obtained in air-lift cultivation, 10 l of the GA solution was added into the airlift bioreactor without the draft tube, which was aerated at 1.0 vvm at 25 ◦ C for 1 h. After separation of the GA-treated cells from the solution by filtration, they were washed with tap water at 4 ◦ C for a few minutes, dried for approximately 24 h at room temperature, and used as whole-cell biocatalyst. 2.5. Measurement of weight of cells immobilized within BSPs The cell concentration immobilized within one BSP was measured as follows: 10 BSPs containing immobilized cells were taken, washed with acetone for 10 min to remove adherent olive oil, and dried for 24 h at 80 ◦ C. The particles plus dried cells were then weighed and treated with an aqueous solution of sodium hypochlorite (approximately 10 vol.%) to remove the immobilized cells. The cell weight was estimated from the difference between the weights. 2.6. Methanolysis reaction
Fig. 1. Dimensions of air-lift bioreactor (mm).
Methanolysis reaction was carried out at 30 ◦ C for 72 h in a 50 ml screw-capped bottle placed inside a reciprocal shaker (150 oscillations/min, amplitude 70 mm). The reaction mix-
M. Oda et al. / Biochemical Engineering Journal 23 (2005) 45–51
ture consisted of 9.65 g of soybean oil, 0.35 g of methanol (one molar equivalent to 9.65 g of soybean oil), 0–3 ml of 0.1 M phosphate buffer (pH 6.8), and 50 BSP-immobilized cells. As at least three molar equivalents of methanol are necessary to fully convert the oil to its corresponding methyl esters, 0.35 g of methanol was successively added to the bottle at 24 and 48 h reaction time, respectively. After the reaction, the whole-cell biocatalyst was separated from the reaction mixture by filtration and added to a new reaction mixture to start the next cycle. 2.7. Gas chromatography/mass spectrometer (GC–MS) and thin-layer chromatography (TLC) analysis Samples (150 l) were taken from the reaction mixture at specified times and centrifuged at 12,000 rpm for 5 min to obtain the upper layer. For GC–MS analysis, 80 l of the upper layer and 20 l of tricaprylin were precisely measured into a 10 ml bottle, to which were added a specified amount of anhydrous sodium sulfate as dehydrating agent and 3.0 ml hexane. Tricaprylin served as the internal standard for GC–MS. A 1.0 l aliquot of the treated sample was injected into a GC–MS (GCMSQP2010; Shimadzu Co., Kyoto, Japan) with a DB-1HT capillary column (0.25 mm × 15 m; J&W Scientific, Folsom, CA, USA) for determination of ME, free fatty acid (FFA), monoglyceride (MG), diglyceride (DG), and TG content in the reaction mixture. The temperatures of the injector and ion source were set at 320 and 180 ◦ C, respectively. The column temperature was maintained at 130 ◦ C for 2.0 min, raised to 350 ◦ C at 10 ◦ C/min, then to 380 ◦ C at 7 ◦ C/min, and maintained at this temperature for 10 min. The isomers of MGs and TGs produced during methanolysis were monitored by TLC as described by Okumura et al. [14] and Ando et al. [17]. The aforementioned 10 l upper layer was mixed with 1.0 ml of hexane and spotted onto a TLC plate, which was a boric acid-impregnated silica gel plate (Silica gel 60 F254; Merck, Darmstadt, Germany). The plate was developed with chloroform/acetone (96/4, v/v), then dried thoroughly, sprayed with a mixture of sulfuric acid and methanol (1:1, w/w), and dried again. The spots were visualized by baking the plate at 180 ◦ C on a hot plate.
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After mixing 24 g of soybean oil and 1.74 g of methanol in a 50 ml screw capped bottle, methanolysis reaction using 3.84 ml of the lipase solution was carried out for 3 h to obtain abundant 2-MGs and 1,2(2,3)-DGs. The resultant mixture was washed with distilled water four times and incubated at 100 ◦ C for 10 min to eliminate and inactivate the lipase in the mixture. To 2.0 g of the resultant reaction mixture was added 0–600 l of water or enzyme solution containing 0.01 g of lipase powder, and the mixture was shaken at 30 ◦ C. After 2 and 16 h incubation, the mixtures were analyzed by TLC.
3. Results 3.1. Comparison of shake-flask and air-lift bioreactor cultivation Fig. 2 shows the methanolysis activity and growth profile of BSP-immobilized R. oryzae cells obtained during shakeflask and air-lift bioreactor cultivation. Methanolysis activity was defined as ME content after 2.5 h methanolysis reaction in the reaction mixture with 15 wt.% water content; the highest values in shake-flask and air-lift bioreactor cultivation, 17 and 24 wt.%, were achieved at 96 and 44 h, respectively. For cell immobilization, similar weights of around 7 mg/BSP were obtained at 96 and 44 h in shake-flask and air-lift bioreactor cultivation, respectively. 3.2. Durability of whole-cell biocatalyst in repeated use for methanolysis The whole-cell biocatalyts obtained in shake-flask and airlift bioreactor culture were compared for their durability in repeated use for methanolysis (Fig. 3). The former lost its methanolysis activity gradually, with ME content dropping to around 60 wt.%; with the latter, however, ME content remained at over 80 wt.% after at least five reactions. Subsequent experiments were therefore carried out using the wholecell biocatalyst from air-lift bioreactor culture.
2.8. Analysis of influence of water content and lipase on acyl migration The effect of water content and lipase on acyl migration of partial glycerides was analyzed using an R. oryzae lipase solution instead of the whole-cell biocatalyst in order to exclude the influences of the cellular constituents other than lipase. Following dissolution of 1.0 g of lipase powder from R. oryzae (F-AP15; Amano Enzyme Inc., Aichi, Japan) in 10 ml of water, the mixture was stirred for 1 h and centrifuged at 3500 rpm for 10 min. The supernatant was used as the lipase solution.
Fig. 2. Methanolysis activity (circle) and growth profile (triangle) of immobilized R. oryzae cells obtained during shake-flask (open) and air-lift bioreactor (closed) culture.
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Fig. 3. Repeated methanolysis reaction using whole-cell biocatalysts obtained in shake-flask (open triangle) and air-lift (closed triangle) culture.
3.3. Effect of water content in reaction mixture on repeated methanolysis Methanolysis reaction was repeated under conditions of varying water content (0, 5, and 15 wt.%) in the reaction mixture (Fig. 4). When no water was added (water content 0 wt.%), ME content reached 75 wt.% in the first cycle, but it dropped sharply in the second cycle to 30 wt.%. ME content at 5 and 15 wt.% water content decreased gradually with increasing the number of reaction cycles, and followed a similar pattern at 10 and 30 wt.% (data not shown). The above results indicate that the ME content profile in repeated methanolysis reaction is comparable at water concentrations of 5 wt.% and over. 3.4. Analysis of intermediate products during methanolysis reaction The reactant components (FFAs, MGs, DGs, and TGs) of methanolysis under conditions of varying water content were analyzed by GC–MS (Fig. 5a) and TLC (Fig. 5b) at the end of each reaction. Increase in water content produced higher FFA content and lower MG content. Although there were almost no MGs, DGs, or TGs present at first at any water
Fig. 5. GC–MS (a) and TLC (b) analysis of repeated methanolysis reaction mixtures with various water contents. Symbols: (closed bar) 1st cycle; (open bar) 5th cycle; (gray bar) 10th cycle; (stripe bar) 20th cycle.
content, the increase in the number of reaction cycles resulted in an increase in levels of these substances, particularly MGs (Fig. 5a). FFA content, on the other hand, was more or less steady throughout, even when methanolysis reaction was repeated 20 times. At all the water contents, the increase in the content of 2-MGs and 1,2-DGs with increasing the number of reaction cycles was marked in comparison with the content of the corresponding isomers, 1(3)-MGs and 1,3-DGs (Fig. 5b). 3.5. Effect of water content and lipase on acyl migration
Fig. 4. Effect of water content on repeated methanolysis reaction. Symbols for water content in the reaction mixture: (open circle) 0 wt.%; (open triangle) 5 wt.%; (closed square) 15 wt.%.
TLC analysis was carried out to confirm that acyl migration from the sn-2 to sn-1(3) position was promoted by R. oryzae lipase at different water contents (Fig. 6a and b). Lane 1 shows methanolysate in 3 h reaction, lane 2 heat-treated methanolysate, and lanes 3–7 and lanes 8–12 samples incubated at 30 ◦ C with various amount of water for 2 and 16 h, respectively.
M. Oda et al. / Biochemical Engineering Journal 23 (2005) 45–51
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time, while those of other components (MEs and TGs) were not affected.
4. Discussion
Fig. 6. Acyl migration of partial glycerides with (b) and without (a) R. oryzae lipase under water content conditions of 0 wt.% (lanes 3 and 8), 5 wt.% (lanes 4 and 9), 10 wt.% (lanes 5 and 10), 15 wt.% (lanes 6 and 11), and 30 wt.% (lanes 7 and 12). Lane 1: initial 3 h methanolysate of soybean oil; lane 2: heattreated sample; lanes 3–7: samples incubated for 2 h; lanes 8–12: samples incubated for 16 h.
In the absence of R. oryzae lipase, although 1(3)-MG content increased slightly with the passage of time, no difference in the progress of acyl migration was found to result from different water contents (Fig. 6a). On the other hand, when samples with various amount of water (Fig. 6b) were incubated under the same conditions as in Fig. 6a, but this time with addition of lipase, contents of 2-MGs and 1,2(2,3)-DGs decreased with increasing water content and incubation time, indicating that R. oryzae lipase promote acyl migration of partial glycerides from the sn-2 to sn-1(3) position. Contents of FFAs increased with increasing water content and incubation
In the present study, scaled-up culture to immobilize R. oryzae cells within BSPs was carried out in an air-lift bioreactor (20 l), and the resultant immobilized R. oryzae cells were used as whole-cell biocatalyst in repeated batch-cycle methanolysis reaction. Faster cell growth and higher methanolysis activity were obtained in air-lift bioreactor cultivation than in shake-flask cultivation (Fig. 1). Dissolved oxygen (DO) is known to affect cell growth and enzyme productivity, and maintenance of DO concentration above a critical DO value, which depends on the kind of microorganism and enzyme, during culture is reported to be important [18–20]. The DO value during airlift bioreactor cultivation of R. oryzae rose sharply from 0.5 to 7.3 ppm at 40 h, after which cell growth and lipase production stopped. Although DO concentration was not measured during shake-flask cultivation, DO limitation was probably the reason for the longer cultivation time and lower methanolysis activity in the shake-flask cultivation. The whole cell biocatalysts obtained in air-lift bioreactor culture had a higher durability in methanolysis reaction than those from shake-flask culture (Fig. 3). The stability of wholecell biocatalyst is generally damaged by organic solvents with low values for logarithm of partition coefficient (log P); the effect, however, depends on cell type [21]. Because various materials are present in the mixture of methanolysis reaction, they may cause impairment of whole-cell biocatalyst. Methanol is known to deactivate lipase particularly [22]. In the present study, since the only microorganism used as whole-cell biocatalyst was R. oryzae IFO 4697, the differences in the durability of the whole-cell biocatalysts used are presumed to have resulted from the diversity of their membrane lipid composition, which is closely related to cellular permeability to various materials. The composition of the cell membrane, whose permeability generally depends on its fluidity, is influenced by environmental factors, such as temperature, pressure, pH, salt concentration, and medium composition; in particular, the fatty material ingested by the cell acts directly on membrane composition. Hama et al. [23] demonstrated that the fatty acid composition of the membrane of R. oryzae cells was easily controllable by addition of various fatty acids to the culture medium. They also found that palmitic acid-enriched cells exhibited significantly greater enzymatic stability than unsaturated fatty acid-enriched cells. Because olive oil is rich in unsaturated fatty acid, the membrane composition of cells cultured with olive oil would have a high degree of unsaturation. As cultivation time becomes longer, proliferating cells take in more unsaturated fatty acid in the form of hydrolysate of olive oil, resulting in increase in the membrane fluidity. The degree of unsaturation of the membrane lipid of shake-flask cultivated cells was actually
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around twice that of air-lift cultivated cells (data not shown), and this is thought to be why whole-cell biocatalyst from the latter showed higher durability in repeated use. Consequently, air-lift bioreactor cultivation is useful to efficiently produce whole-cell biocatalysts with high durability in methanolysis reaction on a large scale. Kaieda et al. [7] demonstrated that, to catalyze methanolysis, R. oryzae lipase requires water in the reaction mixture, and that its stability in the presence of methanol is dependent on this water content. In accordance with the previous report [7,21], the durability of whole-cell biocatalyst in methanolysis reaction was increased by addition of an appropriate amount of water, but ME content decreased gradually even in the presence of water (Fig. 4). To elucidate the reason for the decrease in ME content, the composition of the reactant mixture was analyzed. FFA content was influenced only by the water content, and not by the number of cycle repetitions. When wholecell biocatalyst used ten times for methanolysis reaction was recovered by filtration, washed with water to remove accretion, and used for hydrolysis of soybean oil only, and not methanolysis, its hydrolytic activity was maintained almost intact regardless of the number of reaction cycles (data not shown). In other words, the hydrolytic activity of the wholecell biocatalyst appears to be almost completely unaffected by the number of repeated cycles of methanolysis reaction. Although MG content meanwhile increased greatly with increase in the number of cycle repetitions, it decreased with increasing water content (Fig. 5a). This tendency was found to be pronounced in the case of 2-MG isomer (Fig. 5b). The acyl group of partial glycerides transfers to achieve thermodynamic equilibrium free from lipase influence [7,24–26]. It is presumed that the spontaneous acyl migration rate is ascribable to thermal energy alone, and is not affected by the number of reaction cycles. However, aforementioned results show that the acyl migration rate from 2-MG to 1(3)-MG slowed down with increase in the number of cycle repetitions, namely acyl migration catalyzed by whole-cell biocatalyst was gradually lost. The slowdown of acyl migration rate was prominent at the lower water content. Because the activity of whole-cell biocatalyst to promote acyl migration was, unlike hydrolytic activity, specifically reduced with increase in the number of cycle repetitions, ME content decreased as a result of the marked accumulation of 2-MG in the reaction mixture. Although the acyl migration activity is gradually lost with increasing the number of methanolysis reaction cycles, the whole-cell biocatalyst obtained in air-lift bioreactor cultivation appears to have great potential for industrial biodiesel-fuel production because of its high enzymatic stability.
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Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production Mitsuhiro Odaa , Masaru Kaiedab , Shinji Hamab , Hideki Yamajia , Akihiko Kondoa , Eiji Izumotoc , Hideki Fukudab,∗ a
Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkoudai, Nada, Kobe 657-8501, Japan b Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkoudai, Nada, Kobe 657-8501, Japan c Process Technology and Research Group, Production Technology RD Center, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan Received 25 June 2004; accepted 5 October 2004
Abstract For biodiesel-fuel production by methanolysis of plant oils, Rhizopus oryzae cells producing a 1,3-positional specificity lipase were cultured with polyurethane foam biomass support particles (BSPs) in a 20 l air-lift bioreactor, and the cells immobilized within BSPs were used as whole-cell biocatalyst in repeated batch-cycle methanolysis reaction of soybean oil. The whole-cell biocatalyst had a higher durability in the methanolysis reaction when obtained from air-lift bioreactor cultivation than from shake-flask cultivation. Following repeated methanolysis reaction using the whole-cell biocatalyst, analysis of the reaction mixture composition indicated that monoglycerides (MGs) decreased and free fatty acids (FFAs) increased with increasing water content in the reaction mixture, and that MGs, diglycerides (DGs), and triglycerides (TGs) increased with increasing number of reaction cycles. The isomers of MGs and DGs generated during the 20th methanolysis reaction cycle consisted of 2-MGs and 1,2(2,3)-DGs, respectively. The hydrolytic activity of the whole-cell biocatalyst, on the other hand, was stable regardless of the number of reaction cycles. It was demonstrated thus that the whole cell biocatalyst promotes acyl migration of partial glycerides, and that the facilitatory effect is increased by increase in the water content of the reaction mixture but it is lost gradually with increasing number of reaction cycles. © 2004 Elsevier B.V. All rights reserved. Keywords: Biodiesel fuel; Methanolysis; Rhizopus oryzae; Lipase; Whole-cell biocatalyst; Air-lift bioreactor; Acyl migration
1. Introduction Biodiesel fuel consists of methyl ester (ME) produced by transesterification of triglycerides (TGs) with methanol (i.e. methanolysis). As a fuel with the environmental advantages of renewability, biodegradability, and non-toxicity, it has attracted considerable attention, and is in current production using plant oil in Europe and the USA and waste oil in Japan. A number of processes have been developed for biodieselfuel production involving chemical or enzyme catalysis or supercritical alcohol treatment [1–4]. Although each process ∗
Corresponding author. Tel.: +81 78 803 6192; fax: +81 78 803 6192. E-mail address: [email protected] (H. Fukuda).
1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2004.10.009
has drawbacks and advantages [1], increasing environmental concerns have led to growing interest in the use of enzyme catalysis. There are many reports on biodiesel-fuel production using enzyme catalysis by free or immobilized lipase [5–12]. Immobilized lipase in particular is suitable for continuous biodiesel-fuel production because of its ease of recovery from the reaction mixture. However, the various processes involved result in high cost and can be a barrier to widespread use of enzymatic processing. The use of lipase-producing microbial cells immobilized within porous biomass support particles (BSPs) as whole-cell biocatalyst is effective in improving cost efficiency since the immobilization can be achieved spontaneously during batch cultivation and no purification of lipase is necessary. In a previous study [13], we found that
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glutaraldehyde (GA)-treated Rhizopus oryzae cells immobilized within polyurethane foam BSPs had a high durability in comparison with non-treated cells and were suitable for repeated use. For industrial application of whole-cell biocatalyst to biodiesel-fuel production, a technique for preparing whole-cell biocatalyst in large quantity is needed. In the present study, R. oryzae was cultured in an air-lift bioreactor (20 l), one of whose advantages is the low level of shear stress during cell growth compared to the stirredtank bioreactor. Immobilized cells from shake-flask and airlift cultures were compared for their durability in repeated methanolysis reaction. By analyzing the composition of the resultant reaction mixture, the effect of the number of reaction cycles on the methanolysis activity of the immobilized cells was also investigated.
2. Materials and methods 2.1. Microorganism, culture media, and BSPs All experiments were carried out using the filamentous fungus R. oryzae IFO 4697, which has a 1,3-positional specificity lipase [14–16]. The agar slant was made from 4% potato dextrose agar and 2% agar powder. R. oryzae was grown in basal medium (polypepton 70 g, NaNO3 1.0 g, KH2 PO4 1.0 g, and MgSO4 ·7H2 O 0.5 g in 1 l of distilled water) with olive oil or glucose as carbon source. The pH of the medium was initially adjusted to 5.6 and then allowed to follow its natural course. Six millimeters cubes of reticulated polyurethane foam (Bridgestone Co. Ltd., Osaka, Japan) with a particle voidage of more than 97% and a pore size of 50 pores per linear inch were used as BSPs. 2.2. Air-lift bioreactor Fig. 1 is a diagrammatic illustration of the air-lift bioreactor (20 l) used. The bioreactor is divided into an upper part (200 mm in internal diameter and 350 mm in height) and
a lower part (150 mm in internal diameter and 415 mm in height), and has an inner draft tube of 80 mm diameter and 350 mm height. 2.3. Shake-flask and air-lift bioreactor cultivation For shake-flask cultivation, R. oryzae hyphae and spores grown for 72 h on an agar slant were aseptically inoculated into a Sakaguchi flask containing 100 ml of basal medium with 30 g/l olive oil and 150 BSPs, and cultivated for 90 h at 35 ◦ C on a reciprocal shaker (150 oscillations/min, amplitude 70 mm). For air-lift cultivation, meanwhile, the seed culture was processed for 24 h in a Sakaguchi flask containing 100 ml basal medium with 10 g/l glucose. The resultant culture medium and the R. oryzae cells were then transferred to the air-lift bioreactor containing 10 l basal medium with 30 g/l olive oil and 12,000 BSPs. The bioreactor was aerated at 2.5 vvm at 35 ◦ C. The R. oryzae cells became well immobilized within the BSPs as a natural consequence of their growth during shake-flask and air-lift cultivation. After cultivation, the BSP-immobilized cells were separated from the culture broth by filtration, washed with tap water, dried at room temperature for around 24 h, and crosslinked with GA. 2.4. GA treatment of BSP-immobilized cells The GA treatment was carried out by adding 0.1 vol.% GA solution to BSP-immobilized cells, which were separated from the culture broth, and incubating them at 25 ◦ C for 1 h [13]. In the case of BSP-immobilized cells obtained in air-lift cultivation, 10 l of the GA solution was added into the airlift bioreactor without the draft tube, which was aerated at 1.0 vvm at 25 ◦ C for 1 h. After separation of the GA-treated cells from the solution by filtration, they were washed with tap water at 4 ◦ C for a few minutes, dried for approximately 24 h at room temperature, and used as whole-cell biocatalyst. 2.5. Measurement of weight of cells immobilized within BSPs The cell concentration immobilized within one BSP was measured as follows: 10 BSPs containing immobilized cells were taken, washed with acetone for 10 min to remove adherent olive oil, and dried for 24 h at 80 ◦ C. The particles plus dried cells were then weighed and treated with an aqueous solution of sodium hypochlorite (approximately 10 vol.%) to remove the immobilized cells. The cell weight was estimated from the difference between the weights. 2.6. Methanolysis reaction
Fig. 1. Dimensions of air-lift bioreactor (mm).
Methanolysis reaction was carried out at 30 ◦ C for 72 h in a 50 ml screw-capped bottle placed inside a reciprocal shaker (150 oscillations/min, amplitude 70 mm). The reaction mix-
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ture consisted of 9.65 g of soybean oil, 0.35 g of methanol (one molar equivalent to 9.65 g of soybean oil), 0–3 ml of 0.1 M phosphate buffer (pH 6.8), and 50 BSP-immobilized cells. As at least three molar equivalents of methanol are necessary to fully convert the oil to its corresponding methyl esters, 0.35 g of methanol was successively added to the bottle at 24 and 48 h reaction time, respectively. After the reaction, the whole-cell biocatalyst was separated from the reaction mixture by filtration and added to a new reaction mixture to start the next cycle. 2.7. Gas chromatography/mass spectrometer (GC–MS) and thin-layer chromatography (TLC) analysis Samples (150 l) were taken from the reaction mixture at specified times and centrifuged at 12,000 rpm for 5 min to obtain the upper layer. For GC–MS analysis, 80 l of the upper layer and 20 l of tricaprylin were precisely measured into a 10 ml bottle, to which were added a specified amount of anhydrous sodium sulfate as dehydrating agent and 3.0 ml hexane. Tricaprylin served as the internal standard for GC–MS. A 1.0 l aliquot of the treated sample was injected into a GC–MS (GCMSQP2010; Shimadzu Co., Kyoto, Japan) with a DB-1HT capillary column (0.25 mm × 15 m; J&W Scientific, Folsom, CA, USA) for determination of ME, free fatty acid (FFA), monoglyceride (MG), diglyceride (DG), and TG content in the reaction mixture. The temperatures of the injector and ion source were set at 320 and 180 ◦ C, respectively. The column temperature was maintained at 130 ◦ C for 2.0 min, raised to 350 ◦ C at 10 ◦ C/min, then to 380 ◦ C at 7 ◦ C/min, and maintained at this temperature for 10 min. The isomers of MGs and TGs produced during methanolysis were monitored by TLC as described by Okumura et al. [14] and Ando et al. [17]. The aforementioned 10 l upper layer was mixed with 1.0 ml of hexane and spotted onto a TLC plate, which was a boric acid-impregnated silica gel plate (Silica gel 60 F254; Merck, Darmstadt, Germany). The plate was developed with chloroform/acetone (96/4, v/v), then dried thoroughly, sprayed with a mixture of sulfuric acid and methanol (1:1, w/w), and dried again. The spots were visualized by baking the plate at 180 ◦ C on a hot plate.
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After mixing 24 g of soybean oil and 1.74 g of methanol in a 50 ml screw capped bottle, methanolysis reaction using 3.84 ml of the lipase solution was carried out for 3 h to obtain abundant 2-MGs and 1,2(2,3)-DGs. The resultant mixture was washed with distilled water four times and incubated at 100 ◦ C for 10 min to eliminate and inactivate the lipase in the mixture. To 2.0 g of the resultant reaction mixture was added 0–600 l of water or enzyme solution containing 0.01 g of lipase powder, and the mixture was shaken at 30 ◦ C. After 2 and 16 h incubation, the mixtures were analyzed by TLC.
3. Results 3.1. Comparison of shake-flask and air-lift bioreactor cultivation Fig. 2 shows the methanolysis activity and growth profile of BSP-immobilized R. oryzae cells obtained during shakeflask and air-lift bioreactor cultivation. Methanolysis activity was defined as ME content after 2.5 h methanolysis reaction in the reaction mixture with 15 wt.% water content; the highest values in shake-flask and air-lift bioreactor cultivation, 17 and 24 wt.%, were achieved at 96 and 44 h, respectively. For cell immobilization, similar weights of around 7 mg/BSP were obtained at 96 and 44 h in shake-flask and air-lift bioreactor cultivation, respectively. 3.2. Durability of whole-cell biocatalyst in repeated use for methanolysis The whole-cell biocatalyts obtained in shake-flask and airlift bioreactor culture were compared for their durability in repeated use for methanolysis (Fig. 3). The former lost its methanolysis activity gradually, with ME content dropping to around 60 wt.%; with the latter, however, ME content remained at over 80 wt.% after at least five reactions. Subsequent experiments were therefore carried out using the wholecell biocatalyst from air-lift bioreactor culture.
2.8. Analysis of influence of water content and lipase on acyl migration The effect of water content and lipase on acyl migration of partial glycerides was analyzed using an R. oryzae lipase solution instead of the whole-cell biocatalyst in order to exclude the influences of the cellular constituents other than lipase. Following dissolution of 1.0 g of lipase powder from R. oryzae (F-AP15; Amano Enzyme Inc., Aichi, Japan) in 10 ml of water, the mixture was stirred for 1 h and centrifuged at 3500 rpm for 10 min. The supernatant was used as the lipase solution.
Fig. 2. Methanolysis activity (circle) and growth profile (triangle) of immobilized R. oryzae cells obtained during shake-flask (open) and air-lift bioreactor (closed) culture.
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Fig. 3. Repeated methanolysis reaction using whole-cell biocatalysts obtained in shake-flask (open triangle) and air-lift (closed triangle) culture.
3.3. Effect of water content in reaction mixture on repeated methanolysis Methanolysis reaction was repeated under conditions of varying water content (0, 5, and 15 wt.%) in the reaction mixture (Fig. 4). When no water was added (water content 0 wt.%), ME content reached 75 wt.% in the first cycle, but it dropped sharply in the second cycle to 30 wt.%. ME content at 5 and 15 wt.% water content decreased gradually with increasing the number of reaction cycles, and followed a similar pattern at 10 and 30 wt.% (data not shown). The above results indicate that the ME content profile in repeated methanolysis reaction is comparable at water concentrations of 5 wt.% and over. 3.4. Analysis of intermediate products during methanolysis reaction The reactant components (FFAs, MGs, DGs, and TGs) of methanolysis under conditions of varying water content were analyzed by GC–MS (Fig. 5a) and TLC (Fig. 5b) at the end of each reaction. Increase in water content produced higher FFA content and lower MG content. Although there were almost no MGs, DGs, or TGs present at first at any water
Fig. 5. GC–MS (a) and TLC (b) analysis of repeated methanolysis reaction mixtures with various water contents. Symbols: (closed bar) 1st cycle; (open bar) 5th cycle; (gray bar) 10th cycle; (stripe bar) 20th cycle.
content, the increase in the number of reaction cycles resulted in an increase in levels of these substances, particularly MGs (Fig. 5a). FFA content, on the other hand, was more or less steady throughout, even when methanolysis reaction was repeated 20 times. At all the water contents, the increase in the content of 2-MGs and 1,2-DGs with increasing the number of reaction cycles was marked in comparison with the content of the corresponding isomers, 1(3)-MGs and 1,3-DGs (Fig. 5b). 3.5. Effect of water content and lipase on acyl migration
Fig. 4. Effect of water content on repeated methanolysis reaction. Symbols for water content in the reaction mixture: (open circle) 0 wt.%; (open triangle) 5 wt.%; (closed square) 15 wt.%.
TLC analysis was carried out to confirm that acyl migration from the sn-2 to sn-1(3) position was promoted by R. oryzae lipase at different water contents (Fig. 6a and b). Lane 1 shows methanolysate in 3 h reaction, lane 2 heat-treated methanolysate, and lanes 3–7 and lanes 8–12 samples incubated at 30 ◦ C with various amount of water for 2 and 16 h, respectively.
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time, while those of other components (MEs and TGs) were not affected.
4. Discussion
Fig. 6. Acyl migration of partial glycerides with (b) and without (a) R. oryzae lipase under water content conditions of 0 wt.% (lanes 3 and 8), 5 wt.% (lanes 4 and 9), 10 wt.% (lanes 5 and 10), 15 wt.% (lanes 6 and 11), and 30 wt.% (lanes 7 and 12). Lane 1: initial 3 h methanolysate of soybean oil; lane 2: heattreated sample; lanes 3–7: samples incubated for 2 h; lanes 8–12: samples incubated for 16 h.
In the absence of R. oryzae lipase, although 1(3)-MG content increased slightly with the passage of time, no difference in the progress of acyl migration was found to result from different water contents (Fig. 6a). On the other hand, when samples with various amount of water (Fig. 6b) were incubated under the same conditions as in Fig. 6a, but this time with addition of lipase, contents of 2-MGs and 1,2(2,3)-DGs decreased with increasing water content and incubation time, indicating that R. oryzae lipase promote acyl migration of partial glycerides from the sn-2 to sn-1(3) position. Contents of FFAs increased with increasing water content and incubation
In the present study, scaled-up culture to immobilize R. oryzae cells within BSPs was carried out in an air-lift bioreactor (20 l), and the resultant immobilized R. oryzae cells were used as whole-cell biocatalyst in repeated batch-cycle methanolysis reaction. Faster cell growth and higher methanolysis activity were obtained in air-lift bioreactor cultivation than in shake-flask cultivation (Fig. 1). Dissolved oxygen (DO) is known to affect cell growth and enzyme productivity, and maintenance of DO concentration above a critical DO value, which depends on the kind of microorganism and enzyme, during culture is reported to be important [18–20]. The DO value during airlift bioreactor cultivation of R. oryzae rose sharply from 0.5 to 7.3 ppm at 40 h, after which cell growth and lipase production stopped. Although DO concentration was not measured during shake-flask cultivation, DO limitation was probably the reason for the longer cultivation time and lower methanolysis activity in the shake-flask cultivation. The whole cell biocatalysts obtained in air-lift bioreactor culture had a higher durability in methanolysis reaction than those from shake-flask culture (Fig. 3). The stability of wholecell biocatalyst is generally damaged by organic solvents with low values for logarithm of partition coefficient (log P); the effect, however, depends on cell type [21]. Because various materials are present in the mixture of methanolysis reaction, they may cause impairment of whole-cell biocatalyst. Methanol is known to deactivate lipase particularly [22]. In the present study, since the only microorganism used as whole-cell biocatalyst was R. oryzae IFO 4697, the differences in the durability of the whole-cell biocatalysts used are presumed to have resulted from the diversity of their membrane lipid composition, which is closely related to cellular permeability to various materials. The composition of the cell membrane, whose permeability generally depends on its fluidity, is influenced by environmental factors, such as temperature, pressure, pH, salt concentration, and medium composition; in particular, the fatty material ingested by the cell acts directly on membrane composition. Hama et al. [23] demonstrated that the fatty acid composition of the membrane of R. oryzae cells was easily controllable by addition of various fatty acids to the culture medium. They also found that palmitic acid-enriched cells exhibited significantly greater enzymatic stability than unsaturated fatty acid-enriched cells. Because olive oil is rich in unsaturated fatty acid, the membrane composition of cells cultured with olive oil would have a high degree of unsaturation. As cultivation time becomes longer, proliferating cells take in more unsaturated fatty acid in the form of hydrolysate of olive oil, resulting in increase in the membrane fluidity. The degree of unsaturation of the membrane lipid of shake-flask cultivated cells was actually
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around twice that of air-lift cultivated cells (data not shown), and this is thought to be why whole-cell biocatalyst from the latter showed higher durability in repeated use. Consequently, air-lift bioreactor cultivation is useful to efficiently produce whole-cell biocatalysts with high durability in methanolysis reaction on a large scale. Kaieda et al. [7] demonstrated that, to catalyze methanolysis, R. oryzae lipase requires water in the reaction mixture, and that its stability in the presence of methanol is dependent on this water content. In accordance with the previous report [7,21], the durability of whole-cell biocatalyst in methanolysis reaction was increased by addition of an appropriate amount of water, but ME content decreased gradually even in the presence of water (Fig. 4). To elucidate the reason for the decrease in ME content, the composition of the reactant mixture was analyzed. FFA content was influenced only by the water content, and not by the number of cycle repetitions. When wholecell biocatalyst used ten times for methanolysis reaction was recovered by filtration, washed with water to remove accretion, and used for hydrolysis of soybean oil only, and not methanolysis, its hydrolytic activity was maintained almost intact regardless of the number of reaction cycles (data not shown). In other words, the hydrolytic activity of the wholecell biocatalyst appears to be almost completely unaffected by the number of repeated cycles of methanolysis reaction. Although MG content meanwhile increased greatly with increase in the number of cycle repetitions, it decreased with increasing water content (Fig. 5a). This tendency was found to be pronounced in the case of 2-MG isomer (Fig. 5b). The acyl group of partial glycerides transfers to achieve thermodynamic equilibrium free from lipase influence [7,24–26]. It is presumed that the spontaneous acyl migration rate is ascribable to thermal energy alone, and is not affected by the number of reaction cycles. However, aforementioned results show that the acyl migration rate from 2-MG to 1(3)-MG slowed down with increase in the number of cycle repetitions, namely acyl migration catalyzed by whole-cell biocatalyst was gradually lost. The slowdown of acyl migration rate was prominent at the lower water content. Because the activity of whole-cell biocatalyst to promote acyl migration was, unlike hydrolytic activity, specifically reduced with increase in the number of cycle repetitions, ME content decreased as a result of the marked accumulation of 2-MG in the reaction mixture. Although the acyl migration activity is gradually lost with increasing the number of methanolysis reaction cycles, the whole-cell biocatalyst obtained in air-lift bioreactor cultivation appears to have great potential for industrial biodiesel-fuel production because of its high enzymatic stability.
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