Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188

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Journal of Molecular Catalysis B: Enzymatic

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Screening, immobilization and utilization of whole cell biocatalysts to mediate

the ethanolysis of babassu oil
Grazielle S.S. Andrade ∗ , Larissa Freitas, Pedro C. Oliveira, Heizir F. de Castro
Engineering School of Lorena, University of São Paulo, PO Box 116-12602-810, Lorena, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The screening, biomass growth of lipase-producing fungus isolated from different sources and available
Available online 3 March 2012 at URM (University Recife Mycologia), as well as, the immobilization and utilization of the whole cells
for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor
Keywords: circinelloides (URM 4140, 4182) and Penicillium citrinum URM 4216 were considered to be good intra-
Whole cell cellular lipase producers whereas those from Mucor hiemalis URM 4144 and Mucor piriformis URM 4145
Lipase
were weaker. Fungi biomass containing high lipase activities was immobilized on different biomass
Ethanolysis
support particles (BSPs) and with the exception of Penicillium citrinum URM 4216 all the other fungi
Babassu oil
strains exhibited high lipase activity (20–50 U g−1 ) when immobilized in situ using polyurethane foam
particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanoly-
sis reaction with babassu oil and the highest performance was attained by M. circinelloides URM 4182
giving 83.22 ± 3.68% ester yield in less than 96 h reaction. The biocatalyst operational stability was also
assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 26
days and 2.6 × 10−2 day−1 , for half-life and a deactivation coefficient, respectively. The purified product
(biodiesel) exhibited viscosity (6.63 cSt) close to the value to attend specifications by the ASTM D6751
to be used as biofuel. Results are favorable compared with data already reported in the literature and
demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be used in the
biodiesel synthesis.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction There are two major procedures of enzymatic biocatalyst: (1)

Biodiesel which is derived from triglycerides or free fatty acids
has become increasingly important as a kind of alternative fuel
due to the diminishing petroleum reserves and environmental
regulations [1]. The advantages of biodiesel as fuel are its portabil-
ity, ready availability, renewability, higher combustion efficiency,
lower sulfur and aromatic content, higher cetane number and
higher biodegradability [2]. With an increasing focus on renewable
sources of energy in terms of marketing, usage, distribution and
general acceptance, the opportunities for producers of biodiesel are
promising [3].
There has been considerable research on biodiesel production
from several renewable vegetal oils and fats, particularly through
the biochemical route [1,4–6]. Biocatalysts exhibit advantages over
chemical catalysts in that the overall transesterification process is
less energy intensive and a complex process of catalyst removal
and waste treatment is not required [1].
∗ Corresponding author. Tel.: +55 12 3159 5063; fax: +55 12 3159 50.
E-mail addresses: gra.ss@uol.com.br, grazielle@debiq.eel.usp.br (G.S.S. Andrade).
extracellular lipases in which the enzyme has previously been
recovered from the live-producing microorganism broth and then
purified, having Mucor miehei, Rhizopus oryzae, Candida antarctica
and Pseudomonas cepacia as major producer microorganisms and
(2) intracellular lipase which the enzyme remains either inside or in
the cell-producing walls. Fungi produce intracellular lipases of high
catalytic activity which have desired characteristics for biodiesel
synthesis include strains of Rhizopus spp., R. delemar, R. oryzae, R.
arrhizus, R. chinensis and R. niveus. In both cases the enzyme should
be immobilized in a suitable support matrix to improve its features,
such as increasing the activity, decreasing inhibitions, modulat-
ing selectivity and specificity or improving the enzyme behavior
in synthetic processes. It is important to note that immobilized
extracellular lipase is commercial available by reputable industries
such as C. antarctica lipase B immobilized onto polyacrylate type
matrix, available under the name of Novozym 435® (Novozymes)
or Chirazyme L-2® (Roche). Some lipases are also available in the
cross-linked forms, such as cross-linked enzyme crystals (CLECs)
offered by Altus Biologics Inc. Although these immobilized prepa-
rations are considered to be the most suitable catalysts for several
synthetic reactions their cost is still a limitation for large scale pro-
duction of commodities such as biodiesel. The use of intracellular

1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.02.011
184 G.S.S. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188
lipase (whole-cell biocatalyst) has been identified as a major step
toward the goal of cost effective, sustainable biodiesel production
as the whole-cell lipase substantially reduces cost by avoiding the
complex isolation, purification and immobilization of extracellular
lipase [7].
Whole-cell biocatalysts are prepared by cultivation, and the
enzymes trapped inside the cells are regarded as immobilized and
can be separated easily [8]. Literature examples demonstrate that
immobilized whole cells can efficiently catalyze the methanolysis
of vegetable oils for biodiesel production in solvent-free or organic
solvent systems [3,9]. In addition, whole cells catalysts could be
applied to varied oil feedstock transformation, and there were no
special requirements on the contents of water, phospholipids and
free fatty acids contained in the oil feedstock [10]. Although sev-
eral filamentous fungi have been identified as robust candidates
for whole cell biocatalysis, research works have been concentrated
in the use of R. oryzae strains [11], and therefore it is desirable
to investigate other microbial resources as potential producers of
intracellular lipases of commercial significance.
In this context, the present work aims to investigate the poten-
tial of filamentous fungi strains isolated from different habitats and
available in the culture collection as intracellular lipase producers.
Seven fungi strains were previously selected including two Rhizo-
pus sp. and five Mucor sp. The fungi were screened according to
its intracellular lipase activity, immobilized in different BSPs and
used to catalyze the ethanolysis reaction of babassu oil. The choice
of the starting materials is an exceptional option for the Brazil-
ian biodiesel production, since both babassu oil and ethanol are
readily available in the country. Besides, almost all researchers used
methanol as acyl acceptor that needs to be added in stages to min-
imize its negative effect on the activity of the biocatalyst, and with
ethanol it is expected to overcome this limitation.
2. Experimental
2.1. Materials
Polyurethane foams (Scotch-BriteMR ) having pore size of
0.76 ± 0.11 mm and density of 0.02 ± 0.01 g cm−3 were pur-
chased from local commerce and cut down in cubic pieces
(6-mm). Spherical (average particle diameters of 750–1180 ␮m)
poly-(hydroxybutyrate) (PHB) particles were acquired from PHB
Industrial (São Paulo, Brazil) and Bio-Catalyst Carrier R-630 was
acquired from Manville (Denver, CO). Ethanol 99.8% and tert-
butanol were purchased from Cromoline (Diadema, SP, Brazil).
Babassu oil was a kind gift from Pulcra (Jacareí, SP, Brazil) having
the following composition in fatty acids (wt): 3.5% octanoic, 4.5%
decanoic, 44.7% lauric, 17.5% mirystic, 9.7% palmitic, 3.1% steriac,
15.2 oleic and 1.8% linoleic, with 709.4 g mol−1 average molec-
ular weight. Other characteristics included: acid index (0.65%),
saponification index (238 mgKOH g−1 ), iodine index (25 g I2 g−1 ),
specific mass (0.85 g cm−3 ), free fatty acid (0.33%), oxidation index
(1.82 mEq kg−1 ) and viscosity (29.51 cSt). All other reagents were
of analytical grade.
2.2. Microorganism and culture media
Seven filamentous fungal strains were used in this study: R.
oryzae URM 4692, R. oryzae URM 3231, Mucor circinelloides f.
Circinelloides URM 4140, M. circinelloides f. griseo-cyanus URM
4182, Mucor hiemalis f. Luteus URM 4144, Mucor piriformis URM
4145 and P. citrinum URM 4216. These microorganisms have docu-
mented lipase activity and were purchased from culture collection
URM (University Recife Mycologia) at Federal University of Per-
nambuco (Pernambuco, Brazil). PDA (Potato Dextrose Agar – Difco)
was used as solid culture medium for fungi propagation. The liquid
basal medium used contained in 1 L tap water: polypeptone (Hime-
dia) 70 g; NaNO3 (Vetec) 1.0 g; KH2 PO4 (Synth) 1.0 g; MgSO4 ·7H2 O
(Vetec) 0.5 g and olive oil (Carbonell) 30 g.
2.3. Preparation of whole cells biocatalysts
Erlenmeyer flasks (250 mL) containing 100 mL of the basal
medium were inoculated by aseptically transferring 106 spores of
the filamentous fungi from an agar slant, and incubated for 72 h
at 30 ◦ C on a reciprocal shaker at 170 rpm. Samples were taken
each 24 h and biomass were separated from the culture broth by
filtration, washed with water and acetone and dried under vac-
uum for 24 h. Dry biomass and the filtrated from culture broth were
submitted from analysis of lipolytic activity.
2.4. Preparation of immobilized whole cells biocatalysts
Filamentous fungi were immobilized by placing 100 cubes
(6 mm × 6 mm × 6 mm) (about 0.4 g) of polyurethane foams (PUF)
inside a flask together with the basal medium subjected to prior
sterilization. The other supports, Celite (5.0 g) and PHB (10.0 g) were
added after shaker flask cultivation. At the end of the cultivation,
immobilized cells were separated from the culture broth and dried
under vacuum about 24 h. Both dry biomass and the culture broth
were submitted for analysis of lipolytic activity.
2.5. Biodiesel synthesis
The ethanolysis reactions were performed in closed 250 mL
Erlenmeyer flasks containing 30 g of babassu oil and anhydrous
ethanol at oil-to-ethanol molar ratio of 1:6 and tert-butanol as
solvent at proportional (1:1). The mixtures were incubated with
immobilized whole cells at fixed proportion of 20% (w/w) in rela-
tion to the total weight of reactants involved in the reaction media.
Reactions were carried out at 35 ◦ C for a maximum period of 120 h
under reciprocal shaker (170 rpm). Aliquots were taken to quantify
ethyl esters formed in GC analysis.
2.6. Operational biocatalyst stability
The biocatalyst operational stability was assayed under con-
tinuous run using a fixed bed reactor (PBR) packed with the
immobilized whole cells. Ethanolysis runs were performed at
35 ◦ C, for a 14 days running on substrate consisting of babassu
oil and anhydrous ethanol molar ratio oil to alcohol (1:6) and
tert-butanol as solvent at proportion of 1:1. PBR was a jacketed
glass column (internal diameter = 45 mm; height = 190 mm, and
total volume = 310 cm3 ). The temperature in the reactor was kept
by circulating water in the jacket. The substrate was continuously
pumped (Sj-1211-Hatto) from a reservoir at 35 ◦ C, through sili-
cone tubing, to the bottom end of the bioreactor at flow rate of
0.13 mL min−1 . An amount of 27 g biocatalyst was used, which cor-
responds to a bulk volume of 270 cm3 . Density of the immobilized
cells (dry weight) was about 0.1 g mL−1 . The residence time was cal-
culated according to Levenspiel [12] as described in Eq. (1). Aliquots
were taken every day to quantify ethyl esters formed in GC analysis:
V
= (1)
v0
2.7. Purification of biodiesel
At the end of batch runs the immobilized biocatalyst was sepa-
rated from the reaction medium, and the organic phase was three
times washed with water to remove the remaining free glycerol
 G.S.S. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188 185

Table 1
Biomass concentration and maxima activities (extra and intracellular) attained for
each tested strain.

Strain Biomass (g L−1 ) Lipase activity (U g−1 )

Extracellular Intracellular

R. oryzae 3231 27.5 ± 0.69 4.34 ± 0.81 20.98 ± 0.55

R. oryzae 4692 26.1 ± 0.64 3.16 ± 0.95 20.50 ± 0.57
M. circinelloides 4140 31.5 ± 0.94 2.28 ± 0.25 11.03 ± 0.45
M. circinelloides 4182 33.8 ± 0.83 2.91 ± 0.16 24.00 ± 3.66
M. hiemalis 4145 36.7 ± 0.26 2.85 ± 0.19 2.44 ± 0.60
M. piriformis 4144 12.6 ± 0.56 2.11 ± 0.60 3.33 ± 0.46
P. citrinum 4216 46.5 ± 0.50 3.00 ± 0.31 30.82 ± 0.51

formed as a by-product. Residual ethanol and tert-butanol were

removed by rotated evaporation, and the water remaining in fatty
acid ethyl ester product was removed with addition of sodium sul-
fate salt.

2.8. Analysis

Lipolytic activities were assayed by the olive oil emulsion

method according to the modification proposed by Soares et al. [13]
using substrate concentration at fixed proportion 10:1 oil/water
(w/w). One unit (U) of enzyme activity was defined as the amount
of enzyme that liberates 1 ␮mol of free fatty acid per min under the
assay conditions (35 ◦ C, pH 7.5).
The ethyl esters formed in the ethanolysis reaction was analyzed
in FID gas chromatography (Varian CG 3800, Inc. Corporate Head-
quarters, Palo Alto, CA, USA) using a 5% DEGS CHR-WHP 80/100
mesh 6 ft 2.0 mm ID and 1/8 OD column (Restek, Frankel Com-
merce of Analytic Instruments Ltd., SP, Brazil) following previous
established conditions [14]. Nitrogen was used as the carrier gas
with a flow rate of 25 mL min−1 . The detector and injector tempera-
tures were 190 ◦ C. The column temperature was first set to 90 ◦ C for
3 min and then programmed at 25 ◦ C min−1 to 120 ◦ C for 10 min and
then 170 ◦ C for 15 min. The transesterification yield (%) was defined
as the ratio between the produced and theoretical esters concentra-
tions × 100. Data was collect using Galaxie Chromatography Data
System software version 1.9. Theoretical ester concentrations were
calculated by taking into consideration the babassu oil fatty acid
composition and its initial weight mass in the reaction medium
[14,15].
The absolute viscosity of biodiesel was determined with LVDV-II
cone and plate spindle Brookfield viscosimeter (Brookfield Vis-
cometers Ltd., UK) using a CP 42 cone. A circulating water bath
was used to maintain the temperature at 40 ◦ C during the assays.
The shear stress measurements were taken as a function of shear Fig. 1. Extracellular lipase activity (a) and intracellular lipase activity (b) obtained
from different filamentous fungi strains immobilized in PUF (in situ), Celite (extra
rate, and the dynamic viscosity was determined as a slope constant.
situ) and PHB (extra situ) cultivated with olive oil (3%) at 30 ◦ C and 170 rpm.
Biodiesel samples of 0.5 mL were used, and the measurements were
replicated three times. The density of biodiesel was determined
with DMA 35N EX digital densimeter (Anton Paar). The tempera- citrinum URM 4216 with 46.5 ± 0.5 g L−1 whereas M. piriformis URM
ture was maintaining at 15 ◦ C during the assays. Biodiesel samples 4144 displayed the lowest one achieving only 12.6 ± 0.83 g L−1 .
of 2.0 mL were used, and the measurements were replicated three All mycelia exhibited higher lipolytic activities than values dis-
times. played in the extract broth, suggesting that the lipase produced
by all strains was cell bound. Among the fungal strains, the
3. Results and discussion best lipase producer was P. citrinum URM 4216 exhibiting high
intracellular activity (30.82 ± 0.51 U g−1 ) and low extracellular
3.1. Screening of filamentous fungi with intracellular lipase activity (3.00 ± 0.31 U g−1 ). Similar results were also found for
activity the other strains, with the exceptions for M. piriformis URM 4144
and M. hiemalis URM 4145 which exhibited low intracellular and
Biomass growth followed typical microorganism growth and extracellular lipolyic activities (<4.00 U g−1 ).
for all strains the highest biomass concentration was attained
after 72 h of incubation in a medium containing olive oil at 30 ◦ C. 3.2. Immobilization of fungal strains: BSP selection
Table 1 displays values for biomass concentration together with
extracellular and intracellular lipase activities attained by each To use whole cells as biocatalysts in a convenient form, cells
fungi strain. The highest biomass concentration was obtained by P. should be immobilized in such a way that they resemble ordinary
186 G.S.S. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188
Fig. 2. Micrographs of M. circinelloides 4182 cells surface (a) and cross-section of immobilized biomass on PUF (b).
solid-phase catalysts used conventionally in synthetic chemical
reactions. The five fungi strains with higher intracellular lipase
activities were immobilized in different BSPs in order to select the
carrier that provides the best cell adherence and the highest intra-
cellular lipase activity. To achieve this, three carriers were tested
to perform immobilization either in situ (PUF) or extra situ (PHB
and Celite). Immobilization with PUF was a natural consequence of
the cell growth during cultivation and is the most common proce-
dure to obtain immobilized whole cell. Both PHB and Celite are
cheap matrices which have been already successful applied for
immobilization of several microbial biomass [16]. These supports
were added to the medium after cultivation, providing simple cells
adsorption to the carriers.
Results for intra and extracellular lipase activities are shown in
Fig. 1(a) and (b). Independent of the carrier used, for all strains
values for extracellular lipase activity (Fig. 1(a)) tend to be low
in comparison to intracellular activity, with maximum value of
5.22 ± 0.5 U g−1 in culture broth of R. oryzae URM 4692.
Fig. 1(b) shows a comparison of intracellular lipase activities
attained for all fungi immobilized in the tested carries. The highest
intracellular lipase activity was achieved using PUF for all strains,
as this polymer attached mycelia easily than the other carriers.
Besides, almost all mycelia grow inside and around the PUF parti-
cles, forming a dense and stronger biofilm, which provided a better
substrate transfer and high values of lipase activity. In contrast,
Celite showed low mechanical resistance under strong agitation
and even being added at the end of cultivation, mycelia did not
fully adsorb on this carrier, standing diffused in culture medium.
The same behavior was attained for PHB particles.
Among immobilized cells in PUF (Fig. 1(b)), it is clear the supe-
riority of M. circinelloides URM 4182 as lipase producer in relation
to the other strains, allowing to obtain immobilized cells with
lipolytic activity of 49.94 ± 4.38 U g−1 in 72 h of incubation. Other
immobilized cells had lower intracellular activities attaining values
in the range from 21.08 ± 2.67 to 28.27 ± 1.86 U g−1 . Unsatisfactory
Table 2
Yields and productivities attained in the transesterification reactions of babassu oil with ethanol catalyzed by R. oryzae and M. circinelloides cells immobilized in polyurethane
particles.
Strain Lipase activity (U g−1 ) Total esters (%wt)
R. oryzae 3231 23.64 43.30
R. oryzae 4692 28.27 60.25
M. circinelloides 4140 21.08 13.77
M. circinelloides 4182 49.55 67.62
performance was exhibited by P. citrinum URM 4216, attaining
lipase activity as low as 10.53 ± 0.67 U g−1 . The weak adherence
of P. citrinum URM 4216 strain in PUF could be explained by its
morphology which may limit the pellets adsorption and diffusion
within the polyurethane particles. When P. citrinum URM 4216
strain was cultivated in freely suspended cells, no constraint was
found in the lipase production, however, the addition of PUF par-
ticles in culture medium upset the biomass growth and diffusion
the mycelia within this support, decreasing lipolytic activity of
the immobilized biomass. In contrast, growth morphology of M.
circinelloides and R. oryzae strains is fully entangled filaments that
facilitate the formation of a strong biofilm inside and around the
PUF particles. Fig. 2(a) and (b) shows surface and cross-sectional
micrographs of a M. circinelloides URM 4182 freely suspended and
immobilized cells. Cellular adhesion to the support matrix appears
strong, which means the cells would not be easily released from
the particles even with vigorous agitation of the reaction medium.
These results agree with the results described in the literature
which demonstrated that cells immobilization in reticulated
polyurethane foam is a convenient way to spontaneously obtain
immobilized whole cells [17,18]. Hama et al. [19] performed a com-
parison between intracellular lipase activities in cells grown in a
freely suspended state and cells immobilized within polyurethane
particles. It was observed a similar cell growth profile for both
cultures, but for free cells, both intracellular methanolysis and
hydrolysis activities decreased sharply with increasing cultivation
time, whereas the extracellular hydrolysis activity remained
relatively high throughout. The authors attributed this behavior
to morphology changes in R. oryzae cells after the immobilization
procedure, which strongly inhibited the lipase secretion into the
culture medium. Chen et al. [20] also suggested that differences
in intracellular lipase activities between cells grown in a freely
suspended state and immobilized cells within polyurethane
particles may be due to morphological changes. According to
Adamczack and Bednarski [21], activity of such a preparation is
Yield (%) Productivity (mg g−1 h−1 ) Viscosity 40 ◦ C (cSt)
53.35 3.61 9.94
74.15 5.02 7.03
16.18 1.15 16.55
83.22 5.63 6.63
 G.S.S. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188 187

not uniformly distributed due to the variety of size and shape of 30

the cells obtained in freely suspended culture, and immobilization (a)
can help in achieving lipase with uniform size and activity.
25
Therefore, polyurethane was chosen as the best BSP to immobi-

Ethyl esters (% wt)

lize the selected fungi strain for subsequently used in the babassu
oil ethanolysis. In this set of experiments, P. citrinum URM 4216 20
was not used.
15
3.3. Ethanolysis activity of immobilized whole cells

To perform the ethanolysis activity tests R. oryzae (3231 and 10

4692) and M. circinelloides (4182 and 4140) were immobilized
within PUF, as described in Section 2.4. To avoid reaction reversibil- 5
ity immobilized cells were dried to a level of water content reached
less than 10%. Results are shown in terms of ethyl ester formation
0
as a function of time in Fig. 3. Yields and productivities attained are
0 24 48 72 96 12 0
displayed in Table 2.
Time (h)
The observation of Fig. 3(a)–(c) and Table 2 indicates that all
tested immobilized whole cells were able to form ethyl esters from 30
all fatty acids present in the babassu oil. However, both reaction rate (b)
and yield were dependent on the fungi strain tested. Ethyl esters
concentrations varied in the range from 13.77 to 67.62 wt.%, cor- 25
responding to transesterification yields from 16.18 to 83.22% and

Ethyl esters (% wt)

productivities from 1.15 to 5.63 mg biodiesel g−1 h−1 . 20
The best performance was obtained by M. circinelloides URM
4182 rendering the highest values for transesterification yield
(83.22%) and productivity (5.63 mg of biodiesel g−1 h−1 ), followed 15
by R. oryzae URM 4692 (yield = 74.15%). Slight lower performance
was achieved by R. oryzae URM 3231 (yield = 53.35%). M. circinel- 10
loides URM 4140 gave unsatisfactory results attaining less than 18%
yield (data not shown).
In all reaction systems, a good dispersion of the biocatalyst was 5
visually observed in the substrate during the reaction. This could be
also attributed to the presence of tert-butanol which was used as
0
solvent on the transesterification reactions. This solvent has mod-
0 24 48 72 96 120
erate polarity, allowing the removal accumulated glycerol inside
Time (h)
the polyurethane particles. The accumulated glycerol has a neg-
ative influence at immobilized cell stability, due to mass transfer
limitation. These results were similar to those already published, (c) 30

although the majority of the works used methanol as an acyl donor

which is believed to play a role as a potential inhibitor of the 25
Ethyl esters (% wt)

biomass activity.
In order to confirm the GC results, viscosity analysis was per- 20
formed in the purified biodiesel samples. Viscosity can be used as
a control of transesterification reaction, confirming the formation
of esters from vegetable oils, through the viscosity reduction of 15
the feedstock. Results from Table 2 showed the viscosity measured
for all purified products had a substantially reduction in compar-
10
ison with babassu oil (29.51 cSt). The lowest viscosities (6.63 and
7.32 cSt) were obtained employing M. circinelloides URM 4182 and
R. oryzae URM 4692 as biocatalysts, in agreement with the attained 5
transesterification yields. Similar behavior was found for the reac-
tions catalyze by R. oryzae URM 3231 and M. circinelloides URM
0
4140 immobilized cells which the low ester yields were confirmed
0 24 48 72 96 120
by the high viscosity values (9.94 cSt and 16.55 cSt, respectively). As
the viscosity values obtained are not yet in the range established
Time (h)
by the ASTM D5761 (3.0–6.0 cSt) further optimization tests is still Fig. 3. Ethyl esters formation in the batch ethanolysis reaction of babassu oil cat-
required in order to adjust parameters such as oil to ethanol molar alyzed by immobilized cells: (a) R. oryzae 3231; (b) R. oryzae 4962; (c) M. circinelloides
ratio, biocatalyst amount and water level. 4182. Reaction conditions: 35 ◦ C, 170 rpm (shaker), oil-to-ethanol molar ratio of 1:6,
20% immobilized cells (w/w, based on reaction medium weight). Symbols: : C8;
: C10, 䊉: C12, : C14, : C16, : C18, : C18:1, and : C18:2.
3.4. Operational stability of whole cells in continuous packed bed
reactor (PBR)

Catalytic activities of whole immobilized cells may be decreased

during the repeated batch uses due to the cell exfoliation caused
by vigorous agitation in shaker and other critical parameters that
188 G.S.S. Andrade et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 183–188

70 immobilized cells. These results reveal a good operational stabil-

ity for this biocatalyst, extending its usability in the continuous
operation system.
60
4. Conclusions
Ethyl esters (w/w %)

50
The whole cells from R. oryzae URM 4692 and M. circinelloides
URM 4182 immobilized in polyurethane particles express high
40 transesterification activity in the ethanolysis of babassu oil. Sim-
0,0 ilar ester yields were obtained by both fungi strains which are
favorable compare with results already reported in the literature,
30 -0,1
particularly for M. circinelloides that no data for biodiesel synthe-
ln (A /A)

-0,2
sis is still available. In addition, the high operational stability of
20 the whole cells biocatalyst is rather promising for the applica-
-0,3 K = 0.02666 (day ) tion of lipase-catalyzed transesterification in a fixed bed reactor.
R = 0.98546
Work on this subject is in progress in order to optimize the reac-
10 -0,4
tion parameters to obtain biodiesel samples in accordance with
2 4 6 8 10 12 14
Time (days)
specifications recommended by the ASTM D6751 to be used as
0 biofuel.
0 2 4 6 8 10 12 14
Acknowledgments
Time (days)
The authors gratefully acknowledge FAPESP, CAPES and CNPq
Fig. 4. Continuous ethanolysis tests in PBR running on babassu oil catalyzed by for financial support.
immobilized cells. Reaction conditions: 35 ◦ C, 0.13 mL min−1 of flow rate, oil-to-
ethanol molar ratio of 1:6, 27 g of immobilized cells (deactivation model adjustment
is shown in the inserted box). References

should be taken into consideration following the treatment (e.g.
rinsing) of the biocatalyst after recovery [22]. Alternatively, the bio-
catalyst stability can be assessed in a continuous reactor. In this
work, a packed bed reactor (PBR) operated at an appropriate flow
rate was used to determine the operational stability of the biocata-
lyst (M. circinelloides URM 4182 immobilized cells) which showed
the best performance on the transesterification reaction.
The PBR operated continuously for 14 days at 35 ◦ C for the
ethanolysis reaction of babassu oil under the conditions described
in Section 2 and during the first 6 days, no significant decrease in
the initial activity was observed. The ethyl esters concentrations
varied between 55.0 ± 0.7 and 52.1 ± 0.6 wt%, corresponding to an
average reduction of about 5.3% of the original transesterification
activity (Fig. 4). Subsequently, a progressive decrease in the activ-
ity was detected, and a reduction of 22.7% in the transesterification
activity was obtained after 14 days of operation.
The experimental results showed in Fig. 4 were normalized fol-
lowing Arrhenius equation (Eq. (2)) and fitted to a deactivation
model of first order (box inserted in Fig. 4):
A 259–263.
− ln = Kd ∗ t
Ao
Half-life of the biocatalyst (t1/2 ) was estimated by Eq. (3), in
which t1/2 is the half-life, and Kd is the deactivation coefficient:
0.693
t1/2 =
Kd
A half-life of 26 days and a deactivation coefficient of
2.67 × 10−2 day−1 were estimated for M. circinelloides URM 4182
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