Enzyme and Microbial Technology 52 (2013) 118122

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Enzyme and Microbial Technology

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An integrative process model of enzymatic biodiesel production through ethanol

fermentation of brown rice followed by lipase-catalyzed ethanolysis in a
water-containing system
Daisuke Adachi a , Risa Koda a , Shinji Hama b , Ryosuke Yamada c , Kazunori Nakashima c,d ,
Chiaki Ogino a , Akihiko Kondo a,
a

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan
Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan
c
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan
d
Department of Chemical Engineering, Tohoku University, 6-6-07 Aoba-yama, Sendai, 980-8579, Japan
b

a r t i c l e

i n f o

Article history:
Received 4 October 2012
Received in revised form
14 November 2012
Accepted 15 November 2012
Keywords:
Bioethanol
Biodiesel fuel
Biomass support particles
Ethanolysis
Immobilized cells
Lipase

a b s t r a c t
We attempted to integrate lipase-catalyzed ethanolysis into fermentative bioethanol production. To produce bioethanol, ethanol fermentation from brown rice was conducted using a tetraploid Saccharomyces
cerevisiae expressing -amylase and glucoamylase. The resultant ethanol was distilled and separated into
three fractions with different concentrations of water and fusel alcohols. In ethanolysis using the rst
fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst expressing Fusarium
heterosporum lipase (r-FHL) afforded the highest ethyl ester content of 94.0% after 96 h. Owing to a high
concentration of water in the bioethanol solutions, r-FHL, which works best in the presence of water
when processing ethanolysis, was found to be more suitable for the integrative process than a commercial immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch ethanolysis,
resulting in an ethyl ester content of more than 80% even after the fth batch. Fusel alcohols such as
1-butanol and isobutyl alcohol are thought to decrease the lipase activity of r-FHL. Using this process, a
high ethyl ester content was obtained by simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water concentration. The developed process model, therefore, would contribute to
biodiesel production from only biomass-derived feedstocks.
2012 Elsevier Inc. All rights reserved.

1. Introduction
Biodiesel fuel, fatty acid alkyl esters produced by alcoholysis
of plant oils or animal fats, is expected to serve as an alternative to
fossil fuel [1]. Although an alkaline-catalyzed method has traditionally been used for biodiesel production, increases in environmental
concerns have led to a growing interest in alternatives such as a
lipase-catalyzed method that avoids conventional difculties in the
recovery of glycerol and catalysts such as potassium and/or sodium
salt [24].
Methanol is most frequently used as an acyl accepter in plant oil
transesterication because of its low cost and high reactivity [5,6].
However, methanol has some drawbacks such as toxicity to biocatalysts and petroleum-derived alcohols. Ethanol and 1-butanol
would be an alternative to methanol for biodiesel production
because they are less toxic to biocatalysts and can be produced
through fermentation of sugars from starchy or lignocellulosic

Corresponding author. Tel.: +81 78 803 6196; fax: +81 78 803 6196.
E-mail address: [email protected] (A. Kondo).
0141-0229/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.enzmictec.2012.11.005

biomass using several microorganisms such as yeast, solventogenic clostridia and Escherichia coli [68]. Although ethanolysis and
butanolysis have been widely studied, biodiesel production using
bioethanol or biobutanol obtained by fermentation of biomass has
not been reported. Here, we report the production of biodiesel from
biomass-derived alcohol and oil. To produce a high concentration
of alcohol from biomass, we selected a starchy biomass, brown rice,
which leads to a theoretical ethanol yield during fermentation using
a tetraploid, Saccharomyces cerevisiae, that expresses -amylase
and glucoamylase (MNIV/GS) [9,10]. In the present study, a simple distillation tower was used to distill the fermentation broth.
According to the equilibrium curve of ethanol/water, an ethanol
solution containing more than 95% ethanol cannot be obtained
solely by the distillation of bioethanol because of azeotropy; therefore, another purication step that is associated with high cost is
necessary to further increase the ethanol concentration. Given the
cost increase, we attempted the direct use of a distilled ethanol
solution for enzymatic biodiesel production.
To produce biodiesel from a distilled bioethanol solution, a
recombinant A. oryzae expressing Fusarium heterosporum lipase
(r-FHL) was employed. In a previous study, r-FHL catalyzed

D. Adachi et al. / Enzyme and Microbial Technology 52 (2013) 118122

ethanolysis efciently, resulting in an ethyl ester (EE) content of

more than 90% [11]. Moreover, because r-FHL tolerates a high water
concentration in reaction media [11,12], it has promise as a biocatalyst for ethanolysis when using a bioethanol solution with a
signicant amount of water.
The aim of the present study was to develop an integrative
process model of plant oil ethanolysis catalyzed by r-FHL using
a distilled bioethanol solution from brown rice (see Graphical
Abstract). The effects of the composition of a bioethanol solution and impurities on ethanolysis were investigated for efcient
biodiesel production.
2. Materials and methods
2.1. Strains, media and chemicals
A recombinant tetraploid S. cerevisiae strain expressing -amylase and glucoamylase obtained by -integration and cell fusion (MNIV/GS) [10] was used for
ethanol fermentation. The yeast was aerobically cultivated in SD medium (20 g/L
d-glucose and 6.7 g/L Yeast nitrogen Base w/o amino acids) for 24 h and then in YPS
medium (10 g/L Yeast extract, 20 g/L Bacto-Peptone and 50 g/L Soluble starch) for
72 h. Ethanol fermentation was conducted using a 2 L jar fermenter in the medium
containing 200 g/L brown rice in our form as the only carbon source.
Recombinant A. oryzae which expresses a lipase from F. heterosporum [12] was
grown in dextrin-peptone medium consisting of 20 g/L glucose, 20 g/L polypeptone,
5 g/L KH2 PO4 , 1 g/L NaNO3 , and 0.5 g/L MgSO4 7H2 O.
Rapeseed oil used in biodiesel production was purchased from Wako Pure Chemical Industries (Osaka, Japan).
2.2. Preparation of BSP-immobilized cells
Sakaguchi asks (500 ml) containing 100 ml DP medium, fungal spores, and 300
Biomass support particles (BSPs) were incubated at 30 C for 96 h on a reciprocal
shaker (150 opm). The BSPs used for cell immobilization were 6 mm 6 mm 3 mm
cuboids 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 (ppi).
Fungal cells were spontaneously immobilized within BSPs as a natural consequence
of their growth during shake-ask cultivation.
2.3. Effect of water content on alcoholysis
Three kinds of alcoholysis (methanolysis, ethanolysis and butanolysis) were carried out at 30 C on a thermo block rotator (35 rpm). At the beginning of each form of
alcoholysis, one molar equivalent of each alcohol was added to the reaction mixture.
The compositions of reaction mixtures were variedmethanolysis used rapeseed oil
at 9.65 g and methanol at 0.35 g; ethanolysis used rapeseed oil at 9.5 g and ethanol
at 0.5 g; butanolysis used rapeseed oil at 9.23 g and 1-butanol at 0.77 gwith 100
pieces of BSPs and various amounts of distilled water (0, 0.5, 1.5 or 3.0 g). To fully
convert oil to its corresponding alkyl esters, each one molar equivalent of alcohol
was added stepwise at scheduled times (0, 24, 48, and 72 h).
2.4. Preparation of distilled bioethanol solutions for biodiesel production
To prepare bioethanol for ethanolysis, ethanol fermentation was performed in a
2 L jar fermenter, as reported previously [9]. After fermentation for 96-h, the reaction
mixture was centrifuged at 5000 rpm for 10 min to remove the yeast and residues,
and then the supernatant containing about 8% ethanol was subjected to distillation.
In the distillation step, a separable ask (3 L) and a glass column (length; 800 mm,
external diameter; 25 mm) with McMahon packing were used as a reboiler and as a
distillation tower, respectively. The distillate was separated into three fractions with
different ethanol concentrations. GC was used to analyze the ethanol concentration
of each fraction, as described in Section 2.6.
2.5. Ethanolysis of oil using a distilled bioethanol solution
Either r-FHL or Novozym 435 was used to catalyze the ethanolysis with each of
the distilled bioethanol solutions. The reaction mixture contained 9.5 g of rapeseed
oil, 0.56 g of the rst fraction or 0.737 g of the third fraction corresponding to 0.5 g
of ethanol, and 100 particles of r-FHL or 0.4 g of Novozym 435 with or without the
addition of 0.5 g of water. Bioethanol solution containing one molar equivalent of
ethanol to rapeseed oil was added to the reaction mixture at scheduled times (0, 24,
48, and 72 h).
For the repeated use of r-FHL during the ethanolysis with bioethanol, r-FHL was
recovered from the reaction mixture after one cycle, washed with distilled water,
dried at room temperature, and then added to a fresh reaction mixture for the next
cycle.

119

2.6. Analytical methods

Samples of ethanolysis were obtained from the reaction mixture at intervals
and centrifuged at 12,000 rpm for 5 min for phase separation. The upper oil layer
was analyzed for EE content using a GC-2010 gas chromatograph connected to a ZB5HT capillary column (0.25 mm 15 m; Phenomenex, USA). All of the temperature
conditions were unchanged from those described previously [13]. Chromatographic
peaks were identied via comparison of their retention times with that of a standard
solution. Tricaprylin served as the internal standard for the quantication of the alkyl
esters in the reaction mixture. The detailed procedure for the determination of alkyl
ester content was described in a previous paper [14].
The ethanol concentration of each fraction in a bioethanol solution was analyzed using a GC2010 gas chromatograph (Shimadzu, Kyoto, Japan) connected to a
ZB-WAX plus capillary column (0.25 mm 15 m; Phenomenex, USA). The temperature conditions of the injector and detector were set at 150 and 240 C, respectively.
The column temperature was set at 150 C for 1 min, increased to 230 C at 10 C/min,
and nally maintained at this temperature for 1 min. The concentrations of impurity
alcohols in the bioethanol solution were quantied using a GCMS (GCMSQP2010
Plus; Shimadzu) equipped with a DB-FFAP column (60 m, 0.25-mm internal diameter, 0.5-m lm thickness; Agilent Technologies, Tokyo, Japan). All of the analysis
conditions were unchanged from those described previously [15].
Water content of each bioethanol fraction was measured by a moisture meter
CA-21 (Mitsubishi Chemical Analytech Co., Ltd. Kanagawa, Japan).
2.7. Effect of the various alcohols in a bioethanol solution on lipase activity
To investigate the effect of the various alcohols in a bioethanol solution on lipase
activity, a lipase activity assay of r-FHL using p-nitrophenyl butyrate (pNPB) as
a chromogenic substrate was carried out after an incubation with a 10% ethanol
solution (pH 7.0) containing 3% of each alcohol (1-propanol, 1-butanol, isobutyl
alcohol or 3-methyl-1-butanol). Relative activity was calculated as the ratio of the
hydrolytic activities of r-FHL incubated in an ethanol solution with each alcohol to
those without each alcohol.

3. Results and discussion

3.1. Effect of water concentration on alcoholysis catalyzed by
r-FHL
To investigate the effect of water on plant oil transesterication catalyzed by r-FHL, three kinds of alcoholysismethanolysis,
ethanolysis and butanolysiswere carried out with the addition
of various amounts of water, 03.0 g (Fig. 1). In all cases, the alkyl
ester contents with the addition of water were obviously higher
than those without the addition of water. In a previous study of
methanolysis, a small amount of water was effective for maintaining the lipase stability of immobilized A. oryzae [12,16]. In the
present study, the addition of water also contributed to the high
alkyl ester content in the reaction mixtures. Since a wide range of
water content (530%) provided a sufciently high EE content and
high butyl ester content, we expected that bioethanol and biobutanol solutions with high water concentrations would be applicable
to the enzymatic production of biodiesel. Therefore, bioethanol was
employed for ethanolysis using r-FHL as described elsewhere in this
paper.
3.2. Preparation and analysis of a distilled bioethanol solution
Ethanol fermentation of brown rice was conducted using the
yeast strain (MNIV/GS) and the fermented broth contained more
than 80 g/L of ethanol. The ethanol solution was separated into
three fractions by distillation. The rst fraction contained 89.3%
of ethanol and other peaks were not detected in GCMS analysis (Supplementary data (Fig. S1)). The second fraction with 83.1%
of ethanol contained 1-propanol, 1-butanol, isobutyl alcohol and
3-methyl-1-butanol at 0.78, 0.31, 1.56, and 1.96 g/L, respectively,
whereas, in the third fraction with 67.8% of ethanol, the contents of
these impurities increased to 3.21, 2.10, 12.1, and 37.6 g/L, respectively. The water contents of the rst, second and third fraction
were 8.08, 14.1 and 25.4%, respectively. The detected alcohols (1propanol, 1-butanol, isobutyl alcohol, and 3-methyl-1-butanol),

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D. Adachi et al. / Enzyme and Microbial Technology 52 (2013) 118122

Fig. 1. Time course of various kinds of alcoholysis, (a) methanolysis, (b) ethanolysis, (c) butanolysis with the addition of various amount of water externally, 0 (diamond),
0.5 (square), 1.5 (triangle) and 3.0 g (circle).

referred to as fusel alcohols, are generally found in alcoholic beverages [17] and originate from the metabolism of amino acids
[18]. Since brown rice commonly contains various amino acids,
yeast metabolism of these amino acids might produce the alcohols
present in the ethanol solution. The issue regarding impurities in
an ethanol solution should also be considered when bioethanol is
produced from pretreated lignocellulosic biomass, which releases
several volatiles including formic acid, acetic acid and furfural [19].
3.3. Ethanolysis using a bioethanol solution
To investigate the effect of the composition of a bioethanol solution on biodiesel production, ethanolysis using distilled bioethanol
solutions was performed (Fig. 2). When the rst fraction was used
without the addition of water, the EE content was only 24.7% after
96 h. When 0.5 g of water was added externally, the EE content
was increased to 94.0%. These results suggest that water concentration markedly affects the EE content in ethanolysis when using
a bioethanol solution, as observed in methanolysis [12]. In a similar
manner, the external addition of water also increased the EE content signicantly when using the second fraction (Supplementary
data (Fig. S2)). On the contrary, a sufciently high EE content of
84.3% was obtained using the third fraction without the addition of
water (Fig. 2), and the external addition of water was less effective
(Supplementary data (Fig. S2)). Since the third fraction contained
more water than the rst, a sufcient concentration of water in the
bioethanol solution might have contributed to the high EE content
in the reaction mixture.
Ethanolysis using a bioethanol solution and commercial immobilized lipase (Novozym 435) was also performed and compared
with that using r-FHL. When the rst fraction was used for ethanolysis without the addition of water, the nal EE content was only
9.00% (Fig. 2). Also, other conditions using the rst fraction with
the addition of 0.5 g water and the third fraction without the addition of water did not increase the EE contents; these were only

7.92 and 4.68%, respectively (data not shown). In previous studies,

Novozym 435 effectively catalyzed ethanolysis with the stepwise
additions of ethanol without an organic solvent, resulting in EE
contents of 95 and 84% from tuna oil and soybean oil, respectively
[20,21]. However, the EE contents obtained in the present study
were much lower than those in the previous studies. This result
can be explained by the effect of water in the bioethanol solution
because the presence of more than 0.2% of water in the reaction
mixture signicantly decreased the reaction rate during methanolysis using Novozym 435 [22]. When the bioethanol solution in the
rst fraction was used to attain one molar equivalent of ethanol to
oil, 0.4% of water was found in the reaction mixture. Therefore,
an excess amount of water would decrease the reaction rate in

Fig. 2. Time courses of ethanolysis using 1st fraction without addition of water
(open diamond) and with addition of 5% water (open triangle) or using 3rd fraction
without addition of water (open circle) catalyzed by r-FHL and using 1st fraction
without addition of water catalyzed by Novozym 435 (closed diamond).

D. Adachi et al. / Enzyme and Microbial Technology 52 (2013) 118122

Fig. 3. Recycling of r-FHL during 5 cycles of batch ethanolysis on a thermo block

rotator using 1st fraction with the addition of 0.50 g of water (triangle) and using
3rd fraction without addition of water (circle).

ethanolysis, as was the case with methanolysis. We believe these

results show that for ethanolysis with a bioethanol solution, r-FHL
is more suitable than Novozym 435.
3.4. Recycling of r-FHL for ethanolysis using a bioethanol solution
and the effect of fusel alcohol on lipase activity
We investigated the recycling of r-FHL during 5 cycles of batch
ethanolysis using the rst fraction with the addition of 0.50 g of
water and using the third fraction without the addition of water
(Fig. 3). EE contents of more than 80 and 60% were maintained
after the fth batch using the rst and third fractions, respectively. Although a gradual decrease in the EE content was observed
in both cases, the degree of decline in the EE content using the
third fraction was larger than that using the rst fraction. We
believe the fusel alcohol contained in the third fraction might have
caused the difference. To observe the effect of the alcohols in a
bioethanol solution on the lipase activity of r-FHL, the residual
activities after incubation in a 10% ethanol solution (pH 7.0) with
3% of any one of the alcohols (1-propanol, 1-butanol, isobutyl alcohol and 3-methyl-1-butanol) were investigated (Fig. 4). Among the
four kinds of alcohols, the addition of 1-propanol or 3-methyl-1butanol increased the residual activity of r-FHL compared to that in
only ethanol, whereas 1-butanol or isobutyl alcohol decreased the
residual activity of r-FHL. Therefore, 1-butanol and isobutyl alcohol in the bioethanol solution might have been responsible for the
decrease in the lipase activity of r-FHL. In addition, when all four

alcohols were added to the ethanol solution, the residual activity

of r-FHL was decreased by approximately half compared with that
in only ethanol. Given these results, the bioethanol solution with a
low content of these inhibitory compounds seems to be desirable
for biodiesel production using r-FHL.
According to a report of Biofuel Platform1) , the energy potential of biodiesel and bioethanol were 37.2 MJ/kg and 26.8 MJ/kg,
respectively. In this system, about 11.1 g of biodiesel was produced
using 2.0 g of bioethanol, namely, 413 kJ of energy was generated
using 54 kJ. Moreover, when using bioethanol as a biofuel, complete
removal of water through additional processes including membrane separation is required for increasing the ethanol content
close to 100%. In this system, however, only a simple distillation
is required, leading to a lower cost and lower energy consumption.
Therefore, we think that the process model developed in this study
would be feasible to conduct the process on a large scale. However,
for practical application, further improvement is necessary. Since
reaction condition was not optimized in this study, optimization
of the condition is one of the improvements. For example, in our
previous study, the introduction of continuous production system
using a packed-bed reactor enabled a much shorter reaction time,
resulting in a methyl ester content of more than 85% after 450-min
methanolysis [23]. Other approaches include the enhancement of
promoter activity [24]. For example, our previous study showed
that signicant improvements in the expression level and methyl
ester productivity of lipase-producing A. oryzae are observed when
using a novel plasmid harboring P-enoA142 promoter and the 5 untranslated region of a heat shock protein [25]. The application of
these systems would provide a higher reaction rate for ethanolysis.
4. Conclusions
We successfully integrated lipase-catalyzed ethanolysis with
fermentative bioethanol production from brown rice. Owing to a
high concentration of water in distilled bioethanol solutions, rFHL, which works best in the presence of water when processing
ethanolysis, was found to be more suitable for the integrative
process than a commercial immobilized lipase, Novozym 435.
Moreover, r-FHL maintained a high EE content of more than
80% after the fth batch. In this process, a high EE content was
obtained by simply mixing bioethanol, plant oil and lipase with an
appropriate adjustment of the water concentration. Therefore, the
developed process model would contribute to biodiesel production
from only biomass-derived feedstocks.
Acknowledgements

200

This work was supported by the Japanese Ministry of the Environment for Technical Development of Measures to Prevent Global
Warming (2007). This work was also partly supported by Special
Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research
Areas (Innovative Bioproduction Kobe), MEXT, Japan.

150

Appendix A. Supplementary data

100

Supplementary data associated with this article can be found,

in the online version, at http://dx.doi.org/10.1016/j.enzmictec.
2012.11.005.

250

Relative activity (%)

121

50

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1-propanol 1-butanol Isobutyl 3-methylalcohol 1-butanol

All

Fig. 4. The effect of each alcohol in bioethanol solution on the lipase activity of
r-FHL. The lipase activity was evaluated as described in Section 2.7.

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Web reference
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biodiesel
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