Fuel 135 (2014) 315–321

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Fuel
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Biodiesel production from Acrocomia aculeata acid oil by

(enzyme/enzyme) hydroesterification process: Use of
vegetable lipase and fermented solid as low-cost biocatalysts
Erika C.G. Aguieiras a,⇑, Elisa D. Cavalcanti-Oliveira a, Aline M. de Castro b, Marta A.P. Langone c,
Denise M.G. Freire a,⇑
a
Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, CEP 21949-909, Rio de Janeiro, RJ, Brazil
b
PETROBRAS/CENPES/PDEDS/Biotecnologia, Ilha do Fundão, Av. Horácio Macedo, 950, CEP 21941-915, Rio de Janeiro, RJ, Brazil
c
Departamento de Química Analítica, Instituto de Química, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier, 524, PHLC, sl. 310, CEP 20550-013,
Rio de Janeiro, RJ, Brazil

h i g h l i g h t s

 Biodiesel production by hydroesterification of an acid oil using low-cost lipases.

 Hydrolysis step, catalyzed by a vegetable lipase, produced 99.6% of FFAs after 6 h.
 Dry fermented solids were used as biocatalysts of the esterification step.
 Conversion of 91% of FFAs into ethyl esters was attained in 8 h.
 Biodiesel produced met important Brazilian standards related to biodiesel quality.

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

Article history: The aim of this study was to investigate a new process of enzyme/enzyme hydroesterification for biodie-
Received 30 April 2014 sel production using a low-cost acid oil (10.5 wt.% acidity) from macauba (Acrocomia aculeata) pulp as
Received in revised form 25 June 2014 raw material. The ethyl esters were produced by the hydrolysis of the macauba oil using vegetable
Accepted 27 June 2014
enzyme (VE) obtained from dormant castor seeds followed by esterification of the released free fatty
Available online 15 July 2014
acids (FFAs) with ethanol catalyzed by fermented and dry babassu cake with lipase activity from Rhizo-
mucor miehei. The vegetable enzyme-catalyzed hydrolysis produced 99.6% of FFAs after 6 h in a medium
Keywords:
with high oil concentration (50% v/v) and without organic solvent and emulsifier. For the esterification
Biodiesel
Hydroesterification
reaction, the best result was attained with an ethanol:FFA molar ratio of 2:1 and 15.1 U of dry fermented
Lipase solid per g of FFAs at 40 °C, which yielded 91% of conversion after 8 h in a solvent-free system. In order to
Solid-state fermentation confirm the potential of the fermented solid as biocatalyst, it was confronted with the best commercial
Acrocomia aculeata lipases and was also evaluated for its reuse. Similar conversions were obtained with the commercial
lipases Novozym 435 and Lipozyme RM IM and the fermented solid. The fermented solid was reused
in successive 6-h batches for esterification reactions and conversions of over 60% were maintained for
eight cycles. After two consecutive esterification reactions the resulting biodiesel met important Brazilian
standards such as: density (ASTM D4052), viscosity kinematic (ASTM D445), flash point (ASTM D93), car-
bon residue (ASTM D4530), free glycerol and total glycerol, monoglycerides and triglycerides (ASTM
D6584). The ester content was of 96.7% (esters of fatty acids of 8–18 carbons). To the best of our knowl-
edge, this is the first time that an enzyme/enzyme hydroesterification process using low cost biocatalysts
obtained from vegetable and microorganism using solvent-free media in both reactions is described for
the conversion of an acid and low value oil into biodiesel.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
⇑ Corresponding authors. Address: Federal University of Rio de Janeiro, Chemistry
Institute, Biochemical Department, Brazil. Tel.: +55 21 3938 73 60; fax: +55 21 3938
In recent years, the production of renewable fuels such as bio-
7266.
diesel has become a priority in energy policy strategies at a global
E-mail addresses: erikaaguieiras@gmail.com (E.C.G. Aguieiras), freire@iq.ufrj.br
(D.M.G. Freire). scale. Biodiesel is composed of monoalkyl esters produced from

http://dx.doi.org/10.1016/j.fuel.2014.06.069
0016-2361/Ó 2014 Elsevier Ltd. All rights reserved.
316 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321
vegetable oils or animal fats, has similar properties to petro-diesel
and its burning results in lower emissions of particulates, CO, SOx
and aromatic hydrocarbons [1–3].
Current industrial processes for biodiesel production, that uses
homogeneous alkaline transesterification of edible oils, give high
yield (about 98%) in short reaction time (about 1 h). However, this
process have some drawbacks: difficulty of separating the catalyst
from the glycerol, production of highly alkaline wastewater and
requirement of high-quality/high-price raw materials with low
contents of free fatty acids (FFAs) (acidity less than 0.5%) and water
in order to avoid soap formation. These refined oils are relatively
expensive and their prices correspond to 70–80% of the total bio-
diesel production costs, making it difficult for biodiesel to econom-
ically compete with petro-diesel [3]. Acid catalysis is an alternative
that have not the problems caused by the acidity of the oil since
the acid can simultaneously catalyze esterification and transesteri-
fication. However, despite its insensitivity to FFAs in the feedstock,
the acid-catalyzed transesterification has relatively slower reaction
rate and poor reaction selectivity, is highly energy consuming and
fluids are difficult to handle, causing problems of corrosion in
equipments and producing waste acid, with serious environmental
impact [4,5].
In order to increase the commercial competitiveness of biodie-
sel, to ensure the availability of oil and to improve the use of the
regional resources, it is expected that alternative vegetable
non-edible oil crops will increase their participation as feedstock
for biodiesel production [2,6]. Macauba (Acrocomia aculeata) is a
native oleaginous palm tree of the Brazilian Cerrado and an exam-
ple of candidate crop that can be used as an alternative feedstock
to regional industries in Brazil [1,7]. This palm presents high pro-
ductivity with a potential to produce 4000 t of oil ha 1 and the
oil extracted from macauba pulp is rich in oleic acid, which can
generate a high quality biodiesel with high content of monounsat-
urated esters [1,8]. However, this oil has high acidity and cannot be
used as feedstock for biodiesel production by conventional alkaline
route. In this case, biodiesel synthesis can be carried out by
hydroesterification process, which enables to employ low-quality
raw materials with high concentrations of FFAs that have lower
market price.
Biodiesel production by the hydroesterification route occurs in
two consecutive steps. The first is the hydrolysis of all glycerides
(mono-, di- and triglycerides) that produces FFAs and glycerol
and the second one is the esterification of the FFAs with a short-
chain alcohol to obtain esters (biodiesel) and water. The use of
lipases as catalysts at one or both steps of hydroesterification
brings the advantages of high selectivity, high specificity, mild
operating conditions and high purity of the generate products
(glycerol and esters) [3]. In previous works, our group has reported
the hybrid (enzyme/chemical) hydroesterification process for bio-
diesel production with the hydrolysis reaction catalyzed by lipase
and niobic acid in pellets as catalyst in esterification step [9–11].
Talukder et al. [12] have also studied the hybrid (enzyme/chemi-
cal) hydroesterification process and recently Soares et al. [13]
reported a hybrid (chemical/enzyme) hydroesterification with
the hydrolysis in subcritical water and lipase as catalyst in esteri-
fication step. The (enzyme/enzyme) hydroesterification process,
using commercial lipases in both steps, was studied by Watanabe
et al. [14] and Talukder et al. [15]. However, the present work
stands out for using low-cost lipases to catalyze both steps of the
hydroesterification process.
The aim of this study was to produce biodiesel by enzymatic
hydrolysis followed by enzymatic esterification of the acid oil
from macauba pulp. The hydrolysis reaction was catalyzed by
VE from dormant castor seeds and esterification reaction was
catalyzed by lipase from Rhizomucor miehei in the form of dry
fermented solid (obtained by solid-state fermentation – SSF). In
fermented solids the particles of the solid medium are as sup-
port that keeps the lipases adsorbed on its structure. Thus, the
use of dry fermented solids as catalysts avoids the expensive
steps of lipase extraction, purification and immobilization. More-
over, in SSF are used solid wastes from the production of vege-
table oils (oil cakes) that are inexpensive and plentiful in
countries like Brazil. This new approach is an alternative method
to lower enzyme costs and consequently make the biodiesel
obtained by enzymatic route more cost competitive. Some oper-
ational parameters of the esterification step were evaluated such
as the way of ethanol addition (stepwise or continuous). A com-
parison of the dry fermented solid with the commercial lipases
Novozym 435 and Lipozyme RM IM was performed as well as
the reusability of the dry solid in successive batch reactions.
The biodiesel produced was analyzed according to the standard
methodologies published by ASTM (American Society for Testing
and Materials) and ABNT (Associação Brasileira de Normas Técn-
icas – Brazilian Technical Standards Association).
2. Materials and methods
2.1. Raw material and reagents
The acid oil from macauba (A. aculeata) pulp was obtained from
the fruits processing company PETROVASF (Montes Claros, Brazil).
The water content in the oil was 929 ± 7.12 mg kg 1 and the acidity
was 10.5 wt.%. The macauba oil presented the follow composition
in fatty acids (wt.%): 53.7% of oleic acid (C18:1), 19.1% of palmitic
acid (C16:0), 18.8% of linoleic acid (C18:2), 4.1% of palmitoleic acid
(C16:1), 1.3% of stearic acid (C18:0) and 1.3% of linolenic acid
(C18:3n3). Furthermore, the macauba oil also contained medium
chain fatty acids such as 0.1% of capric acid (C10:0), 1.2% of lauric
acid (C12:0) and 0.4% of miristic acid (C14:0). This indicates that
there was a contamination with kernel oil during the oil extraction
step. The average molar mass of macauba oil fatty acids, calculated
from the oil composition, was 274.6 g mol 1. The castor bean seeds
were supplied by Embrapa Cotton Research Center (Campina
Grande, Brazil) and stored at 4 °C until use. The babassu cake, a
solid residue from the babassu oil industry, was kindly provided
by Tobasa S.A. (Tocantinópolis, Brazil). All other reagents were of
analytical grade.
2.2. Biocatalysts
The commercial immobilized lipases Lipozyme RM IM (lipase
from R. miehei) and Novozym 435 (lipase B from Candida
antarctica) were provided by Sigma Aldrich (Brazil). The enzyme
extract from dormant castor bean seeds (Ricinus communis L.)
was produced by triturating and washing the dormant castor seeds
in acetone, according to the methodology described by Cavalcanti
et al. [16]. The acetone powder obtained was stored at 4 °C until
use and named as vegetable enzyme (VE). The fermented solid
was obtained by SSF of babassu cake, an agroindustrial byproduct
selected due to high lipase production previously reported by our
group [17,18], using the mesophilic strain of R. miehei (IDAC acces-
sion number 071113-01). Fermentations were carried out in tray-
type bioreactors containing 15 g of babassu cake (particles below
1.18 mm) moistened to 65 wt.% and sterilized. The babassu cake
was inoculated with 107 spores g 1 and cultivated at 30 °C under
humidified air injection. Whole trays were taken every 24 h for
up to 120 h to determine the profile of lipase production. After
incubation, the fermented solids were dried in a lyophilizer until
a moisture content of less than 3 wt.%, stored at 4 °C until use
and named dry fermented solid.
 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321
2.3. Enzyme activity determination
Hydrolytic activity of the dry fermented solids was measured
using p-nitrophenyllaurate (pNP-laurate) as substrate according
to Gutarra et al. [18]. After fermentation and lyophilization the
enzymes were extracted with phosphate buffer (0.1 mol L 1, pH
7.0) as described by Gombert et al. [19]. The supernatant was
used for hydrolytic activity determination. One unit (U) of
hydrolytic activity was defined as the amount of enzyme that
releases 1 lmole of p-nitrophenol per minute under the assay
conditions.
Lipase activity of the dry fermented solid (0.2 g) was deter-
mined by titrimetric method using olive oil 5% (w/v) as substrate,
according to Gutarra et al. [18]. The reaction time was 20 min.
Lipase activity of VE was determined using tributyrin 5% (w/v) as
substrate, according to Cavalcanti et al. [16]. Lipase activity was
determined by titration of the FFAs released by enzyme action,
according to Freire et al. [20]. One unit (U) of lipase activity was
defined as the enzyme amount that releases 1 lmole of fatty acids
per minute under the assay conditions.
Esterification activity was measured by the consumption of
oleic acid in the esterification reactions with ethanol (oleic
acid:ethanol molar ratio of 1) with an enzyme amount of 3 wt.%
for the commercial lipases or 8.6 wt.% for the dry fermented solid
at 40 °C. The reactions were carried out in closed 20 mL batch
reactors magnetically stirred and thermostated. At fixed intervals,
triplicates samples of 100 lL were removed from the medium,
dissolved at acetone/ethanol 1:1 (40 mL) and residual FFAs were
analyzed by titration with NaOH 0.04 mol L 1. One esterification
unit (U) was defined as the enzyme amount that consumes
1 lmole of fatty acid per minute under the experimental condi-
tions [21]. Hydrolytic, lipase and esterification activities were
expressed as units per gram of solid (U g 1).
The esterification capacity of the fermented solid was also
determined measuring the consumption of oleic acid after 8 h
under the same experimental conditions described above. The oleic
acid conversion was determined by titration of the remaining FFAs
and was defined as the number of moles of fatty acids consumed
per mole of fatty acids fed to the system.
2.4. Enzymatic hydrolysis of acid oil from macauba pulp
The hydrolysis reactions to produce the FFAs were carried out in
1 L thermostated reactors under mechanical stirring during 24 h at
30 °C. The reaction medium was composed of macauba oil (50% v/
v), 0.1 mol L 1 sodium acetate buffer pH 4.0 (50% v/v) and 2.5% (w/
v) of VE (7.8 U per g of oil) as biocatalyst. At the end of the reaction,
the FFAs were extracted with hexane according to the methodol-
ogy described by Sousa et al. [10]. The percentage of FFAs (w/w)
produced, expressed as oleic acid, was determined measuring the
acidity of the oil [10].
2.5. Enzymatic esterification of free fatty acids
Esterification reactions were carried out in closed 20 mL batch
reactors magnetically stirred and thermostated. The medium was
solvent-free and composed of substrates (macauba oil hydrolyzate
and ethanol) and enzyme (detailed at Section 3). Reaction progress
was monitored by taking samples at fixed intervals that were
analyzed for residual FFAs content and ethyl ester content. As an
alternative, it was investigated the continuous feeding of ethanol
using a peristaltic pump (Minipuls EVOLUTION GILSON) at 54,
27.5 and 13.5 lL min 1 flow rates. The reactions were carried out
with molar ratio ethanol:FFAs of 1:1, 15.1 U of dry fermented solid
per g of FFAs at 40 °C during 6 h.
317
2.6. Reuse of the fermented solid
For the study of the dry fermented solid reuse, the esterification
batch reactions were repeated for ten cycles, each one of 6 h. The
first batch was carried out with 11.7 g of mixture (molar ratio eth-
anol:FFAs of 1:1) and 15.5 U of dry fermented solid per g of FFAs at
40 °C. Ethanol was added at 0, 1 and 2 h. After the cycle, the fer-
mented solid was washed with 80 mL of ethanol, vacuum filtered
and placed in a desiccator for 24 h. The reaction product was con-
centrated in a rotary evaporator for conversion and ester yield
determination while the fermented solid was reused in a new
batch.
2.7. Fatty acid ethyl esters content determination
The fatty acid ethyl esters (FAEEs) content was determined on a
GC-2010 (Shimadzu Co.) equipped with a flame ionization detector
(FID) and an Omegawax capillary column (30 m  0.25 mm 
0.25 lL). The detector and injector were set at 250 °C and 260 °C,
respectively. The oven program was as follows: 200 °C for 5 min,
then heated up at 20 °C min 1 to 260 °C and kept constant at
260 °C for 6 min. Helium was used as carrier gas at a 2 mL min 1
flow rate. Samples of 20 lL were diluted in 480 lL of an internal
standard solution of methyl heptadecanoate (9 mg mL 1) in
heptane and 1 lL was injected with a split ratio of 1:20. The ester
content was quantified using the peak area of the internal
standard.
2.8. Biodiesel production and characterization
For biodiesel characterization, firstly, 1.2 L of macauba oil was
hydrolyzed as described in Section 2.4. Esterification reactions of
FFAs were carried out in two steps in 1 L closed and thermo-
stated reactors under mechanical stirring. The first reaction
was carried out for 48 h and the medium was composed of eth-
anol and FFAs in a molar ratio of 2:1 in order to displace the
reaction equilibrium towards ester formation and 17.4 U of dry
fermented solid per g of macauba oil hydrolyzate. One sixth of
the total ethanol volume was added at times 0, 1 and 2 h and
1/2 was added at 4 h. At the end of the reaction, the product
was extracted with hexane, filtered in filter paper, dried over
anhydrous sodium sulfate, concentrated in a rotary evaporator
and heated at 110 °C to evaporate any traces of solvent and
water. The product (acidity equal to 6.45%) was then used in a
second esterification reaction, in order to convert the residual
FFAs. The conditions of this reaction were: ethanol and FFAs
molar ratio of 10:1, with 2/5 of the total ethanol volume added
at 0 h and 1/5 added at 1, 2 and 4 h, and 17.4 U of dry fer-
mented solid per g of biodiesel (6.45% of acidity). After 24 h,
the product was extracted with hexane and was subjected to
the same steps of the first reaction. At fixed intervals, acidity,
water content and ethyl ester yield were determined. Other
analyses were: specific mass at 20 °C, flashpoint, kinematic vis-
cosity at 40 °C, carbon residues, water content, free glycerol,
total glycerol, monoglycerides, diglycerides, triglycerides and
methanol or ethanol contents, and stability to oxidation at
110 °C. These analyses were kindly performed at Laboratory of
Green Technologies (GREENTEC) located in the Federal University
of Rio de Janeiro that is a laboratory licensed by ANP (National
Agency of Petroleum, Natural Gas and Biofuels). The biodiesel
analyses were carried out according to the standard methodolo-
gies published by ASTM and ABNT. The results were compared
with the values established by ANP Resolution N°14 of 11/05/
2012 which establishes the specifications necessary for the sale
of biodiesel in Brazil.
318 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321
3. Results and discussion
3.1. Enzymatic hydrolysis of acid oil from macauba pulp
Hydrolysis reactions of acid oil from macauba (10.5 wt.% acid-
ity) were carried out using vegetable enzyme (VE) extract from
dormant castor seeds as biocatalyst (7.8 U per g of oil). This acid
lipase was chosen due to its hydrolytic potential earlier recognized
by other authors [16,22], although they have not used this enzyme
to catalyze reactions in a large volume and with high oil concentra-
tion. After 1 h of reaction, it was obtained 76.7% of FFAs (productiv-
ity of 384 g/L h of FFAs) and percentages of 99.6% and 99.9% of FFAs
were attained in 6 and 24 h, respectively. Although several studies
have been reported the use of lipases from seeds and microorgan-
isms in hydrolysis reactions of different oils, many authors use
organic solvent in the reaction medium to dissolve the oil, which
requires a step of separation and recovery at the end of the reaction
[15,23]. In some cases, emulsifiers were used to increase the inter-
facial area and the rate of hydrolysis [24,25] or the oil content used
was very low (5% m/v) [26]. A high oil content (50%) and medium
without solvent or emulsifier was used by Talukder et al. [12] and
Cavalcanti-Oliveira et al. [9] to hydrolyze waste cooking oil using
Candida rugosa lipase and soybean oil using Thermomyces lanugino-
sus lipase (TL 100L), respectively. Sousa et al. [10] presented inter-
esting results with lipases from vegetable seeds. The vegetable
enzyme extract from germinated physic nut seeds (Jatropha curcas)
was able to hydrolyze different raw materials used for biodiesel
synthesis. Complete hydrolysis of physic nut oil was achieved with
50% (v/v) oil in Tris–HCl buffer and 2.5% (m/v) crude extract con-
taining lipase, without adding organic solvent or emulsifier.
The results of this study show the potential application of the
VE of castor seeds to hydroesterification processes for biodiesel
production. Plant lipases are interesting due to its low cost, ease
of purification, wide availability from natural sources and excellent
hydrolytic activity [27]. Moreover, hydrolysis reaction was carried
out with high oil concentration (50% v/v), low concentration of a
non-commercial lipase and without organic solvent and emulsifier.
These factors are very important for the feasibility of a process for
production of a low value product as biodiesel.
3.2. Production of fermented solid
To catalyze the esterification reactions, it was chosen the fer-
mented solid with lipase activity produced by SSF over babassu
cake without supplementation. To evaluate the profile of lipase
production, the lipase activity and the conversion of oleic acid into
ethyl esters in the fermentation times of 24, 48, 72, 96 and 120 h
were determined. Lipase activity enhanced until 72 h of fermenta-
tion and then stabilized showing similar values for 72, 96 and
120 h (Supplementary data (Fig. S1)). At 72 h the lipase activity
was 30.4 ± 3.2 U g 1. It was observed the same behavior for the
oleic acid conversion. At 72 h of fermentation the conversion
obtained after 8 h of esterification reaction was 31 ± 1.4%. The fer-
mentation time of 72 h was chosen for the production of fermented
solid that would be used in later studies.
For industrial use, a catalyst must have characteristics such as
manufacturing batch reproducibility and storage stability. We
monitor the fermented solid production for six independent fer-
mentations (carried out in different months) and the results
showed a low variability (10%) for the hydrolytic activity, with
an average of 41.1 ± 4.1 U g 1 (obtained after 72 h fermentation).
The hydrolytic activity of the fermented solid was also measured
before and after lyophilization process and it did not change
(42 U g 1). The storage stability of the dry fermented solid was
evaluated by measuring the lipase activity every month during
one year of storage at freezer ( 20 °C), refrigerator (4 °C) and at
room temperature (25 °C). The biocatalyst showed 100% of its ini-
tial activity during 9 months in freezer, refrigerator or at room
temperature. These results showed that the dry fermented solid
is highly stable and can be stored for a long period.
3.3. Optimization of enzymatic esterification of free fatty acids
The dry fermented solid obtained by SSF in babassu cake and
containing lipase from R. miehei was used as catalyst in the
esterification reaction of the FFAs from macauba oil hydrolyzate.
Ethanol was selected as acyl acceptor because it is less toxic than
methanol and due to its high production in Brazil from renewable
resources [3]. The reaction medium was solvent-free which has
several advantages such as increase of volumetric capacity and
reduction of environmental issues (toxicity of the organic solvent)
and processing costs (recovery and losses). Initially it was observed
that it would be better to use ethanol in excess in order to displace
the reaction equilibrium towards ester formation and add ethanol
in steps to avoid lipase deactivation due to a high initial alcohol
concentration. It was evaluated the reaction conducted with etha-
nol and FFAs molar ratio of 2:1, with the alcohol added in equal
parts at times 0, 1, 4, 24, 25 and 28 h, and with 4.25 U (hydrolytic
activity) of dry fermented solid per g of FFAs at 40 °C, during 72 h.
Conversions of 75.3%, 91.3% and 92.2% were attained after 24, 48
and 72 h, respectively. Blank reactions were carried out using
babassu cake or autoclavated fermented solid and the conversions
were lower than 3% after 96 h. To improve the conversion, the con-
centration of fermented solids was increased from 4.25 to 15.1 U
per g of FFAs. It was observed that the rate of production of ethyl
esters increased significantly and conversions of FFAs above 85%
were attained in 6 h. The addition of 15.1 U of hydrolytic activity
per g of FFAs was used for the following studies.
In order to improve the reaction rates and reduce the reaction
times, it was investigated the stepwise ethanol addition in shorter
time intervals as well the continuous feeding of ethanol. The reac-
tions were carried out with a molar ratio ethanol:FFAs of 1:1. In the
continuous feeding, 1/3 of the ethanol volume was added at the
beginning of the reaction (0 h) and, after that, the remaining 2/3
was continuously fed by the pump. The results were compared
to those obtained with stepwise ethanol addition in equal parts
at times 0, 1 and 2 h or 0, 1 and 4 h. It can be seen at Fig. 1 that,
with the exception of the condition in which ethanol was added
at times 0, 1 and 4 h, all other conditions studied showed similar
profiles of fatty acids conversion. When ethanol was added at 0,
1 and 4 h it can be seen a large reduction in the rate of reaction
Fig. 1. Kinetic of esterification reaction of FFAs from macauba oil hydrolyzate with
stepwise or continuous ethanol addition. The reaction was conducted with molar
ratio ethanol:acid = 1:1 and 15.1 U of dry fermented solid per g of FFAs at 40 °C.
 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321
Fig. 2. Kinetic of esterification reactions of FFAs from macauba oil hydrolyzate with
ethanol catalyzed by lyophilized fermented solid, Novozym 435 or Lipozyme RM
IM. Ethanol was added at 0, 1 and 2 h (R ethanol:acid = 1:1) using 14 U of
esterification activity per g of FFAs at 40 °C.
at 2 h, probably due to a lack of substrate for the enzyme. The con-
version rate increased only after the addition of the third aliquot of
ethanol (at 4 h). This results show that the stepwise ethanol addi-
tion strategy has an important effect on the time course of the
reaction. After 6 h the conversions were similar for all conditions
studied.
In view of this result, the stepwise ethanol addition in shorter
time intervals (0, 1 and 2 h) was chosen for later studies. As shown
in Fig. 1, the conversion was high (>80%) after 4 h and the reaction
rate has decreased due to ethanol exhaustion. In order to try to
increase the reaction rate it was evaluated the addition of excess
of ethanol (final molar ratio ethanol:acid = 2:1) employing two
feed conditions: (i) 1/6 of the total ethanol volume was added at
0, 1, 2, 4, 5 and 6 h; or (ii) 1/6 of the total ethanol volume was
added at 0, 1 and 2 h and 1/2 was added at 4 h. The conversions
attained after 6 h were similar to that obtained with a final molar
ratio of 1:1 and conversion of 91% was achieved after 8 h for the
two conditions studied.
3.4. Evaluation of the fermented solid efficiency
3.4.1. Comparison of the fermented solid with commercial lipases
The biodiesel synthesis catalyzed by the dry fermented solid
was compared with those reactions carried out with the commer-
cial immobilized lipases Novozym 435 and Lipozyme RM IM. As
shown in Fig. 2, the kinetic of the reaction catalyzed by the fer-
mented solid was similar to those obtained with commercial
lipases. After 6 h the reactions catalyzed by the three biocatalysts
reached the same conversion (83%). At 1 h, the productivities were
2.1, 2.7 and 2.8 g of FFAs consumed h 1, for Novozym 435,
Lipozyme RM IM and fermented solid, respectively. These results
show that the fermented solid is a very promising biocatalyst for
biodiesel synthesis.
Despite of the fact that previous works had recognized the
potential of using lipolytic fermented solid as biocatalyst [13,28–
31] our results stand out from the others due to the shorter reac-
tion times. Fernandes et al. [28] obtained an ester yield of 94% after
18 h in esterification reactions of oleic acid with ethanol catalyzed
by lyophilized fermented solids containing Burkholderia cepacia
lipase in an organic reaction media. In another work, Salum et al.
[29] studied the ethanolysis of soybean oil using the fermented
solid in packed-bed reactor and obtained 100% of conversion after
96 h. Conversion of 92% was attained after 31 h by Soares et al. [13]
using fermented solids as biocatalyst to convert the FFAs into ethyl
esters. Liu et al. [30] used fermented solid with lipase from
319
Burkholderia cenocepacia to catalyze the ethanolysis of soybean
oil in a medium with t-butanol and obtained ester yield of 86% in
96 h. In a solvent-free system, conversion was only 14% after 96 h.
Typically, the biocatalytic route for esterification and transeste-
rification has been carried out using commercial immobilized
lipases that are easily recovered and reused. However, this
approach is expensive when compared with in situ produced bio-
catalysts used in this work, because immobilized commercial
lipases require steps of lipase extraction, concentration, purifica-
tion and immobilization [28]. The present work demonstrates, for
the first time, a hydroesterification process in which an enzymatic
hydrolysis of an acid oil (macauba acid oil) is followed by an enzy-
matic esterification step, both carried out using low-cost enzymes
obtained from plant and by SSF.
3.4.2. Operational stability of the fermented solid in batch reactor
One of the main drawbacks for enzymatic production of biodie-
sel is the high cost of the lipases. In this context, enzyme lifetime is
an important key to make the production of a commodity like bio-
diesel possible by enzymatic route [6]. The longer an enzyme can
be reused the lower is its contribution to the overall price of the
product. Therefore, the reuse of fermented solids was investigated
in this work in successive esterification batch reactions of 6 h.
Ethanol was chosen as washing solvent in view of the possibility
of its use as substrate for the reaction after enzyme recovery.
Fig. 3 shows the fatty acid conversion and the ester yield attained
after each cycle. It can be seen an increase of about 10% in conver-
sion and ester yield in the second reaction batch. This increase can
be explained due to the contact of the fermented solid with the
esters and fatty acids in the first reaction, which may have caused
an effect of pretreatment of the biocatalyst. This effect would result
in a better diffusion of substrates as previously suggested by other
authors [32–35]. According to Samukawa et al. [33], the penetra-
tion of water into the support matrix is suppressed due to the
impregnation of the carrier with ester or oil, which results in lower
water content and higher penetration of the substrate into the car-
rier matrix during the reaction. The fermented solid remained
active after ten cycles and the values of conversion and ester yield
remained above 95% of the initial values during eight cycles. In
addition, both ethyl ester yield and fatty acids conversion had
similar values indicating that consumption of fatty acids was really
for the production of FAEE. Hydrolytic activity of the fermented
solid was measured after ten cycles and the value obtained was
39.7 ± 7.6 U g 1 (94.5% of its initial activity). These results show
that the fermented solid is highly stable and acts as a support that
keeps the lipase adsorbed on its structure.
Fig. 3. Effects of the lyophilized fermented solid reuse on fatty acids conversion and
ester yield. Each reaction was conducted for 6 h, using macauba oil hydrolyzate and
ethanol as substrates (ethanol added at 0, 1 and 2 h (R ethanol:acid = 1:1)) and
15.5 U of dry fermented solid per g of FFAs at 40 °C.
320 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321

Table 1
Analysis of biodiesel obtained by enzymatic hydroesterification of the macauba acid oil.

Resolution ANP N°14 (11/05/2012)

Properties Unity Result Min. Max. Method
Density 20 °C kg (m3) 1 872.2 850 900 ASTM D4052
Water content, max. mg kg 1 219 a
ASTM D6304
Viscosity kinematic 40 °C mm2 s 1 5.01 3.0 6.0 ASTM D445
Flash point, min. °C 151 100 ASTM D93
Carbon residue, max. wt.% 0.039 0.05 ASTM D4530
Oxidation stability 110 °C h 0.95 6 – EN 14112
Ester content wt.% 95.9b 96.5 – EN 14103
Methanol or ethanol, max. wt.% 0.32 – 0.2 EN 14110
Free glycerol, max. wt.% 0.0058 – 0.20 ASTM D6584
Total glycerol, max. wt.% 0.1024 – 0.25 ASTM D6584
Monoglycerides, max. wt.% 0.1516 – 0.80 ASTM D6584
Diglycerides, max. wt.% 0.3841 – 0.20 ASTM D6584
Triglycerides, max. wt.% 0.0022 – 0.20 ASTM D6584
a
The limit of 380 mg kg 1 will be accepted 60 days after the Resolution publication. From January 1st to December 31, 2013 will be allowed a maximum of 350 mg kg 1
and from January 1st, 2014, the maximum limit will be of 200 mg kg 1.
b
Considering only the ethyl esters of fatty acids with more than 12 carbons in the chain. If it were considered the esters of fatty acids of 8–12 carbons this value is 96.7% of
esters.

Liu et al. [30] reported the reusability of the B. cenocepacia fer-
mented solids in ethanolysis of soybean oil over 288 h, with 66.9%
of its original activity. Fermented solid was reused in packed-bed
reactor, and conversions of over 84% were attained for six cycles
in esterification reactions of 48 h [13]. According to Soares et al.
[13] the higher operational stability of the fermented solids for
esterification reaction than that observed for transesterification
one is due to the absorption of glycerol, produced only in transe-
sterification reactions, onto the biocatalyst that causes mass trans-
fer limitations. This problem is avoided in hydroesterification
process.
3.5. Biodiesel production and characterization
For the biodiesel production and characterization, the FFAs pro-
duced by hydrolysis of macauba oil with castor been lipase were
used in two consecutive esterification reactions with ethanol and
catalyzed by the R. miehei fermented solid. After the first esterifica-
tion reaction (48 h), the product showed 93.6% conversion, an ester
yield of 89.7% and an acidity of 6.45% (Supplementary data
(Fig. S2)). High amount of FFAs can lead to corrosion in engine,
which in turn reduces engine efficiency. So, the product of the first
step (acidity equal to 6.45%) was used as reagent for a second
esterification reaction, in order to consume the residual FFAs and
reduce its acidity. After 24 h the acidity was reduced to 1.52%, with
a water content of about 1600 mg kg 1 (Supplementary data
(Fig. S3)). Although the product acidity had been reduced from
6.45% to 1.52%, it is still higher than allowed by the resolution
ANP N°14 (0.25% or 0.50 mg KOH g 1). Chemical neutralization
using sodium carbonate was evaluated to reduce the biodiesel
acidity, but it was not efficient due to gel formation. It is notewor-
thy that there are only a few works reporting the chemical neutral-
ization of biodiesel due to the difficulty of separation of FFAs
without promoting the hydrolysis of the methyl/ethyl esters.
To evaluate the other characteristics of biodiesel obtained by
enzymatic hydroesterification route, the product was analyzed
according to the methods described in ANP Resolution 14 from
11/05/2012. Table 1 shows the properties of the biodiesel pro-
duced and the respective specifications published by ANP. It can
be seen that the biodiesel produced by enzyme/enzyme hydroeste-
rification process was within several limits established by the stan-
dards related to biodiesel quality. The values of specific mass and
kinematic viscosity indicate good lubricating property of the bio-
diesel product. The high flash point (151 °C) ensures safer storage
and transportation of the fuel. The ester content did not met ANP
specification by standard EN 14103 method because this method
ignores the esters of fatty acids with less than 14 carbons in the
chain. However, the ester content would meet the minimum limit
if it were also considered the esters of fatty acids of 8–12 carbons
(96.7%). Since these fatty acids were derived from the contamina-
tion of pulp oil by the kernel oil (the oil from the macauba pulp
has only fatty acids with more than 16 carbons in the chain) we
can further assume that the ester content will be in a accordance
with the minimum limit established by the EN 14103 if were used
raw material without contamination. Ethanol, water and diglyce-
rides contents were slightly above the limit. The excess of ethanol
and water can easily be removed from biodiesel by distillation, as it
is made at industrial scale. The relatively high diglycerides content
is related to incomplete hydrolysis of triglycerides during hydro-
lytic step and a longer hydrolysis reaction could solve this problem.
The low oxidative stability, probably due to excess of FFAs, can be
solved by formulation with additives. Thus, although some param-
eters are not within the established limits, most of them can be
improved by the use of relatively simple processes. It is notewor-
thy that it is very important that the crude biodiesel product
should be purified by downstream steps to achieve high-purity
and quality biodiesel fuel that can be suitably used in diesel
engines [36].
These results show that our product (with 1.5% acidity) can be
employed as a blend. Considering a large-scale process, it could
represent an important contribution for regional use of resources
and alternative vegetable oil crops to soybeans such as macauba.
4. Conclusions
The production of cheap biocatalysts, the development of low-
cost and environmentally friendly processes, and the exploration
of alternative potential feedstocks are the gains of this study. The
enzyme/enzyme hydroesterification process using low-cost bio-
catalysts in both reactions and macauba acid oil as raw material
is described for the first time in this work. The use of this acid oil
to biodiesel production does not compete with the food industry,
allows a diversification of the oil crops used for biodiesel produc-
tion in Brazil and provides the Social Seal to biodiesel producers
that buy this oil from family farmers. The hydrolysis step was car-
ried out with a low vegetable lipase concentration (2.5% m/v), high
oil concentration (50% v/v) and without organic solvent and emul-
sifier, and produced 99.6% of FFAs after 6 h. In the esterification
 E.C.G. Aguieiras et al. / Fuel 135 (2014) 315–321
reaction, the FFAs were esterified to biodiesel with a conversion of
91% at 8 h, using dry fermented solid as biocatalysts. The reactions
carried out with the dry fermented solid showed similar conver-
sions to those conducted with commercial lipases and this alterna-
tive biocatalyst could also be recovered and reused for ten times.
The resulting fuel properties met important Brazilian standards.
The enzyme/enzyme hydroesterification process described in this
study appears to be a promising alternative to the traditional pro-
cess of biodiesel production and can contribute to make the enzy-
matic biodiesel economically feasible.
Acknowledgments
This research received financial support from FAPERJ, CNPq,
PETROBRAS, FINEP and PRH-ANP/MCTI. The authors are also grate-
ful to Dr. Donato Alexandre Gomes Aranda, of the ‘‘Laboratory of
Green Technologies (GREENTEC)’’ localized in Federal University
of Rio de Janeiro, to kindly provide his laboratory for biodiesel
analyses.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.fuel.2014.06.069.
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