Bioprocess and Biosystems Engineering (2018) 41:573–583

https://doi.org/10.1007/s00449-018-1892-5

RESEARCH PAPER

Optimization of biodiesel synthesis by esterification using a fermented

solid produced by Rhizopus microsporus on sugarcane bagasse
Vanderleia Botton1 · Leandro Piovan2 · Henry França Meier1 · David Alexander Mitchell3 · Jesús Cordova4 ·
Nadia Krieger2

Received: 22 September 2017 / Accepted: 4 January 2018 / Published online: 20 January 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse
enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was
used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as
a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO)
in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at
a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-
esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work
foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

Keywords  Lipases · Solid-state fermentation · Rhizopus microsporus · Esterification · Biodiesel

Introduction (MOR) as catalysts [2]. However, chemical catalysis has

Biodiesel is a renewable fuel produced as a substitute for
petrodiesel [1]. Industrially, it is produced mainly via chemi-
cal synthesis by transesterification, using triacylglycerols
and short-chain alcohols, such as methanol or ethanol, with
metal hydroxides (MOH) or alkoxides from alkali metals
Electronic supplementary material  The online version of this
article (https://doi.org/10.1007/s00449-018-1892-5) contains
supplementary material, which is available to authorized users.
* Nadia Krieger
nkrieger@ufpr.br
1 biodiesel monoesters and the glycerol, rendering the purifi-
Departamento de Engenharia Química,
Universidade Regional de Blumenau, Blumenau,
Santa Catarina 89030‑000, Brazil
2
Departamento de Química, Universidade Federal do Paraná,
Cx. P. 19081 Centro Politécnico, Curitiba, Paraná 81531‑980,
Brazil
3
Departamento de Bioquímica e Biologia Molecular,
Universidade Federal do Paraná, Cx. P. 19046, Centro
Politécnico, Curitiba, Paraná 81531‑980, Brazil
4
Departamento de Quimica, CUCEI, Universidad de
Guadalajara, Blvd. Marcelino García Barragán 1421,
44430 Guadalajara, Jalisco, Mexico
several disadvantages, such as the necessity of removing
the catalyst and the salt formed at the end of the process,
which leads to additional purification steps not only of the
biodiesel itself, but also of the by-product, glycerin. It is also
necessary to treat the alkaline wastewater generated during
the purification process [3].
Another drawback for industrial biodiesel production
through alkali-catalyzed transesterification is the high cost
of raw materials, since it is necessary to use materials with
low moisture contents and low levels of free fatty acids.
Water can favor the hydrolysis of the triacylglycerols and of
the fatty acid esters, and the free fatty acids can form soaps
that not only consume the catalyst, but also emulsify the
cation process more difficult [4].
Low-quality materials containing high free fatty acid con-
tents and high water contents can be processed by an alterna-
tive process, hydro-esterification. This process involves two
steps. In the first step, the fatty materials are hydrolyzed
to release free fatty acids; water and free fatty acids in the
raw material do not interfere with this step. In the second
step, the fatty acids are recovered and esterified. Since the
glycerol is produced and removed in the hydrolysis step, it
does not interfere with this esterification step and does not

13
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574
contaminate the final biodiesel product [5, 6]. Both steps can
be catalyzed either chemically or enzymatically [5, 7, 8].
In fact, the enzymatic synthesis of biodiesel using lipases
(EC 3.1.1.3) has been the focus of many studies over the last
10 years and is already implanted in industries in the United
States of America and China [9, 10]. However, more wide-
spread industrialization of enzymatic biodiesel is hampered
by the costs of lipase production and the relatively low sta-
bility of the lipases in the reaction medium [11]. To address
these problems, recently, our research group proposed to
produce lipases by solid-state fermentation (SSF) and then to
dry the fermented solids containing the lipases and add these
solids directly to solvent-free reaction media to produce bio-
diesel, in both esterification [12, 13] and transesterification
reactions [14, 15]. The main advantages of this process are
that the substrates for lipase production are agroindustrial
residues, such as sugarcane bagasse, and that there is no
need for extraction, recovery and immobilization of the
lipases. These combined advantages can decrease the cost
of the enzymatic route of biodiesel significantly, rendering
it more competitive with the chemical route.
However, there are still many challenges for the applica-
tion of the dried fermented solids as catalysts for biodiesel
synthesis, including the scaling-up of the SSF process to
produce fermented solids and improving the productivity
of the enzymatic process in solvent-free media. Our best
results for transesterification and esterification with etha-
nol in solvent-free reaction media have been obtained with
the fermented solids produced using Burkholderia cepacia
(denoted BCFS). As examples of transesterification, Salum
et al. [16] obtained a conversion of soybean oil of 95% in
46 h, using BCFS produced with a mixture of sugarcane
bagasse and sunflower seed meal, while Galeano et al. [14]
obtained a conversion of palm oil of 89% in 30 h, using
BCFS produced with sugarcane bagasse impregnated with
soybean oil. As examples of esterification, using the same
BCFS as Salum et al. [16], Soares et al. [13] obtained a
conversion of fatty acids derived from soybean soapstock
acid oil (FA-SSAO) of 92% in 31 h, while Dias et al. [12]
obtained a conversion of olein of 88% in 24 h.
Although we have achieved high ester yields with BCFS
in esterification and transesterification reactions, the use of
these solids could be problematic in industrial processes.
To have sufficient catalyst for a large-scale biodiesel pro-
duction process, it would be necessary to produce hundreds
of kilograms of BCFS by SSF. Since various Burkholderia
strains are opportunistic pathogens, large-scale fermenta-
tions undertaken with strains of this genus would require the
implementation of adequate containment systems, thereby
increasing process costs significantly. To avoid these prob-
lems, recently we studied the possibility of producing lipases
in SSF using Rhizopus microsporus, considering that vari-
ous species of the genus Rhizopus are GRAS, being used to
13
Bioprocess and Biosystems Engineering (2018) 41:573–583
produce several Asian fermented foods [17]. A fermented
solid produced in Erlenmeyer flasks using Rhizopus micro-
sporus (denoted RMFS, for “Rhizopus microsporus fer-
mented solid”) gave a conversion of 68% in 72 h for the
transesterification of corn oil with ethanol in a solvent-free
system [15]. We then scaled up the production of lipases by
Rhizopus microsporus, using a substrate composed of 7.5 kg
of wheat bran and 7.5 kg of sugarcane bagasse (dry masses)
in a pilot-scale SSF bioreactor [18]. The dried fermented
solid (denoted RMFS) gave a 68% conversion in 48 h for
the esterification of oleic acid with ethanol in a solvent-free
system [18].
The conversions that we have obtained to date using the
RMFS are far below the values that are necessary for bio-
diesel production. Therefore, in the present work we used
response surface methodology to optimize the esterifica-
tion of fatty acids using RMFS in a solvent-free system.
We started using oleic acid, as a model fatty acid, and then
investigated the esterification of a lower quality raw mate-
rial, namely FA-SSAO, previously used by Soares et al. [13].
If the performance of RMFS can improved to be similar to
that of BCFS, then this will make it easier to produce the fer-
mented solid in large-scale SSF bioreactors, since expensive
containment systems will not be necessary.
Materials and methods
Reagents and suppliers
The reagents used were ethanol (99.5%) and urea from
Synth (São Paulo, Brazil), lactose from Nuclear (São
Paulo, Brazil), ­K2HPO4 from Qhemis (São Paulo, Brazil),
­M gSO 4·7H 2O, methanol (99.8%), 1-propanol (99.5%),
1-butanol (99.4%), 1-pentanol (98%), 1-hexanol (98%),
n-heptane (99.5%) and n-hexane (99.5%) from Vetec (Rio
de Janeiro, Brazil), tricaprylin (99%), molecular sieve
(3 Å, 8–12 mesh) and methyl heptadecanoate (99.8%) from
Sigma–Aldrich (Missouri, EUA). The sugarcane bagasse
used to prepare the SSF media was kindly donated by Com-
panhia Melhoramentos Norte do Paraná (Paraná, Brazil).
The commercial soybean oil Lisa produced by Cargill (São
Paulo, Brazil) was used to induce lipase production. The
substrates for the esterification reactions were oleic acid
(90%) from Sigma-Aldrich (Missouri, EUA) and a mixture
of free fatty acids obtained from the hydrolysis of soybean
soapstock acid oil (FA-SSAO) in subcritical water, prepared
according to Soares et al. [13], and kindly donated by Ubal-
dino Rodrigues Soares e Cia. Ltda. (Paraná, Brazil). The
fatty acid composition of FA-SSAO was 16.5% palmitic,
4.2% stearic, 33.4% oleic, 44.2% linoleic and 1.7% others,
with an average molecular weight of 277.3 g/mol, an acid
value of 195.5 mg KOH/g and a saponification value of
Bioprocess and Biosystems Engineering (2018) 41:573–583 575
199 mg KOH/g [13]. All other reagents were of analytical
grade and had the purity specifications recommended by the
methods used, and were obtained from Brazilian suppliers.
Fungal strain and inoculum
A Rhizopus strain was originally isolated in Guadalajara,
Mexico, and previously characterized as Rhizopus micro-
sporus CPQBA 312-07 DRM [19]. A spore suspension was
prepared by growing the fungus on potato dextrose agar at
30 °C for 7 days and harvesting the spores with a Tween 80
solution (0.01% w/v). The spore concentration in the suspen-
sion was determined using a Neubauer chamber.
Production of fermented solid with esterification
activity
To produce the fermented solid, the sugarcane bagasse was
washed three times with tap water, followed by drying at
80 °C for 24 h and sieving to obtain particles between 0.85
and 2.0 mm.
SSF was performed in 250-mL Erlenmeyer flasks con-
taining 4  g of sugarcane bagasse impregnated with an
emulsion containing 0.8 g soybean oil and 20 mL of culture
medium, which contained (per liter) 4 g urea, 5 g lactose,
5 g ­K2HPO4, 1 g M
­ gSO4·7H2O and 4 mL oligoelement solu-
tion (containing, per liter, 10 g EDTA, 2.0 g ­MnCl2·4H2O,
2.8 g ­CoSO4·7H2O, 1.5 g ­CaCl2·2H2O, 0.2 g ­CuCl2·2H2O
and 0.3 g ­ZnSO4·7H2O, pH 4.0) [15, 20]. The pH of the oli-
goelement solution was adjusted to 7.0 with 10% HCl (v/v).
Erlenmeyer flasks containing the substrate were plugged
with cotton wool and autoclaved at 121 °C for 15 min. After
cooling, spore suspension was added to give 3 × 107 spores/g
of dry solid substrate. The initial moisture content, 75–80%
(w/w, wet basis), was measured in an infrared moisture bal-
ance (Gehaka, model IV 2000, São Paulo, Brazil). The flasks
were incubated for 18 h at 40 °C.
After fermentation, RMFS was lyophilized for 24 h at
− 45 °C and 0.1 mbar in a Jouan LP3 Lyophilizer (Allerød,
Frederiksborg, Denmark). For the esterification reactions,
RMFS was not further processed and was maintained at
− 18 °C in sealed plastic bags.
Determination of lipase activities
The hydrolytic activity of the dry RMFS was assessed by
the titrimetric method. A tricaprylin emulsion was prepared
with gum arabic (3% w/v), 2 mmol/L ­CaCl2, 2.5 mmol/L
Tris–HCl and 150 mmol/L NaCl [21]. For each assay, 20 mL
of this emulsion and 250 mg of RMFS were mechanically
stirred in a thermostated vessel, at 37 °C for 5 min, with the
free fatty acids released during the reaction being titrated
with 0.05 mol/L NaOH in a pH-Stat (Metrohm, model 718
Stat Titrino, Herisau, Switzerland) set at pH 7.0. One unit
of activity (U) was defined as the release of 1 µmol of fatty
acid per minute, under the assay conditions.
The tricaprylin-hydrolyzing activity of RMFS was
183 ± 6 U/g. This activity remained stable during storage
over the period of experimentation. Errors of all the analyses
represent standard deviations of the mean.
For the esterification activity, reactions were carried out
in 50-mL screw-capped bottles containing 5 mL of a mixture
containing 210 mmol/L of ethanol and 70 mmol/L of fatty
acid (i.e., an alcohol to acid molar ratio of 3:1) in n-heptane
and 500 mg of fermented solid. These bottles were incubated
on a rotary shaker at 200 rpm and 40 °C. At fixed intervals,
100-µL samples of the mixture were removed and analyzed
for residual free fatty acids by the Lowry-Tinsley method
[22]. Alkyl-ester production was calculated by consumption
of free fatty acids from the reaction mixture. One unit (U) of
esterification activity equals 1 µmol of alkyl-ester produced
per minute, under the assay conditions. The esterification
activity of RMFS was 15 U/g. This activity remained stable
during storage over the period of experimentation.
Both hydrolytic and esterification activities were
expressed in units of activity per gram (U/g) of dry fer-
mented solid (RMFS).
Esterification reactions in solvent‑free medium
Reactions were carried out in a solvent-free system, in
50-mL flasks (with polytetrafluoroethylene caps) containing
30 mmol of substrate, 8.5 g oleic acid or 8.3 g FA-SSAO and
the amount of alcohol necessary to obtain the desired molar
ratio (MR) (i.e., an alcohol to acid molar ratio in the range
of 1:1–15:1). The mixture was pre-incubated for 10 min on
a rotary shaker at 200 rpm at the stated temperature, and the
reactions were started by adding 1.8 g of dry RMFS (i.e.
20% by mass in relation to the fatty acids). This amount of
dry RMFS contained 329 U tricaprylin-hydrolyzing activity
and 27 U of esterification activity. The alcohol was added in
two equal aliquots, at 0 and 24 h; the quoted MR represents
the total moles of alcohol added per mole of initial fatty
acid. Samples were collected at intervals for gas chromatog-
raphy (GC) analysis. After the reaction, the fermented solid
was separated from the reaction medium by filtration using
Whatman no 1 filter paper. The reactions were evaluated at
different temperatures (40–50 °C). When molecular sieves
were added to the reaction medium, they were previously
dried at 300 °C for 3 h and then stored in a desiccator at
room temperature. Water and molecular sieve contents are
expressed as percentages of the initial weight of the sub-
strates, ethanol and oleic acid.
A ­22 factorial design with three central points was used
to optimize the conditions for esterification of both oleic
acid and FA-SSAO [23]. The data obtained were fitted to a
13

576
first-order polynomial equation. The goodness-of-fit of the
model was evaluated by the coefficient of determination (R2)
and an analysis of variance (ANOVA). The regression analy-
ses, statistical tests and response surfaces were determined
using the software STATISTICA version 10.0 (StatSoft Inc.,
Tulsa, OK, USA). The contrast coefficients, which allow the
determination of the effect of each independent variable,
were calculated by Eq. (1).
( )
2 Σyi + − Σyi −
Ci = , (1)
n 20
where Ci is the contrast coefficient of independent variable i,
the yi+ values are the values obtained for the response vari-
able (i.e., percentage conversion) when independent variable
i is at its highest value; the yi− values are the values obtained
for this response variable when independent variable i is at
its lowest value, and n is the number of experiments.
Gas chromatography analysis
Alkyl-ester content was determined using a GC-2010
(Shimadzu Co., Kyoto, Japan) equipped with a hydrogen
flame ionization detector and a SGE HT-5 capillary column
(0.32-mm internal diameter, 25-m length and 0.1-mm film
thickness). Prepared sample (50 mg) was diluted in 1 mL
of an internal standard solution of methyl heptadecanoate
(10 mg/L) in n-heptane. Then 1 µL was injected, with a
split ratio of 1:50, using N
­ 2 as the carrier gas. The injector
and detector were set at 250 °C. The oven program was as
follows: 120 °C for 2 min, heating at 10 ℃/min to 180 °C,
180 °C maintained for 3 min, heating at 5 °C/min to 230 °C,
230 °C maintained for 2 min. Peaks in the chromatograms
were identified by comparison of the retention times with a
standard solution. The ester content (in mass percent) was
determined relative to the peak area of the internal standard
[24].
Results
Effect of the chain length of the alcohol
RMFS was used to catalyze the esterification of oleic acid
with alcohols varying from 1 to 6 carbon atoms. The alco-
hols were added in two equal aliquots (0 and 24 h) to give
an overall MR of 3:1 (Fig. 1). After 48 h, high conversions
(above 85%) were obtained with all alcohols except for
methanol (63%), with the highest conversion (almost 100%)
being obtained for propanol. Poor performance of lipases
with short-chain alcohols, especially methanol, has been
attributed to lower solubility of short-chain alcohols in the
reaction medium, which can cause mass transfer problems
13
Bioprocess and Biosystems Engineering (2018) 41:573–583
100
90
80
70
Conversion (%)
60
50
40
30
10
0
0 12 24 36 48
Time (h)
Fig. 1  Profiles for the esterification of oleic acid with different alco-
hols in a solvent-free medium catalyzed by the fermented solid pro-
duced by Rhizopus microsporus. Key: (circle) methyl-oleate; (square)
ethyl-oleate; (filled triangle) 1-propyl-oleate; (filled down pointing
triangle) 1-butyl-oleate; (asterisk) 1-pentyl-oleate; (open triangle)
1-hexyl-oleate. Conditions: 90 mmol alcohol and 30 mmol oleic acid
with 1.8 g fermented solid, incubated at 40 °C and 200 rpm. Alcohols
were added in two equal aliquots at 0 and 24 h
[25] and to competitive inhibition caused by the higher
interaction of the small alcohols with the active site of the
enzyme [26–28] and to denaturation [29–31].
These results show that RMFS can be used to catalyze
esterification of various alcohols in solvent-free media.
Some of the longer-chain alcohol esters, such as the hexyl-
ester, are environmentally friendly products that can be used
in the biofuel industry to substitute mineral oil, which is a
petroleum derivative [32–34].
Although longer-chain alcohols gave higher conver-
sion percentages, the use of ethanol to produce biodiesel
has several advantages and is of strategic interest in Bra-
zil, where it is abundant and is produced from renewable
sources. Besides, ethanol is less toxic than methanol and
less expensive than longer-chain alcohols. Therefore, ethanol
was chosen for the optimization of esterification reactions.
Kinetic profile of the esterification of oleic acid
with ethanol
When the esterification of oleic acid with ethanol was fol-
lowed for 72 h, the conversion reached 85% at 30 h and
then remained constant (Fig. 2). To determine whether the
reaction had stopped due to denaturation of the lipase, the
residual esterification activity of RMFS was determined.
The residual activity fell to 65% in the first 6 h and then
slowly decreased to around 55% at 72 h. Since the RMFS
maintained a significant residual activity throughout the
reaction, we can conclude that 85% conversion represents
Bioprocess and Biosystems Engineering (2018) 41:573–583 577

100 100 A 100

Residual esterification activity (%)

90 90
90
80 80
80
70 70
Conversion (%)

70
60 60
60

Conversion (%)
50 50
40 40 50
30 30 40
20 20 30
10 10 20
0 0
10
0 12 24 36 48 60 72
Time (h) 0
Control 5 10 20 30
Water content (wt%)
Fig. 2  Profile for the ethyl-esterification of oleic acid and residual
esterification activity of the fermented solid produced by Rhizopus B 100
microsporus. Key: (open triangle) conversion in ethyl-oleate; (filled 90
triangle) residual activity of the fermented solid. Conditions: 1.8  g 80
of dry fermented solids in 90 mmol of ethanol and 30 mmol of oleic
acid (molar ratio of 3:1). Ethanol was added in equal aliquots at 0 70
and 24  h. Incubation was at 40  °C and 200  rpm. Residual activities 60
were determined relative to an initial esterification activity of 15 U/g. Conversion (%)
Errors bars represent the standard error of the mean. In all cases, the 50
standard error of the mean was smaller than the symbol size 40
30
the equilibrium value. This value is in the range of previ- 20
ously reported equilibrium conversions reported for enzy- 10
matic production of ethyl-oleate at 45 °C [35, 36].
0
Control 5 (0 h) 5 (24 h)
Effect of water content on the synthesis Molecular sieve content (wt%)
of ethyl‑oleate
Fig. 3  Effect of water content on the ethyl-esterification of oleic acid
Since water plays an essential role in synthesis reactions catalyzed by the fermented solid produced by Rhizopus microsporus.
catalyzed by lipases, the influence of water on the ethyl- a Addition of water. b Addition of type 3 Å molecular sieve (at 0 or
24  h). Control: addition of neither water nor molecular sieve. Con-
esterification of oleic acid was investigated. ditions: 1.8  g of dry fermented solids in 90  mmol of ethanol and
When 5% of water was added to the reaction medium 30  mmol of oleic acid. Ethanol was added in two equal aliquots at
(Fig. 3a), the conversion at 48 h decreased to 39%, which is 0 and 24  h. Incubation at 40  °C and 200  rpm for 48  h. Water and
much lower than the value of 85% obtained with no added molecular sieve contents are expressed as percentages of the initial
weight of the substrates, ethanol and oleic acid. Values are plotted as
water. With higher percentages of water (10, 20 and 30%), means of triplicate analyses ± the standard errors of the mean
the conversion increased to around 60%. In these cases, a
second phase formed in the reaction medium at the begin-
ning of the reaction; a second phase did not form during the the percentage conversions at 48 h increased with further
whole reaction period for the experiments with no added increases of the water content in the medium. However,
water and 5% water. In the case of 5% water, the water may since there was more water in the system than was the case
have been entirely sorbed by the fermented solid, creating with no added water, the formation of a second aqueous
an aqueous phase in the microenvironment of the enzyme phase in the experiments with 10–30% added water was
[37]. This phase tends to be rich in ethanol, favoring enzyme not able to give the high final conversion of 85% that was
deactivation. On the other hand, when a second aqueous obtained with no added water.
phase forms, the ethanol and also the water produced in the With the addition of 5% of molecular sieve at the begin-
reaction are partially removed from direct contact with the ning of the reaction, the percentage conversion at 48 h was
enzyme, reducing deactivation [31, 38–40]. Additionally, 92%. The conversion was even higher, 96%, when the molec-
since water is a product of the esterification reaction, its ular sieve was added after 24 h of reaction (Fig. 3b). The
removal to the aqueous phase shifts the equilibrium of the higher conversions obtained with the addition of molecu-
reaction towards synthesis. These factors would explain why lar sieves in comparison to the control (without molecular

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578 Bioprocess and Biosystems Engineering (2018) 41:573–583

sieves, 85%) are probably due to the removal of the water Table 2  Contrast coefficients (Ci) calculated from the results of the
formed during the reaction, dislocating the position of equi- factorial experiment used to optimize the esterification catalyzed by
the fermented solid produced by Rhizopus microsporus 
librium towards synthesis [41]. For both times of addition,
higher percentages of molecular sieves (up to 20%) were Variable Contrast coefficient (percentage points for
studied, but produced no further increase in the percentage conversion)
conversion (data not shown). Oleic acid (%) FA-SSAO (%)

Ta − 6.1 ± 0.4 − 5.0 ± 0.9
MRb + 4.3 ± 0.4 + 5.3 ± 0.9
Improvement of the esterification conditions
T × MR − 0.3 ± 0.4 + 3.1 ± 0.9

In this step, the conditions for esterification were optimized
both for oleic acid and for FA-SSAO [13]. We undertook
these studies without the addition of molecular sieves, to
minimize the process costs.
Ethanol was added in two equal aliquots, at 0 and 24 h.
The independent variables studied were the temperature and
the ethanol to fatty acid MR; they were varied according to a
­22 factorial design with three replicates at the central point.
The response variable was the percentage conversion at 48 h.
Table 1 shows the real and coded values of the independent
variables and the experimental and predicted conversions.
The predicted percentage conversions for oleic acid and
FA-SSAO (Ŷ with the appropriate subscript) were obtained
using Eqs. (2) and (3), respectively.
Ŷ oleic acid = 88.4286 − 3.25T + 2.25MR.
Ŷ FA−SSAO = 80.7143 − 2.50T + 2.50MR,
where T and MR represented the coded values for the inde-
pendent variables temperature and molar ratio, respectively.
For both substrates, oleic acid and FA-SSAO, both T and
MR were statistically significant, given that the absolute
values of the contrast coefficients (|i|) were higher than the
standard deviation of the central point (Table 2). The inter-
action term (T × MR) was significant for FA-SSAO, but not
for oleic acid.
The standard deviation for the central point was 0.4 for oleic acid and
0.9 for FA-SSAO
a
 T = temperature (℃)
b
 MR = molar ratio of ethanol to fatty acid
In the case of oleic acid, the ANOVA indicated that the
first-order polynomial model was an adequate representation
of the dependence of the percentage conversion on the sig-
nificant variables, with a good coefficient of determination
(R2 = 0.98) (Table S1). The good adjustment of the model to
the experimental data is also shown by the F value, which
was much higher than the tabulated value (F2,4,0.05). Moreo-
ver, the value of the mean square ratio (lack of fit/pure error)
was not statistically significant.
(2) On the other hand, for FA-SSAO, the ANOVA gave a low
coefficient of determination (R2 = 0.81) for the dependence
(3)
of the percentage conversion on the significant variables
(Table S2). The poor fit of the model to the experimental
data is also shown by the F value, which was similar to the
tabulated value (F2, 4, 0.05). Nevertheless, the value of the
mean square ratio (lack of fit/pure error) was not statistically
significant.
Since the regression equation was significant in the case
of oleic acid esterification, a response surface was generated
(Fig. S1). The percentage conversion at 48 h increased with

Table 1  Experimental Trial Independent variables Oleic acid conversion at 48 h (%) FA-SSAO conversion at 48
conditions and results for the 2­ 2 (coded value) h (%)
factorial design experiment used
to optimize the esterification Ta (℃) MRb Experimental Predicted Experimental Predicted
catalyzed by the fermented
solid produced by Rhizopus 1 40 (− 1) 1:1 (− 1) 88.9 89.4 81.9 80.7
microsporus  2 50 (1) 1:1 (− 1) 83.1 82.9 73.8 75.7
3 40 (− 1) 5:1 (1) 93.5 93.9 84.1 85.7
4 50 (1) 5:1 (1) 87.1 87.4 82.2 80.7
5 45 (0) 1:3 (0) 88.9 88.4 81.3 80.7
6 45 (0) 1:3 (0) 89.0 88.4 82.0 80.7
7 45 (0) 1:3 (0) 88.3 88.4 80.3 80.7

Run 5 was performed in triplicate, incubation for 48 h, giving sample standard errors for the central point
of 0.4 for oleic acid and 0.9 for FA-SSAO
a
 T = temperature (ºC)
b
 MR = molar ratio of ethanol to fatty acid

13
Bioprocess and Biosystems Engineering (2018) 41:573–583 579
decreasing temperature and increasing molar ratio. The maxi-
mum conversion, 93.5%, was obtained at the highest MR (5:1)
and the lowest temperature (40 °C). Although the regression
equation was not significant in the case of FA-SSAO esteri-
fication, the highest conversion (Table 1), 84.1%, was also
obtained in these conditions.
The response surface for oleic acid (Fig. S1) suggests that
a higher MR could further increase the conversion, so we
studied this possibility, comparing the conversions at 48 h, for
reactions done at 40 °C. In the case of oleic acid, increasing
the MR from 5:1 to 10:1 increased the conversion from 93 to
98% (p < 0.05); however, at a MR of 15:1, the conversion was
only 92%. In the case of FA-SSAO, increasing the MR did
from 5:1 to 15:1 did not increase the conversion significantly
(p > 0.05) (Fig. 4).
Comparison of specific rates obtained RMFS
and BCFS
To determine whether 48 h was the best reaction time, kinetic
profiles were obtained for the ethyl-esterification of oleic acid
and FA-SSAO for MR 10:1 and 40 °C, using RMFS. The high-
est conversions were indeed obtained at 48 h, 98% for oleic
acid and 86% for FA-SSAO (Fig. S2).
The following equation (Eq. 4), which is an empirically
simplified version of the Ping Pong Bi Bi equation, was used
as a tool in the estimation of the initial rate of consumption of
the fatty acid (rFA, mM/h):
−kCFA CEt
rFA = ( ), (4)
D1 CW + D2 CFA + CFA CEt
100
90
80
70
Conversion (%)
60
50
40
30
20
10
0
FA-SSAO Oleic acid
Fatty acids
Fig. 4  Effect of high molar ratios of ethanol to fatty acid on the ethyl-
esterification catalyzed by the fermented solid produced by Rhizopus
microsporus. Key: molar ratio of ethanol to fatty acids (FA-SSAO
and oleic acid): (black bar) 5:1, (dark gray bar) 10:1 and (light gray
bar) 15:1. Conditions: 1.8 g of dried fermented solid and 30 mmol of
fatty acids. The ethanol added in two equal aliquots at 0 and 24  h.
Incubation at 40 °C and 200 rpm for 48 h. Values are plotted as the
means of triplicate analyses ± the standard errors of the mean
where CFA, CEt and CW are the concentrations (in mmol/mL)
of fatty acid, ethanol and water, respectively, while k, D1, D2
and D3 are fitting constants.
The curves plotted in Fig. 5 represent the best fits of
Eq. (4) to the data, obtained through non-linear regression.
The specific initial rates (i.e., per gram of dry fermented
solid) for the consumption of free fatty acids (oleic acid
and FA-SSAO) by RMFS were compared with the results
obtained by Soares et al. [13] for the ethyl-esterification of
FA-SSAO by BCFS (fermented solid produced by B. cepa-
cia LTEB11) (Table 3). BCFS has a much higher specific
initial rate than does RMFS.
Discussion
In this work, the esterification reaction catalyzed by a dry
fermented solid produced by a non-pathogenic fungus,
Rhizopus microsporus, was studied in a solvent-free system
using two starting materials: oleic acid, as a model system,
and FA-SSAO, a mixture of fatty acids produced by physical
hydrolysis of low cost lipids. With an ethanol to fatty acid
MR of 10:1, we obtained a 98% conversion of oleic acid,
after 48 h at 40 °C. Under these conditions, the conversion
of FA-SSAO was 85%. The lower conversion obtained for
FA-SSAO is probably due to its high content in linoleic acid
(44.2%) and a relatively low content of oleic acid (33.4%); in
comparison, the commercial oleic acid that we used contains
Fig. 5  Profiles for fatty acid consumption used in the determination
of specific rates. (open circle) (dashed line) Ethyl-esterification of
FA-SSAO catalyzed by the fermented solid produced by Rhizopus
microsporus. (open square) (dotted line) Ethyl-esterification of oleic
acid catalyzed by the fermented solid produced by Rhizopus micro-
sporus. (filled circle) (solid line) Ethyl-esterification of FA-SSAO
catalyzed by dried fermented solid produced by Burkholderia cepa-
cia LTEB11 [13]. The curves were used to determine initial rates,
which, in turn, were used to calculate the specific initial rates given
in Table 3
13

580
Table 3  Specific initial rates Data Microorganism
of fatty acid consumption in
the esterification reactions
catalyzed by the fermented
solids This work Rhizopus microsporus
This work Rhizopus microsporus
[13] Burkholderia cepacia LTEB11
a
 This was calculated as the initial rate (i.e., tangent at zero time) of the corresponding curve in Fig. 5, mul-
tiplied by the total reaction volume and divided by the mass of dry fermented solids
over 90% oleic acid. This hypothesis is supported by the
results of Botton [42], who determined the esterification
activity of RMFS using different fatty acids (C8, C12, C14
C16, C18:1 and C18:2) and reported similar esterification
activities for all studied fatty acids (15 U/g), except for lin-
oleic acid, for which RMFS presented an activity 40% lower
than for the other fatty acids. These results suggest that if
residual materials with oleic acid contents higher than that
of FA-SSAO were to be used, higher conversions could be
obtained.
Our esterification reaction, which gave conversions of
85–98% in 48 h, had a twofold higher ester productivity
than the ethanolysis of corn oil reported by Zago et al. [15],
where a conversion of only 68% was obtained in 72 h. Our
esterification reaction could be used as the second step of a
hydro-esterification process, in which fatty-acid-rich resid-
ual materials would be used to produce free fatty acids in the
initial hydrolysis step. Such a strategy is likely to be more
feasible than the transesterification process, increasing the
potential of the enzymatic route to compete with the chemi-
cal route for biodiesel synthesis [5, 6].
The 98% conversion of oleic acid that we obtained using
RMFS in a solvent-free system in the present work for a MR
of 10:1 exceeds the minimum value specified for biodiesel
(96.5%) [43], suggesting that the reaction products can be
used as biodiesel with minimal processing. The conver-
sion of 86% in 48 h for the esterification of FA-SSAO using
RMFS is comparable to the conversions reported by Dias
et al. [12], for the production of ethyl-oleate using BCFS, by
Meng et al. [44], for the ethanolysis of soybean oil using the
lipase from Yarrowia lipolytica immobilized on fabric mem-
branes, and by Aguieiras et al. [7], for the esterification of
fatty acids obtained as byproducts of the refining of soybean
and palm oil, catalyzed by a dry fermented solid produced
by SSF of a strain of Rhizomucor miehei on babassu cake
(Table 4). This suggests that RMFS can be used to catalyze
the esterification of different fatty acid mixtures obtained
from residual raw materials.
RMFS was used to synthesize not only ethyl esters of
oleic acid, but also esters of alcohols with longer chains.
This opens other alternatives for the use of RMFS in indus-
trial processes. Long-chain-alcohol esters (C-3 and longer)
can be used as biodiesel [51], but they also have industrial
13
Bioprocess and Biosystems Engineering (2018) 41:573–583
FA Specific rate of fatty acid
consumption mmol/(h
mg)a
Oleic Acid 480
FA-SSAO 502
FA-SSAO 1888
application as biolubricants, plastic film plasticizers and
additives for diesel, biodiesel and hydraulic fluids [52–54].
These esters of long-chain alcohols are of great interest
because they are biodegradable and produced from renew-
able sources, and can replace similar compounds produced
from non-renewable sources [55–57].
In the case of ethyl-esterification of FA-SSAO, Soares
et al. [13] obtained a higher conversion (91% in 30 h)
using BCFS than we achieved with RMFS (86% in 48 h)
(Table 4). BCFS is undoubtedly more efficient than RMFS.
However, RMFS would be much safer to use in large-scale
SSF processes for the production of fermented solids and
in large-scale esterification processes. In fact, the viability
of scaling-up the production of RFMS in a pilot bioreactor
was already demonstrated by Pitol et al. [18]. Although
they only obtained a 67% conversion of oleic acid (using
the same reaction conditions as in this work), the costs
of the culture medium (a 1:1 mixture of wheat bran and
sugarcane bagasse) were 62% cheaper than the costs of
the medium used in the current work. Our current work
suggests that if the reaction conditions were optimized for
this alternative RMFS, it would be possible to obtain high
conversions, but more cheaply.
Conclusions
In this work, we optimized biodiesel synthesis by ethylic
esterification, catalyzed by fermented solids containing
lipases of R. microsporus, using two substrates, oleic acid
and a mixture of fatty acids obtained by hydrolysis of
soybean acid oil (FA-SSAO). In solvent-free media, we
obtained conversions of 98% for oleic acid and 86% for
FA-SSAO. The fermented solid also catalyzed ester syn-
thesis with long-chain alcohols, producing other esters of
industrial interest. The use of a fermented solid produced
by a non-pathogenic microorganism, together with the
possibility of using low-quality hydrolyzed fatty raw mate-
rials, could render the scale-up of the enzymatic synthe-
sis of biodiesel via hydro-esterification more feasible, and
more competitive with the chemically catalyzed processes.
Bioprocess and Biosystems Engineering (2018) 41:573–583 581

Table 4  Reports on biodiesel production by esterification and transesterification catalyzed by lipases in solvent-free systems
Microorganism Enzymatic prepara- Reaction medium Time and tempera- Molar ratio Conversion (%) References
tion/support ture

Rhizopus microspo- Sugarcane bagasse Ethanol/oleic acid 48 h; 40 °C 10:1 98 (This work)
rus and nutrient solu-
tion
Rhizopus microspo- Sugarcane bagasse Ethanol/FA-SSAO 48 h; 40 °C 10:1 86 (This work)
rus and nutrient solu-
tion
Rhizopus microspo- Sugarcane bagasse Ethanol/corn oil 72 h; 44 °C 3:1 68 Zago et al. [15]
rus and nutrient solu-
tion
Rhizopus microspo- Sugarcane bagasse Ethanol/oleic acid 48 h; 40 °C 1.5:1 67 Pitol et al. [18]
rus and wheat bran
Aspergillus ory- Whole-cell Methanol/FA-palm 6 h; 30 °C 1.5:1 90 Adachi et al. [45]
zae whole-cell and soybean oil
expressing Candida
antarctica B lipase
(r-CALB)
Aspergillus ibericus Crambe oil and Butanol/decanoic 24 h; 37 °C 1:1 100 Oliveira et al. [46]
MUM 03.49 sesame oil cake acid
Palm oil fruit Palm oil fruit cake Methanol/residual oil 36 h; 35 °C 6:1 92 Suwanno et al. [47]
containing the from palm oil mill
lipase effluent
Yarrowia lipolytica Fabric membrane Ethanol/FA-soybean 3 h; 30 °C 1:1 85 Meng et al. [44]
oil
Serratia marcescens Whole-cell Methanol/FA- waste 72 h; 30 °C 4:1 97 Li et al. [48]
YXJ-1002 grease
Rhizomucor miehei Dry babassu (Attalea Ethanol or methanol/ 24 h; 45 °C 2:1 80–85 Aguieiras et al. [7]
speciosa) cake FA-soybean and
palm oils
Rhizomucor miehei Dry babassu (Attalea Butanol/macauba 70 h; 40 °C 5.5:1 80 Todeschini et al. [8]
speciosa ) cake (Acrocomia acu-
leata) acid oil
Rhizomucor miehei Dry babassu (Attalea Ethanol/FA-macauba 8 h; 40 °C 2:1 91 Aguieiras et al. [5]
speciosa ) cake (Acrocomia acu-
leata) oil
Burkholderia con- Sugarcane bagasse Ethanol/palm oil 48 h, 50 °C 4.5:1 90 Galeano et al. [14]
taminans LTEB with soybean oil
Burkholderia lata Sugarcane bagasse Ethanol/olein 24 h; 45 °C 1.5:1 88 Dias et al. [12]
LTEB11 and sunflower seed
meal
Burkholderia cepacia Sugarcane bagasse Ethanol/FA-SSAO 31 h; 50 °C 3:1 92 Soares et al. [13]
LTEB11 and sunflower seed
meal
Burkholderia cepacia Sugarcane bagasse Ethanol/soybean oil 46 h; 50 °C 3:1 95 Salum et al. [16]
LTEB11 and sunflower seed
meal
Burkholderia ceno- Sugarcane bagasse Ethanol/soybean oil 96 h; 45 °C 4:1 86 Liu et al. [49]
cepacia and sunflower seed
meal
Burkholderia ceno- Sugarcane bagasse Ethanol/soybean oil 96 h; 44.2 °C 4.3:1 91 Liu et al. [50]
cepacia and sunflower seed
meal

FA fatty acid, FA-SSAO fatty acids from soybean soapstock acid oil

13

582 Bioprocess and Biosystems Engineering (2018) 41:573–583

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