Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B

Bioresource Technology xxx (2012) xxx–xxx

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Bioresource Technology

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Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzaewhole-cell biocatalyst highly expressing Candida antarctica lipase B

Daisuke Adachi a, Shinji Hama b, Kazunori Nakashima c,d, Takayuki Bogaki e, Chiaki Ogino a,Akihiko Kondo a,⇑a Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japanb Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japanc Department of Chemical Engineering, Tohoku University, 6-6-07 Aoba-yama, Sendai 980-8579, Japand Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japane Ozeki Co., Ltd. 4-9 Imazu Dezaike-cho, Nishinomiya-shi, Hyogo 663-8227, Japan

h i g h l i g h t s

" Enzymatic production of biodiesel from plant oil hydrolysates." The catalyst was an immobilized recombinant fungus expressing CALB (r-CALB)." Esterification using r-CALB attained a methyl ester content over 90% after 6 h." Stepwise additions of methanol and a little water were unnecessary." A methyl ester content over 90% was maintained during 20 batch cycles.

a r t i c l e i n f o

Article history:Received 20 April 2012Received in revised form 21 June 2012Accepted 26 June 2012Available online xxxx

Keywords:Biodiesel fuelBiomass support particlesEsterificationImmobilized cellsLipase

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.06.092

⇑ Corresponding author. Tel./fax: +81 78 803 6196.E-mail address: [email protected] (A. Kondo).

Please cite this article in press as: Adachi, D., ehighly expressing Candida antarctica lipase B. B

a b s t r a c t

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocata-lyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed.Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysateswere subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification,r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalentsof methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintain-ing the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALBcan be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester con-tent of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALBshows promise for biodiesel production from plant oil hydrolysates.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction energy consumption and easy product purification (Fukuda et al.,

Biodiesel fuel, fatty acid methyl esters produced by plant oilmethanolysis, is expected to serve as an alternative to fossil fuel(Clark et al., 1984). Alkaline-catalyzed methanolysis, which hasbeen widely employed, suffers from complicated downstream pro-cesses (Fukuda et al., 2001) and also requires rigorous feedstockspecifications such as low contents of water and fatty acids (Maand Hanna, 1999). Since necessities for the feedstock specificationsaccount for the high cost of biodiesel, the application of low-costfeedstock has received considerable interest in research.

The lipase-catalyzed process has the potential to solve theaforementioned drawbacks. Except for advantages such as low

ll rights reserved.

t al. Production of biodiesel froioresour. Technol. (2012), http:

2001), most lipases catalyze esterification, and convert free fattyacids present in unrefined oils into the product. Among variouslipases, lipase B from Candida antarctica (CALB) is a versatile en-zyme that has been studied in a wide range of applications, whichincludes kinetic resolution (Fransson et al., 2006; Lou and Zong,2006) and ester synthesis (McCabe and Taylor, 2002; Larios et al.,2004). The application to biodiesel production has been studiedextensively using immobilized CALB commercially available asNovozym435 (Du et al., 2004; Samukawa et al., 2000; Shimadaet al., 1999; Talukder et al., 2009). Moreover, to produce biodieselenzymatically from various feedstocks, a 2-step process was previ-ously proposed – hydrolysis of acylglycerols using Candida rugosalipase, followed by methyl esterification of the resultant hydroly-sates using CALB (Talukder et al., 2010; Watanabe et al., 2007).In this process, unrefined oils with various fatty acids and water

m plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst//dx.doi.org/10.1016/j.biortech.2012.06.092

http://dx.doi.org/10.1016/j.biortech.2012.06.092

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2 D. Adachi et al. / Bioresource Technology xxx (2012) xxx–xxx

can be used as a feedstock, and a short reaction time can be ob-tained because esterification proceeds more than 10 times fasterthan transesterification using CALB as a catalyst (Watanabe et al.,2002). The development of an efficient esterification process usingplant oil hydrolysates is, therefore, necessary to reduce the overallcost in enzymatic biodiesel production. However, the cost of lipaseproduction is a drawback to the practical application of theprocess.

To overcome the obstacles associated with complex proceduresfor lipase purification, the direct use of lipase-producing cells aswhole-cell biocatalysts was attempted. Thus far, an Aspergillus ory-zae whole-cell biocatalyst carrying lipase-encoding genes has beendeveloped (Adachi et al., 2011; Hama et al., 2008, 2009; Kaiedaet al., 2004; Takaya et al., 2011). Of various strains, a recombinantA. oryzae strain expressing Fusarium heterosporum lipase (r-FHL) isa promising candidate for enzymatic biodiesel production. In pre-vious work using the strain, transesterification of triglyceride(i.e., methanolysis) yielded a conversion of more than 90% after96 h with stepwise additions of methanol and 5% water (Hamaet al., 2008). Although the applications of CALB to whole-cell bioca-talysis in A. oryzae (Tamalampudi et al., 2007), Escherichia coli(Narita et al., 2006), and Saccharomyces cerevisiae (Tanino et al.,2007) were reported, their potential for biodiesel production re-mains unknown probably because of problems inherent to meth-anolysis such as lipase inactivation by insoluble methanol(Shimada et al., 1999). In the present study, to further enhancethe performance of whole-cell biocatalysts, CALB was expressedin A. oryzae using a plasmid containing a P-enoA142 promoter(Tsuboi et al., 2005) and the 50-untranslated region of a heat shockprotein (Koda et al., 2006) that improved the transcription andtranslation efficiencies of a heterologous gene.

The aim of this study was to develop a process for esterificationof plant oil hydrolysates using a recombinant A. oryzae highlyexpressing CALB (r-CALB). Plant oils were hydrolyzed by using C.rugosa lipase, as reported previously (Talukder et al., 2010; Watan-abe et al., 2007), and the resultant hydrolysates were subjected toesterification using r-CALB immobilized within biomass supportparticles (BSPs). In esterification, several reaction conditions,including additions of methanol and water, were investigated be-cause they are crucial for conventional methanolysis throughwhole-cell biocatalysis. Here, the advantage of the proposed pro-cess using r-CALB compared to that using r-FHL is shown. Long-term lipase stability using soybean and palm oils was also investi-gated. The proposed process using r-CALB was compared withesterification and transesterification using r-FHL, which is a prom-ising biocatalyst in related research fields (Takaya et al., 2011).

2. Methods

2.1. Strains, media and chemicals

A. oryzae NS4 (niaD�, sC�) derived from the wild-type strainRIB40, was used as a recipient for transformation. E. coli DH5áwas used for the construction and propagation of plasmids.

Reticulated polyurethane foam BSPs (Bridgestone Corp., Osaka,Japan) measuring 6 mm � 6 mm � 3 mm cuboids were used toimmobilize A. oryzae. Powder CRL was from Meito Sangyo Co.,Ltd. (Aichi, Japan). Soybean and palm oils were obtained fromWako Pure Chemical Industries (Osaka, Japan).

2.2. Construction of lipase expression vectors

The CALB gene, which encodes a lipase from Candida antarctica,was amplified from pNAN8142-PPM-CALB (Tamalampudi et al.,2007) by PCR using the primers CALB-F1 (50-TCGCAAACATGAAGC-

Please cite this article in press as: Adachi, D., et al. Production of biodiesel frohighly expressing Candida antarctica lipase B. Bioresour. Technol. (2012), http:

TACTCTCTCTGACCC-30) and CALB-R1 (50-CACATGCATGCTCAGGGGGTGACGATGCCG-30). An amplified fragment was digested withrestriction enzyme SphI and inserted into pSENSU, containingP-enoA142 (Tsuboi et al., 2005) and 50UTR of Hsp12 (Koda et al.,2006), which was digested using restriction enzymes PmlI and SphI.The resultant plasmid was designated pSENSU-CALB. The construc-tion of pSENSU-FHL for expressing F. heterosporum lipase (FHL) wasdescribed in a previous paper (Takaya et al., 2011).

2.3. Transformation of A. oryzae

Transformation of A. oryzae was carried out according to a pro-cedure developed by Gomi et al. Gomi et al. (1987). A. oryzae pro-toplasts were prepared using Yatalase (Takara, Shiga, Japan) frommycelia grown at 30 �C for 48 h in dextrin-peptone medium, whichconsisted of 2% dextrin, 1% polypeptone, 0.5% KH2PO4, and 0.05%MgSO4�7H2O. The transgene copy number of transformants wasdetermined using real-time PCR. The recombinant A. oryzae strainscarrying pSENSU-CALB and pSENSU-FHL were designated r-CALBand r-FHL, respectively.

2.4. Preparation of A. oryzae whole-cell biocatalysts

Each recombinant A. oryzae strain was grown at 30 �C for 5–6 days on a CD agar plate, and spores were harvested with 5 ml of0.01% Tween 80. The spore solution was aseptically inoculated intoa 500 ml Sakaguchi flask containing 300 BSPs in 100 ml of DP med-ium and cultivated at 30 �C on a reciprocal shaker at 150 oscillationsper min. After cultivation for 96 h, the fungal cells immobilized onthe BSPs were collected by filtration, washed with distilled water,and lyophilized for 48 h. The lipase-expressing cells thus obtainedwere used as whole-cell biocatalysts for esterification.

The amount of immobilized cells on the BSPs was calculated bymeasuring the weight of cells before and after cell removal bytreatment with 10 vol.% sodium hypochlorite solution (Oda et al.,2005). The hydrolytic activity of each whole-cell biocatalyst wasdetermined using hydrolysis of p-nitrophenyl butyrate (pNPB) asa chromogenic substrate.

2.5. Two-step methyl ester conversion of oil

Hydrolysis of oil using C. rugosa lipase (CRL) was conducted in a1 L conical flask at 30 �C for 24 h on a reciprocal shaker at 150 rpm.The reaction mixture contained 200 g of soybean oil, 100 g of dis-tilled water and 9.0 g of powder CRL. In case of palm oil, hydrolysiswas carried out at 50 �C. These hydrolysates were centrifuged at10,000g for 5 min. The supernatants were subjected to esterifica-tion using r-CALB as a catalyst in a screw-capped bottle containing8.84 g of each hydrolysate, 1.74 g of methanol in total (1.5 Mequivalent to fatty acids (FA) in hydrolysate), and 100 particles ofimmobilized r-CALB. The reaction condition was set at 30 �C for24 h either on a reciprocal shaker at 150 rpm or on a thermo blockrotator at 35 rpm.

2.6. Analytical methods

Samples were obtained from the reaction mixture of esterifica-tion at specified times and centrifuged at 12,000 rpm for 5 min.The upper oil layer was analyzed using a GC-2014 gas chromatogra-phy system (Shimadzu, Kyoto, Japan) equipped with a ZB-5HT IN-FERNO capillary column (0.25 � 15 m; Phenomenex, USA). Thetemperature conditions of the injector and detector were set at320 and 380 �C, respectively. The column temperature was set at130 �C for 2 min, increased to 350 �C at 10 �C/min, then to 370 �Cat 7 �C/min, and finally maintained at this temperature for 10 min.The contents of ME and other components {FA, mono-glyceride



D. Adachi et al. / Bioresource Technology xxx (2012) xxx–xxx 3

(MG), di-glyceride (DG), tri-glyceride (TG)} in the reaction mixturewere calculated based on the standard curves using each standardsolution.

The water content of samples was measured using a moisturemeter CA-21 (Mitsubishi Chemical Analytech Co., Ltd., Kanagawa,Japan).

2.7. Recycling of r-CALB for esterification

Repeated batch esterification was performed using r-CALB on areciprocal shaker and on a thermo block rotator (NISSIN, Tokyo, Ja-pan). The reaction mixture contained 8.84 g of hydrolysate, 1.74 gof methanol (1.5 M equivalent to FA in hydrolysate), and 100 par-ticles of r-CALB. After one cycle of esterification, immobilized cellswere recovered from the reaction mixture and directly added to afresh reaction mixture for the next cycle. Residual cell weight (%)and residual activity (%) were calculated as follows.

Residual cell weight ð%Þ ¼ Cell weight immobilized on a BSP after the 20th batch ðmgÞCell weight immobilized on a BSP before esterification ðmgÞ � 100

Residual activity ð%Þ ¼ Hydrolytic activity of the immobilized cell after the 20th batch ðU=BSPÞHydrolytic activity of the immobilized cell before esterification ðU=BSPÞ � 100

3. Results and discussion

3.1. Catalytic performance of r-CALB

In the present study, the strains r-CALB and r-FHL harboredpSENSU-CALB and pSENSU-FHL in 3 and 2 copies, respectively.The catalytic properties of the strains such as immobilized cellweight and hydrolytic activity were first investigated. As shownin Table 1, the lipase hydrolytic activity per BSP was similar be-tween the two strains.

The hydrolysate of soybean oil containing 91.9% FA, 0.691% MG,1.52% DG, 2.26% TG and 0.636% water was subjected to the nextesterification. The esterification was conducted using r-CALB as acatalyst with or without the addition of 0.50 g water (Fig. 1(a)and (b)). To evaluate the catalytic performance of r-CALB in ester-ification, r-CALB was compared with another strain, r-FHL, whichhas exhibited high transesterification activity in an aqueous sys-tem (Takaya et al., 2011) (Fig. 1(c) and (d)). Methanol was addedto adjust the amount to 0.5, 1.0, 1.5 or 2.0 M equivalents to FA inthe hydrolysate. As shown in Fig. 1, both strains showed similarperformance when 0.5 M equivalent of methanol was added tothe reaction mixture. However, by increasing the amount of meth-anol to more than 1.0 M equivalent, the difference in the ME con-tent was obviously widened. When r-CALB was used, an MEcontent of more than 90% was achieved by adding more than1.0 M equivalent of methanol, but increasing the amount of meth-anol with r-FHL resulted in a low ME content. Both r-CALB and

Table 1Catalytic performance of recombinant A. oryzae cells immobilized on BSPs.

Immobilized cellweight on a BSP(mg-dry cell/BSP)a

Hydrolytic(U/g-dry ce

r-CALB 3.12 ± 0.18 14.1 ± 0.45r-FHL 2.53 ± 0.15 16.7 ± 0.28

a Each data are expressed as mean ± standard deviation (n = 3).


r-FHL can catalyze esterification because methanol is miscible withFA; however, the reaction rate in esterification using r-FHL wasmuch lower than that using r-CALB (Fig. 1). Therefore, a highamount of residual methanol in the reaction mixture may beresponsible for the inactivation of r-FHL.

Since the stepwise addition of methanol is not suitable forindustrial biodiesel production, there is considerable advantagein esterification using r-CALB, which does not require such labori-ous handling.

Although a small amount of water is effective for transesteri-fication of triglyceride using a whole-cell biocatalyst (Hama et al.,2008, 2009), this was not the case in esterification using r-CALB(Fig. 1(b)). Lipase generally shows activity at the water/lipidinterfaces; therefore, a certain amount of water could increasethe activity. Meanwhile, the active site of CALB is a narrow fun-nel where no interfacial reaction occurs (Martinelle et al., 1995).Due to characteristics that differ from other lipases, water con-

tent might have little effect on the esterification activity of r-CALB.

In addition, the reaction rate in esterification using r-CALB wasmuch higher than that in transesterification using r-FHL. The MEcontent reached 90% after a 6-h esterification, whereas it took24 h to yield 90% in transesterification using r-FHL, as reportedpreviously (Takaya et al., 2011). In a preliminary experiment,transesterification was carried out using r-CALB under the sameconditions as in a previous paper (Hama et al., 2008). Unexpect-edly, the ME content was only 11.3% after a 96-h transesterification(data not shown). Hydrolysis of soybean oil was also performedusing r-CALB by the addition of 10 wt.% water to oil at 30 �C for24 h; however, the fatty acid content was only 2.41% (data notshown). Therefore, despite the possible versatility of CALB, theactivity of r-CALB was specialized towards esterification. A previ-ous study (Kato et al., 2007) reported on the low hydrolytic activityof CALB toward triglycerides with long chain fatty acids. Althoughtransesterification activity of CALB was extensively studied usingan immobilized lipase (Novozym435), r-CALB showed low transe-sterification activity. Since the characteristics of CALB is affected bythe surface of a lipase-immobilized carrier (Secundo and Carrea,2002), the characteristics of the cell surface might led to the activ-ity of r-CALB specialized toward esterification. Although the phe-nomenon is of interest from the viewpoint of enzymology,further study is necessary to explore the reasons. In esterificationusing r-CALB, the highest ME content, 95.5%, was obtained after a24-h esterification when 1.5 M equivalents methanol was added

activity of the cellll)a

Hydrolytic activity of lipases per BSP(U/BSP)a

0.0879 ± 0.00230.0845 ± 0.0017



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(a) (b)

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Fig. 1. Comparison of the time course of esterification of fatty acids from soybean oil using r-CALB (a and b) and r-FHL (c and d). The following equivalent of methanol wasadded to FA in the hydrolysate: 0.5 M equivalent (circle); 1 M equivalent (square); 1.5 M equivalents (diamond); or 2 M equivalents (triangle). The reactions were performedwithout the addition of water (a and c) or with the addition of 0.50 g of water externally (b and d).

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Fig. 2. Effect of methanol addition on three cycles of repeated batch esterification using r-CALB. Methanol was added stepwise at 1–4 times to adjust the total amount ofmethanol to 1.5 M equivalents to FA in the hydrolysate: (a) 0.375 M equivalent methanol was added at 0, 24, 48 and 72 h; (b) 0.5 M equivalent of methanol was added at 0, 24and 48 h; (c) 0.75 M equivalent of methanol was added at 0 and 24 h; and (d) 1.5 M equivalents of methanol was added at 0 h. The reactions were performed without theaddition of water (diamond) and with the addition of 0.50 g of water (circle).


Please cite this article in press as: Adachi, D., et al. Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalysthighly expressing Candida antarctica lipase B. Bioresour. Technol. (2012), http://dx.doi.org/10.1016/j.biortech.2012.06.092


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Rotation systemShaking system

Fig. 3. Comparison of final ME content during 20 cycles of repeated batchesterification of soybean oil hydrolysate using r-CALB on a reciprocal shaker(diamond) and a thermo block rotator (circle).


to the reaction mixture. This amount of methanol was thus em-ployed in the subsequent experiments.

3.2. Effect of the addition of methanol and water on lipase stabilityduring esterification

In a previous study of soybean oil transesterification, the step-wise addition of methanol and a small amount of water was effec-tive for maintaining the lipase stability of whole-cell biocatalysts(Hama et al., 2008, 2009). To investigate the effect of methanoland water on the lipase stability of r-CALB, three cycles of batchesterification of soybean oil hydrolysate were carried out usingr-CALB as a catalyst in a reciprocal shaker (Fig. 2(a)–(d)). During re-peated esterification, an appropriate amount of methanol wasadded 1–4 times to adjust the total amount to 1.5 M equivalentsto FA in each reaction cycle with or without 0.50 g of water. Asshown in Fig. 2(d), an ME content of more than 90% was retainedduring three batches when 1.5 M equivalent of methanol wasadded in a single step to the reaction mixture. These results suggestthat the stepwise addition of methanol and a small amount of waterare unnecessary for maintaining the lipase stability of r-CALB dur-ing esterification. Because transesterification using r-FHL requiredthese laborious operations to maintain lipase stability, esterifica-tion using r-CALB seems to be superior to transesterification using

Table 2Detailed data on the reaction product and the r-CALB in repeated esterification using each

Hydrolysate from soybean oil (shakingsystem)

Hydrolysasystem)

1 cycle 20 cycle 1 cycle

ME (wt.%)a 91.9 ± 0.95 81.8 ± 0.61 94.5 ± 0.FA (wt.%)a 0.916 ± 0.14 9.86 ± 0.79 0.746 ± 0.MG (wt.%)a 0.356 ± 0.10 0.515 ± 0.12 0.342 ± 0.DG (wt.%)a 0.698 ± 0.11 1.13 ± 0.10 0.624 ± 0.TG (wt.%)a n.d.d 2.24 ± 0.16 n.d.Water (wt.%)a 0.175 ± 0.049 0.336 ± 0.11 0.156 ± 0.Residual cell weight (%)a,b 84.4 ± 2.92 65.8 ± 4.41 90.1 ± 3.Residual activity (%)a,c 60.2 ± 4.3

a Each data are expressed as mean ± standard deviation (n = 3).

b Residual cell weight (%) =Cell weght immobilized on a BSP after the 20<ce:sup>th<=ce:sup> batch ðmgÞCell weight immobilized on a BSP before esterification ðmgÞ �

c Residual activity (%) =Hydrolytic activity of the immobilized cell after the 20<ce:sup>th<=ce:sup> batch ðU=Hydrolytic activity of the immobilized cell before esterification ðU=BSPÞ

d Not detected.


r-FHL. On the basis of these results, the stepwise additions of meth-anol and a small amount of water were eliminated in the subse-quent experiments.

3.3. Long-term esterification using r-CALB

To reduce the production cost in industrial applications,whole-cell biocatalysts should be recycled for an extended periodof time. Twenty cycles of batch esterification on a reciprocal sha-ker were thus carried out using r-CALB. As shown in Fig. 3, thefinal ME content in each cycle decreased gradually, but an MEcontent of more than 80% was maintained even after the 20thbatch cycle, suggesting the high lipase stability of r-CALB duringlong-term esterification. However, serious physical damage wasobserved on r-CALB after the 20th cycle and further recyclingseemed to be difficult. To avoid this attrition, a mild agitation iseffective but insufficient agitation could cause a decrease in thereaction rate. A technology based on free-fall mixing using a hor-izontally placed drum was thus introduced. Such a reaction sys-tem could be applied to achieve a mild and effective agitationin ethanol production for high-solid concentrations (Jørgensenet al., 2006). In a rotation system, an improvement in the ME con-tent was observed during 20 batches of esterification (Fig. 3). TheME content at the 20th batch cycle was 8.5% higher than that inthe shaking system. Given the improved performance, an attritionof the surface of r-CALB seemed to be suppressed in this rotationsystem. As shown in Table 2, the residual cell amounts immobi-lized on BSP after 20 batches of esterification were 65.8 ± 4.41%and 80.9 ± 3.46% in the shaking and rotation systems, respec-tively. Moreover, the residual lipase activities per BSP in the shak-ing and rotation systems were 60.2 ± 4.3% and 79.4 ± 3.7%,respectively. These results suggest that a suppression of thedetachment of the cells in a rotation system contributes to animprovement in the final ME content during long-term esterifica-tion. In an adsorptive immobilization of lipase, the effect of lipasedetachment from carrier on the catalytic process was discussed ina previous study (Zhang et al., 2012). Thus, a suppression of thedetachment of the cells and lipases seems to be a crucial factorfor maintaining the lipase activities of r-CALB as well as of animmobilization lipase.

In addition, the initial reaction rate and the final ME content inthe first batch were similar between shaking and rotation systems(Data not shown), which suggests that the rotation system doesnot produce a negative impact on the reaction efficiency. There-fore, the developed rotation system is suitable for esterificationusing r-CALB.

reaction system.

te from soybean oil (rotation Hydrolysate from palm oil (rotationsystem)

20 cycle 1 cycle 20 cycle

61 90.3 ± 0.95 90.5 ± 1.4 85.8 ± 2.0052 3.18 ± 0.25 2.66 ± 0.33 5.31 ± 0.9214 0.565 ± 0.033 0.515 ± 0.057 0.513 ± 0.064063 0.955 ± 0.094 1.81 ± 0.37 2.54 ± 0.43

n.d. n.d. n.d.052 0.228 ± 0.025 0.185 ± 0.024 0.229 ± 0.01621 80.9 ± 3.46 89.9 ± 4.11 78.4 ± 3.48

79.4 ± 3.7 75.8 ± 3.5

100.BSPÞ � 100.



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Fig. 4. (a) Recycling of r-CALB during 20 cycles of batch esterification of palm oilhydrolysate on a thermo block rotator. (b) Comparison of initial reaction rateprofiles during esterification using r-CALB and hydrolysates from soybean oil(circle) and palm oil (triangle).


3.4. Application of r-CALB to an esterification of hydrolysate from palmoil

Palm oil, which has a fatty acid composition that differs fromsoybean oil, is an attractive feedstock for biodiesel production be-cause it is one of the most abundant and cheapest vegetable oilsavailable. A repeated esterification using palm oil hydrolysate ina thermo block rotator was thus investigated. Palm oil was hydro-lyzed using powder CRL and the resulting hydrolysate contained88.4% FA, 0.548% MG, 3.03% DG, 2.26% TG and 0.583% water. Thehydrolysate became solidified at room temperature; therefore, itwas fused completely at 50 �C prior to esterification. As shownin Fig. 4, the final ME content in the first batch was 4% lower thanthat using the hydrolysate from soybean oil. One explanation is anFA content in the hydrolysate from palm oil that was lower thanthat from soybean oil. In addition, the palm oil hydrolysate wassolidified easily at 30 �C because it contained more palmitic acid.Thus, a decreased diffusion of the substrate could be another rea-son. However, a final ME content of more than 85% after 20batches was maintained using palm oil hydrolysate (Fig. 4(a)). Inaddition, the initial reaction rate using palm oil hydrolysate wassimilar to that using soybean oil hydrolysate (Fig. 4(b)). These re-sults show that the developed reaction system using r-CALB is alsoeffective in the esterification of hydrolysate using palm oil as afeedstock.

In esterification using r-CALB, the ME content reached 90%after 6 h under optimum conditions. In previous studies of the


esterification of plant oil hydrolysate using commercial CALB(Novozym 435), an ME content of 93% was obtained using thehydrolysate from acid oil after 10 h without an organic solvent(Watanabe et al., 2007). In another study, the ME contentreached 98% after 2 h using the hydrolysate from crude palmoil in the presence of isooctane (Talukder et al., 2010). Althoughthe variety of the reaction conditions makes a fair comparisondifficult, the reaction rate in the developed system is comparableto those in previous studies using commercial CALB. Since a lowreaction rate in enzymatic transesterification is one of the majorobstacles for industrialization, the developed reaction system isconsidered promising for biodiesel production through whole-cellbiocatalysis.

4. Conclusions

An immobilized recombinant A. oryzae carrying pSENSU-CALBwas employed for esterification of plant oil hydrolysates. The MEcontent reached 90% after a 6-h esterification without stepwiseadditions of methanol and small amount of water. In a rotator,the stability of r-CALB during long-term esterification was im-proved, resulting in a final ME content over 90% even after the20th batch. Palm oil was also successfully adapted for the system.Therefore, the presented reaction system using r-CALB shows pos-sibility for biodiesel production from plant oil hydrolysates.

Acknowledgements

This work was partially supported by Regional Innovation Cre-ation R&D Programs, the Ministry of Economy, Trade and Industry(METI) and Special Coordination Funds for Promoting Science andTechnology, Creation of Innovation Centers for Advanced Interdis-ciplinary Research Areas (Innovative Bioproduction Kobe), MEXT,Japan. The generous gift of the fungal expression vectors pSEN-SU-CALB and pSENSU-FHL from Ozeki Co., Ltd. (Hyogo, Japan) arealso gratefully acknowledged.

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Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B