Abstract As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae) IFO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanolysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium. Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction mixture reached 90% -the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is considered to be promising for biodiesel fuel production in industrial applications

Akihiko Kondo

Hideki Fukuda

Kazuhiro Ban

Masaru Kaieda

Takeshi Matsumoto

CiteSeerX

Biochemical Engineering Journal 8 (2001) 39–43
Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae
cells immobilized within biomass support particles
Kazuhiro Ban a, Masaru Kaieda b, Takeshi Matsumoto c,
Akihiko Kondo b, Hideki Fukuda c,∗
a Fujikin Incorporated, 90 Sinke-Higashi, Higashi-Osaka 577-0026, Japan
b Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
c Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
Received 28 September 2000; accepted 14 November 2000
Abstract
As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae)
IFO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanol-
ysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis
activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium.
Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells
were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with
stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction
mixture reached 90% — the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is
considered to be promising for biodiesel fuel production in industrial applications. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Biodiesel fuel; Rhizopus oryzae; Methanolysis; Whole cell biocatalyst; Biomass support particles
1. Introduction
In recent years, methyl esters (MEs) produced by
methanolysis of plant oils, commonly referred to biodiesel
fuel, have become viewed as promising renewable sources
of fuel, since they emit much lower levels the of sulfur ox-
ides, halogens, and soot in their exhaust gas than petroleum
diesel fuel. Utilization of lipase as a catalyst for biodiesel
fuel production has great potential compared with chemical
methods using an alkaline catalyst because no complex op-
erations are needed, not only for the recovery of glycerol
but also in eliminating of the catalyst and salt.
We have already reported that several lipases efficiently
catalyzed the methanolysis of soybean oil without an or-
ganic solvent [1–4]. In a very low-water-content system
(i.e. a microaqueous system) plant oil methyl esters (MEs)
could be efficiently synthesized with immobilized Candida
antarctica lipase [1–3]. In contrast, the extracellular lipase
from Rhizopus oryzae (R. oryzae) catalyzed methanolysis
∗ Corresponding author. Tel.: +81-78-803-6192; fax: +81-78-803-6206.
E-mail address: fukuda@cx.kobe-u.ac.jp (H. Fukuda).
in the presence of 4–30% water in the starting materials,
whereas, the lipase was nearly inactive in the absence of wa-
ter [4]. With theses characteristics R. oryzae lipase is con-
sidered to be a potentially effective enzyme when waste oil
is used as the substrate, since this inevitably contains a cer-
tain amount of water. However, the use of extracellular R.
oryzae lipase as a catalyst requires complicated recovery,
purification, and immobilization processes for the enzyme,
which are disadvantages for industrial applications.
Fukuda and co-workers [5–7] have reported a promis-
ing process for the industrial interesterification of fats and
oils. Unlike conventional methods using extracellular lipase
obtained by recovery from the culture broth, acetone-dried
cells of R. chinensis immobilized within polyurethane foam
particles (BSPs) could be used directly as a whole cell bio-
catalyst.
In the present study, in order to utilize immobilized R.
oryzae cells within BSPs as a whole cell biocatalyst, the
culture conditions for lipase production were first examined,
and the effects of pre-treatment of the cells and the water
content in the reaction mixture on methanolysis were then
investigated.
1369-703X/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1 3 6 9 -7 0 3X(00 )00133 -9
40 K. Ban et al. / Biochemical Engineering Journal 8 (2001) 39–43
2. Materials and methods
2.1. Microorganism and media
All experiments were carried out using R. oryzae IFO
4697, which has a 1,3-positional specifity lipase. The basal
medium used contained in 1 l tap water: polypepton 70 g;
NaNO3 1.0 g; KH2PO4 1.0 g, and MgSO4·7H2O 0.5 g. To
examine the effect of substrate-related compounds, olive oil,
oleic acid, oleyl alcohol, methyl caprate or Tween-80 was
added to the basal medium. Glucose was also added when
necessary.
2.2. Biomass support particles (BSPs) and shake-flask
cultivation
Sakaguchi flasks (500 ml) containing 100 ml of the basal
medium were inoculated by aseptically transferring spores
from an agar slant, and incubated for 80–90 h at 35◦C on
a reciprocal shaker (150 oscillations/min; amplitude 70 mm)
with BSPs. The R. oryzae cells became well immobilized
within the BSPs as a natural consequence of their growth
during shake-flask cultivation. The BSPs used for immobi-
lization were 6-mm cubes of reticulated polyurethane foam
(Bridgestone Co., Ltd., Osaka) with a particle voidage be-
yond 97% and a pore size of 50 pores per linear inch. Immo-
bilization was effected by placing 150 particles inside a flask
together with the medium subjected to prior sterilization.
2.3. Preparation of cells immobilized within BSPs
BSP-immobilized cells were separated from the culture
broth by filtration. After washing them with tap water,
the BSPs were treated with an organic solvent (methanol,
ethanol, isopropanol or acetone) by immersion for 0–30 min,
or left untreated, and then, dried under a vacuum for about
48 h. Immobilized cells with a water content of approx-
imately 5% were obtained and used as a methanolysis
catalyst. The filtrate from the culture broth was used for
analysis of the hydrolysis activity.
2.4. Enzyme reaction
The methanolysis and hydrolysis reactions were carried
out at 35◦C in a 50-ml screw-cap bottle with incubation on a
reciprocal shaker (150 oscillations/min; amplitude 70 mm).
The compositions of the reaction mixtures were as follows:
for the methanolysis, soybean oil 9.65 g, 0.1 M acetate buffer
(pH 5.6) 0–3.0 ml, and methanol 0.35 g, were added with 50
BSPs to a screw-cap bottle; for the hydrolysis reaction, 0.1 M
acetate buffer (pH 5.6) 9.0 ml, 0.1 M CaCl2 1.0 ml, olive oil
2.0 g, and culture broth 0.5 ml were added to a screw-cap
bottle. These two mixtures were incubated for 2.5 and 1 h,
respectively. One molar equivalent of methanol was 0.35 g
against 9.65 g soybean oil.
To fully convert the oil to its corresponding MEs, when
the ME content in the reaction mixture reached approxi-
mately 30 and 60%, 0.35 g of methanol was added twice.
In this case, the reaction mixture was incubated for 72 h.
The methanolysis products were analyzed by capillary gas
chromatography (cGC) as described below.
2.5. Analysis
The biomass concentration within a BSP was measured as
follows. Twenty particles were taken and washed with ace-
tone several times to remove substrate-related compounds
and they were then dried for 24 h at 100◦C. The particles
plus dried cells were weighed and treated with an aqueous
solution of sodium hypochlorite (approximately 10% v/v) to
remove biomass. The cleaned particles were rinsed, dried,
and reweighed. The biomass was estimated from the differ-
ence between the weights. Water content in the BSPs was
determined by Karl Fischer titration (Kyoto Electronics Mfg.
Co., Ltd., Japan).
The ME and free fatty acid (FFA) contents in the reaction
mixture were quantified using a GC-18A gas chromatograph
(Shimadzu Corp., Kyoto) connected to a DB-5 capillary
column (0.25 mm × 10 m; J&W Scientific, Folsom, CA).
Samples (150 l) were taken from the reaction mixture at
specified times and centrifuged to obtain the upper layer.
For cGC analysis, 100 l of the upper layer and 20 l
tricaprylin were precisely measured into a 10-ml bottle, to
which a specified amount of anhydrous sodium sulfate as
a dehydrating agent and 3.0 ml hexane were added. Tri-
caprylin served as the internal standard for cGC. A 1.0-l
aliquot of the treated sample was injected into a GC-18A
gas chromatograph connected to a DB-5 capillary column
(0.25 mm × 10 m) to determine the ME and FFA contents
in the reaction mixture. The ME and FFA contents were,
respectively, determined as the ratio of MEs and FFAs con-
verted to the reaction mixture without water and glycerol.
The column temperature was held at 150◦C for 0.5 min,
raised to 300◦C at 10◦C/min, and maintained at this temper-
ature for 3 min. The temperatures of the injector and detector
were set at 245 and 250◦C, respectively.
The detailed hydrolysis procedure of Fukumoto et al. [8]
was followed, with 1 mmol of fatty acids liberated per min
being defined as 1 unit (U).
3. Results and discussion
3.1. Effects of substrate-related compounds added
in the culture medium on methanolysis activity
To examine the effects of substrate-related compounds
on methanolysis activity, cells were cultivated with BSPs in
the presence of various substrate-related compounds. The
concentration of each compound was 30 g/l, and glucose
was added at 3 g/l, to the basal medium. The BSPs were
K. Ban et al. / Biochemical Engineering Journal 8 (2001) 39–43 41
Table 1
Effects of substrate-related compounds on lipase production by immobilized R. oryzae cells
Substrate-related compound Cells immobilized within BSPs Extracellular hydrolysis activity (U/ml)
Cell concentration (mg/BSP) Methyl ester content (wt.%)
Oleic acid 7.09 10.4 1.30
Olive oil 8.52 12.8 1.62
Oleyl alcohol 0.99 1.4 1.25
Methyl caprate 2.21 0.2 0.10
Tween 80 3.34 5.0 0.50
None 3.12 1.2 1.87
not treated with an organic solvent. Table 1 shows the ME
contents after 2.5-h methanolysis with 15% water content in
the reaction mixture.
Except for methyl caprate, the ME contents in the pres-
ence of each substrate-related compound was greater than
that without them. Two of the compounds, olive oil and oleic
acid, greatly enhanced the ME content. However, the extra-
cellular hydrolysis activity was higher without the addition
of a compound than with any of them. The reason may be
that these compounds act to maintain lipase within the cells
to some degree. On the basis of the results, olive oil was
selected as the most suitable compound to enhance intracel-
lular methanolysis activity using cells immobilized within
BSPs.
Fig. 1 shows surface and cross-sectional micrographs of a
6-mm cubic particle with immobilized cells cultivated in the
presence of olive oil. The cells can be seen to have formed
a dense film near the particle surface, which would severely
limit the amount of oxygen in the center. Cellular adhesion
to the support matrix appears strong, which means the cells
Fig. 1. Micrographs of surface (a) and cross-section (b) of a BSP.
would not be easily released from the particles even with
vigorous agitation of the reaction mixture.
To determine the optimal olive oil concentration, its initial
concentration (with 3 g/l of glucose) was varied between 0
and 50 g/l (Fig. 2). The ME content increased with the olive
oil concentration up to 30 g/l, but beyond this concentration,
no change in ME content was observed. Consequently, 30 g/l
olive oil was used in the subsequent experiments.
3.2. Effect of glucose concentration added in the
culture medium on methanolysis activity
Fig. 3 shows the effect of the initial glucose concentration
on the methanolysis activity. Olive oil and glucose were
added to the culture medium at 30 and 3 g/l, respectively. The
ME content was high an initial glucose range from 0 to 5 g/l,
but beyond this it decreased gradually. A similar trend was
also slightly apparent for the numbers of cells immobilized
within the BSPs. It is thought that the presence of a large
amount of glucose causes a reduction in lipase production,
42 K. Ban et al. / Biochemical Engineering Journal 8 (2001) 39–43
Fig. 2. Effect of olive oil concentration added in the culture medium
on methyl ester content. Symbols: () methyl ester content; () cell
concentration.
Fig. 3. Effect of glucose concentration added in the culture medium
on methyl ester content. Symbols: () methyl ester content; () cell
concentration.
and it was clear that no glucose was necessary to enhance
the methanolysis activity of the immobilized R. oryzae cells.
This trend is almost identical to the case of intracellular
lipase production from R. chinensis for the interesterification
of fats and oils [5].
3.3. Treatments of BSPs-immobilized cells with
organic solvents
To investigate the effects of organic solvents on cell
permeabilization, BSP-immobilized cells were treated with
three various alcohols (methanol, ethanol, or isopropyl al-
cohol) or acetone (all >99% pure). Fig. 4 shows the effects
of the treatment time on the ME content after incubation
for 2.5 h. In the cases of isopropyl alcohol and acetone,
there was almost no decrease in the ME content even with
30 min treatment. In contrast, with ethanol or methanol re-
sulted in a rapid decrease in the ME content; with methanol
Fig. 4. Treatments of BSPs-immobilized cells with organic solvents. Sym-
bols: () acetone; () isopropanol; () ethanol; () methanol.
almost all catalytic activity was lost. These findings indi-
cate that the use of non-treated cells is preferable to obtain
a highly active, whole cell biocatalyst, and also that the
concentration of methanol in the reaction mixture should
be kept at a low level since methanol directly attacks the
enzyme [1].
We previously reported that permeabilization of recombi-
nant Saccharomyces cerevisiae cells with alcohol solutions
resulted in significantly effective whole cell biocatalysts for
the conversion of methylglyoxal to S-lactoylglutathione [9].
However, unlike the production of S-lactoylglutathione, in
which a non-permeable substrate was used, in the methanol-
ysis of oil a certain amount of methanol is itself utilized as
a substrate, resulting in a high degree of substrate perme-
ation into R. oryzae cells without any treatment for the cell
membrane.
3.4. Effect of water content in the reaction mixture
on methanolysis activity
To complete the bioconversion of soybean oil to the cor-
responding ME, at least three molar equivalents of methanol
are necessary. Since a high methanol concentration causes
irreversible denaturation [1], a molar amount of methanol
equal to that of oil was added twice at the times indicated
by the arrows in Fig. 5, which shows the time courses of
methanolysis with stepwise additions of methanol under
an increasing water content. When methanolysis was car-
ried out with 0.4–3.0 ml acetate buffer (4.0–30 wt.% water
by substrate weight), the ME content reached approxi-
mately 80–90 wt.% after 72-h reaction. The highest ME
content, 91.1 wt.%, was attained when the reaction mixture
contained 1.5 ml buffer solution (15 wt.% water by sub-
strate to weight). In contrast, with 0.3 ml buffer solution
(3.0 wt.% water by substrate weight) the ME content was
only 54.4 wt.%, and with no addition of buffer solution the
ME content failed to improve after reaching approximately
33 wt.%. An insufficient amount of water probably results
K. Ban et al. / Biochemical Engineering Journal 8 (2001) 39–43 43
Fig. 5. Time courses of methyl ester content in methanolysis with
BSP-immobilized cells in the presence of () 0, () 2.0, () 3.0, ()
4.0, () 10, () 15, () 20, or () 30% water in the reaction mixture.
Methanol was added twice at the times indicated by the arrows.
in irreversible inactivation of lipase, which may be due to
denaturation of the enzyme by methanol.
We previously reported that although the extracellu-
lar lipase from R. oryzae cells is considered to exhibit
1(3)-regiospecifity, acyl migration can occur spontaneously
in a water-containing system [4]. The ME content using
cells immobilized within BSPs reached the same level as
that using the extracellular lipase. This clearly demonstrates
that acyl migration occurred with the intracellular lipase of
the immobilized cells as it did in the case of the extracellular
lipase. Our findings suggest that R. oryzae cells immobi-
lized within BSPs can effectively catalyze the methanolysis
reaction as a whole cell biocatalyst in water-containing
systems.
In industrial applications using BSP-immobilized cells,
they need to stable enough for repeated use. Thus, the
stability of whole cell biocatalysts is currently under inves-
tigation.
4. Conclusion
In this study, to utilize immobilized R. oryzae cells within
BSPs as a wole cell biocatalyst for biodiesel fuel produc-
tion, both the culture conditions for lipase production and
methanolysis conditions were investigated. To enhanced
the methanolysis activity of the immobilized cells olive oil
or oleic acid added in the culture broth was significantly
effective, but no glucose was necessary. The high methyl
ester content over 90% obtained in water-containing systems
was probably attributable to the spontaneous acyl migration.
Acknowledgements
The authors wish to express their thanks to Dr. H. Yanagi,
Department of Chemical Science and Technology, Kobe
University, for his taking micrographs of cells in BSPs. This
work was financed by New Energy and Industrial Tech-
nology Development Organization (NEDO), Japan.
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Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles

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Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles

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