Biochemical Engineering Journal 81 (2013) 1523

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Biodiesel production from soybean soapstock acid oil by hydrolysis in

subcritical water followed by lipase-catalyzed esterication using a
fermented solid in a packed-bed reactor
Diniara Soares a , Andrei Ferreira Pinto b , Alan Guilherme Goncalves c ,
David Alexander Mitchell a , Nadia Krieger b,
a

Departamento de Bioqumica e Biologia Molecular, Universidade Federal do Paran, Cx. P. 19046 Centro Politcnico, Curitiba 81531-980, Paran, Brazil
Departamento de Qumica, Universidade Federal do Paran, Cx. P. 19081 Centro Politcnico, Curitiba 81531-980, Paran, Brazil
c
Departamento de Farmcia, Universidade Federal do Paran, Av. Lothario Meissner, 3400, Jardim Botnico, Curitiba, Paran, Brazil
b

a r t i c l e

i n f o

Article history:
Received 19 February 2013
Received in revised form 30 June 2013
Accepted 26 September 2013
Available online 5 October 2013
Keywords:
Biodiesel
Hydroesterication
Lipases
Burkholderia cepacia
Solid-state fermentation
Packed-bed reactor

a b s t r a c t
We investigated a new hydroesterication strategy for the production of biodiesel from low-value oil
feedstocks: complete hydrolysis of the feedstock to fatty acids in subcritical water, followed by the use of
a packed-bed reactor, containing a fermented solid with lipase activity, to convert the fatty acids to their
ethyl esters. The fermented solids were produced by cultivating Burkholderia cepacia LTEB11 for 72 h on
a 1:1 mixture, by mass, of sugarcane bagasse and sunower seed meal. The esterication of fatty acids
obtained from soybean soapstock acid oil was studied in the packed-bed bioreactor, in a solvent-free system, with the best results being a 92% conversion in 31 h, obtained at 50 C. When the packed-bed reactor
was reused in successive 48-h esterication reactions, conversions of over 84% of the fatty acids to esters
were maintained for ve cycles at 50 C and for six cycles at 45 C. Unlike previous hydroesterication
processes that have used lipase-catalyzed hydrolysis followed by chemically-catalyzed esterication, our
process does not expose the lipases to contaminants present in low quality feedstocks such as soapstocks.
This advantage opens the possibility of operating the packed-bed esterication reactor in continuous
mode.
2013 Published by Elsevier B.V.

1. Introduction
Biodiesel is currently being produced as a substitute for
petrodiesel, however, it is not economically competitive, with its
use requiring either subsidies or government policies, such as
in Brazil, where all petrodiesel is currently required to contain
5% biodiesel [1]. It is composed of esters of short-chain alcohols
(methyl or ethyl alcohols) and long-chain fatty acids. The feedstocks used in most commercial biodiesel production processes
are derived from triacylglycerols of edible vegetable oils [2]. However, these oils are relatively expensive, and since the feedstock
in biodiesel synthesis corresponds to 5085% of total production
costs, it is desirable to use low-cost starting materials, in order
to increase the commercial competitiveness of biodiesel [35].
Potential low-value feedstocks include animal fat from sewage and
residual oil of domestic, industrial or commercial origin.
Current industrial processes for the production of biodiesel from
vegetable oils use alkaline transesterication, which gives high

Corresponding author. Tel.: +55 41 33613470; fax: +55 41 33613006.

E-mail address: nkrieger@ufpr.br (N. Krieger).
1369-703X/$ see front matter 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.bej.2013.09.017

yields (98%) in a short reaction time of about 1 h. However, alkaline transesterication requires starting materials with low levels
of moisture and free fatty acids and therefore is not appropriate for
the production of biodiesel from low-value feedstocks, which contain signicant amounts of free fatty acids or water. Free fatty acids
react with the alkaline catalyst, producing soaps. This decreases the
reaction yield and makes the separation of products difcult. Too
much water favors hydrolysis over transesterication. Additionally, the alkaline catalyst contaminates both the glycerol and the
biodiesel that are produced. Its removal from the biodiesel requires
several washings with water. This not only generates large amounts
of wastewater, but also gives rise to residual water in nal product
[6,7].
Several strategies can be used to overcome problems caused by
the presence of fatty acids in low-value feedstocks. It is possible to
carry out the process using catalysis with an inorganic acid [810],
a heterogeneous catalyst [11,12] or lipases [4,1316], since all can
simultaneously catalyze esterication and transesterication. It is
also possible to use a two-step process. In the rst step, the free fatty
acids are converted to esters using an acid catalyst (such as sulfuric
acid), with the remaining acylglycerols in the feedstock then being
converted to biodiesel esters by alkaline transesterication [17,18].

16

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

All of these processes transesterify the acylglycerols and esterify

the free fatty acid, while avoiding the formation of soaps. However,
acid catalysts are highly corrosive to equipment, while both heterogeneous catalysts and lipases are expensive and transesterication
rates are relatively slow [7]. Also, if there is a signicant amount of
water in the reaction medium then it will compete with the alcohol,
promoting the hydrolysis of the acylglycerols.
Other strategies have also been tried. Haas et al. [8] rst carried out an alkali-catalyzed saponication of soybean soapstock,
which converts both free fatty acids and the fatty acids of acylglycerols into soaps. The soaps were recovered and then acidied
to produce free fatty acids, which were then esteried in an
acid-catalyzed process. On the other hand, Wang et al. [19] acidied the soapstock, causing it to separate into an aqueous phase
and an acid oil phase containing free fatty acids and acylglycerols. This acid oil phase was then subjected to an acid-catalyzed
esterication/transesterication process. However, both of these
processes involve homogeneous catalysis with acids, which must
be separated from the product by successive washes, generating
wastewater contaminated with catalyst and reaction products.
Recently, the production of biodiesel by hydroesterication of
oils has been proposed [2023]. The process involves two steps.
In the rst step, triacylglycerols are hydrolyzed completely to fatty
acids and glycerol. In the second step, the fatty acids recovered from
the rst step are esteried with an alcohol to give the corresponding
ester and water. This process is advantageous for biodiesel production when low-value feedstocks are used. Since the rst step
is a hydrolysis step, the water content and the free fatty acid content of the feedstock do not interfere with nal yields. Additionally,
the aqueous glycerol produced in the hydrolysis step is more pure
than that obtained in the alkali-catalyzed transesterication process [21,22].
Although both the hydrolysis and esterication steps in a
hydroesterication process can be carried out chemically, several
authors have investigated the potential of using commercial or
plant lipases to catalyze the hydrolysis step [2123]. The present
work takes a different approach. We produce fatty acids by hydrolysis in subcritical water of several low-value feedstocks and we then
use lipases, produced by Burkholderia cepacia LTEB11, to catalyze
esterication with ethanol. Ethanol was selected as the alcohol
because it is less toxic than methanol and is abundantly available
in Brazil. In order to minimize the costs of the lipases, we use them
in the form of dried fermented solids, obtained by solid-state
fermentation.
2. Materials and methods
2.1. Raw materials
Soybean oil, soybean soapstock acid oil, beef tallow and waste
cooking oil were donated by the company Ubaldino Rodrigues
Soares e Cia. Ltda. (Ponta Grossa, Paran, Brazil). All other reagents
were of analytical grade.
2.2. Hydrolysis of fat feedstocks
Hydrolysis of the different feedstocks (Table 1) was carried out
in the pilot plant of the company Ubaldino Rodrigues Soares e Cia.
Ltda. (Ponta Grossa, Paran, Brazil). The process involves continuous hydrolysis of the feedstocks in the presence of subcritical water,
in a pressure tower at 60 atm and 250 C [24]. In this process, fatty
material and water react in a countercurrent ow in the absence
of catalyst. The free fatty acids produced are discharged from the
top of the tower and the water/glycerol mixture is discharged from
the bottom of the tower. The free fatty acids from each feedstock

were distilled and their compositions (Table 1) were determined

by gas chromatography [25]. All free fatty acid preparations contained less than 0.5% (w/w) water. Saponication and acid values
were determined according to the methods of the American Oil
Chemists Society (AOCS Ofcial Method Cd 3-25 and Ca 5a-40)
[26,27].
2.3. Microorganism
B. cepacia LTEB11 was maintained at 18 C in Luria Bertani (LB)
medium with 50% (w/v) glycerol. A stock culture was transferred
to a LB agar plate and incubated for 48 h at 29 C. Isolated colonies
were transferred to 30 mL of LB medium in a 250-mL Erlenmeyer
ask and then incubated on a rotary shaker at 29 C and 200 rpm
for 810 h, which represents mid-exponential phase. This culture
broth was used as inoculum for the solid-state fermentation.
2.4. Solid-state fermentation
The fermented solid was obtained by solid-state fermentation
(SSF) of a mixture of sugarcane bagasse and sunower seed meal
(1:1, w/w on a dry basis). Sugarcane bagasse was donated by Usina
de lcool Melhoramentos (Jussara, Paran, Brazil) and sunower
seed was purchased in the local market. Sugarcane bagasse and
sunower seed were milled, separately, in a knife mill, followed by
sieving to obtain particles ranging between 0.85 and 2.36 mm. The
SSF was done in 2000-mL Erlenmeyer asks, each containing 80 g of
milled dry substrate. Phosphate buffer solution (0.1 mol L1 , pH 7.0)
was added to obtain 75% moisture (w/w, wet basis). The moisture
content was determined in an infrared moisture balance (Gehaka IV
2000, So Paulo, Brazil). Flasks were plugged with cotton wool and
autoclaved at 121 C for 20 min. After cooling, the substrates were
inoculated with 8 mL of inoculum and incubated at 29 C. During
the fermentation, the hydrolytic and esterication activities of the
fermented solids were determined every 24 h. After incubation, the
fermented solids were dried (see Section 2.5) to a moisture content of less than 10% (w/w on a wet basis) and stored at 4 C. The
dry fermented solids were then used directly in all esterication
reactions.
2.5. Drying and preparation of the fermented solid
Three different drying processes were evaluated. In the rst,
frozen fermented solids were lyophilized for 24 h at 101 mbar and
40 C in a lyophilizer (Jouan LP3, Virginia, USA). In the second,
the fresh fermented solids were dried in a column made with two
integrated polyvinyl chloride tubes (each 50 cm height and 4.3 cm
diameter). The lower tube was lled with activated silica to dry
the air and the top contained 200 g of fresh fermented solids. Air at
approximately 25 C was blown at 20 L min1 into the bottom of the
column. In the third, 200 g of fresh fermented solids was placed in a
fan-forced laboratory oven (Nova tica 400-3ND, So Paulo, Brazil)
at 30 C. During the drying, the moisture content of the solids was
determined with the infrared moisture balance, and the hydrolytic
activity of the dried fermented solids was compared with that of
the fresh fermented solids. Values are expressed as the means of
triplicate analyses the standard error of the mean.
In reactions where n-hexane was used as the solvent, in order to
avoid interferences in the determination of the esterication activity, the fermented solids were delipidated after drying to remove
lipids deriving from the fermentation. Delipidation involved three
washes with n-hexane (10 mL per gram of fermented solids). In
each washing, the mixture was agitated vigorously for 10 min at
200 rpm and 25 C. The solution was then ltered and the retained
solids were dried in a vacuum desiccator at room temperature. In
the solvent-free reaction system, the delipidation was unnecessary

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

17

Table 1
Characterization of free fatty acids from hydrolysis in subcritical water.
Composition (% of free fatty acids)
FA-SO
Myristic
C14:0
C16:0
Palmitic
Palmitoleic
C16:1
Stearic
C18:0
Oleic
C18:1
Linoleic
C18:2
Others
Average molecular weight (g mol1 )
Acid value (mg KOH g1 )
Saponication value (mg KOH g1 )

7.9
0.6
4.8
30.1
52.6
4.0
278.6
191.3
196

FA-SSAO
16.5
4.2
33.4
44.2
1.7
277.3
195.5
199

FA-WCO

FA-BT

2.1
22.0
1.5
4.4
35.6
32.6
1.8
274.3
190.9
198

5.9
30.5
5.9
10.0
45.3
0.8
1.6
269.6
199.3
203

Fatty acids from soybean oil (FA-SO), soybean soapstock acid oil (FA-SSAO), waste cooking oil (FA-WCO) and beef tallow (FA-BT).

because the lipid content in the fermented solids was negligible

compared to the lipid content of the reaction mixture.
2.6. Activity measurement with the fermented solid
The hydrolytic activity of the dry fermented solids was
assessed by the titrimetric method. The solution contained triolein
(67 mmol L1 ), gum arabic 3% (w/v), CaCl2 (2 mmol L1 ), TrisHCl
(2.5 mmol L1 ) and NaCl (150 mmol L1 ) in distilled water [28]. It
was emulsied with a blender at high speed, initially for 5 min and
then for an additional 1 min immediately before use. For each assay,
20 mL of emulsion and 100 mg of fermented solids were placed in a
glass vessel maintained at 40 C. The free fatty acids released during
the reaction were titrated for 5 min in a Metrohm 718 STAT Titrino
potentiometric titrator (Metrohm, Herisau, Switzerland) with the
pH being maintained at 7.0 through the addition of a 0.05 mmol L1
NaOH solution. One unit (U) of hydrolytic activity was dened as
the release of 1 mol of fatty acid per minute, under the assay
conditions.
For esterication activity, reactions were carried out in 12 mL
screw-capped bottles containing 10 mL of a mixture containing
70 mmol L1 of fatty acid, 210 mmol L1 of ethanol (i.e. an acid to
alcohol molar ratio of 1:3) in n-hexane and 800 mg of delipidated
fermented solids. These bottles were incubated on a rotary shaker
at 200 rpm and 40 C. At xed intervals, 50 L samples of the mixture were removed and analyzed for residual free fatty acids by
the LowryTinsley method [29]. Ethyl ester production was calculated by consumption of free fatty acids from the reaction mixture.
One unit (U) of esterication activity equals 1 mol of ethyl ester
produced per minute, under the assay conditions.
Both hydrolytic and esterication activity were expressed in
units of activity per gram of dry fermented solid (U gdfs1 ).
2.7. Stability of the fermented solid
The stability of the dry fermented solids was evaluated by
measuring the hydrolytic activity before and after incubation in
different systems. The fermented solids were incubated for 48 h in
the following media: (a) ethanol, (b) n-hexane, (c) 70 mmol L1 of
oleic acid and 210 mmol L1 of ethanol in n-hexane and (d) oleic
acid and ethanol in a 1:3 molar ratio (i.e. solvent-free reaction mixture). For these tests, screw-capped bottles containing 500 mg of
fermented solids and 10 mL of reaction mixture were incubated on a
rotary shaker at 200 rpm and 40 C. After incubation, the fermented
solids were washed four times, each time with 10 mL of n-hexane,
to remove the substrates and reaction products. All samples were
then ltered and dried in a vacuum desiccator at room temperature. The residual hydrolytic activity, determined by the titrimetric
method using triolein as substrate, was expressed as percentage of
the initial hydrolytic activity.

2.8. Preliminary studies of biodiesel synthesis

Preliminary tests were done to dene the amount of fermented
solids in the reaction mixture and to compare the reaction in the
presence and absence of n-hexane as solvent. The reactions were
carried out in 12 mL screw-capped bottles with 10 mL of reaction
mixture (oleic acid and ethanol in a molar ratio of 1:3) and the
stated amount of fermented solids, with incubation at 200 rpm and
40 C. At xed intervals, 50 L samples were collected and analyzed
for residual free fatty acids by the LowryTinsley method [29]. Ester
(ethyl oleate) production was calculated by consumption of free
fatty acids from the reaction mixture.
2.9. Solvent-free esterication in a packed-bed reactor
The packed-bed reactor was made of a glass column (2.7 cm
internal diameter and 21 cm high) with an external jacket that
received water from a water bath at the stated temperature (Fig. 1).
The column was packed with 12 g (on a dry basis) of fermented
solids, with the solids being poured in through a funnel and then
lightly pressed with a glass rod. Undertaking this procedure with
this amount of fermented solids gave a bed height of 16 cm, such
that the bulk density of the bed was 130 g L1 . The reservoir (4.4 cm
internal diameter and 9 cm high) was loaded with 100 g of fatty
acids from soybean soapstock acid oil (FA-SSAO) (on the basis of
a molar mass of 277 g, this corresponds to 361 mmol) and 50 g of
ethanol (corresponding to 1089 mmol). A peristaltic pump (Stenner 45MHP10, Florida, USA) pumped the reaction mixture into the
bottom of the bed at a ow rate of 5 mL min1 . Samples of the reaction mixture were collected from the top of the packed-bed reactor
and washed with saturated NaCl solution to separate the excess of
ethanol. The upper organic fraction was collected and dried over
anhydrous Na2 SO4 and centrifuged. The conversion of the fatty
acids into ester was monitored by measuring the acid value determined by titration [27]. The content of ester was determined by GC
analysis.
For the study of the reutilization of the fermented solids in the
packed-bed reactor, the reaction was repeated for eight cycles, each
of 48 h. The experimental procedure, the amount of the fermented
solids and reaction mixtures used were as described above. After
each cycle, the reaction mixture was collected from the reactor and
replaced with a new mixture. Reaction yields after each cycle were
expressed as fractions of the yield obtained at the end of the rst
batch.
2.10. GC analysis
Ethyl ester content was determined using a GC-2010 (Shimadzu
Co., Kyoto, Japan) equipped with a hydrogen ame ionization detector and a SGE HT-5 capillary column (0.32 mm internal diameter,

10

120
100

80
6
60
4
40
2

20
0

0
0

24

48

72

96

Esterification activity (U gdfs-1)

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

Hydrolytic activity (U gdfs-1)

18

120

Time (h)
Fig. 2. Activity of the fermented solids from Burkholderia cepacia LTEB11 in solidstate fermentation. Key: () hydrolytic activity; () esterication activity. Values
plotted represent the mean of duplicate asks the standard error of the mean.

Fig. 1. Schematic representation of the packed-bed reactor system. Key: (1) reservoir; (2) reaction mixture fed to reactor; (3) peristaltic pump; (4) glass column
packed with fermented solids; (5) reaction mixture removed from reactor; (6) sampling port; (7) water jacket.

25 m length and 0.1 mm lm thickness). Prepared sample (56 mg)

was diluted in 1 mL of an internal standard solution of methyl
heptadecanoate (1 mg mL1 ) in n-heptane. Then 1 L was injected,
with a split ratio of 1:50, using N2 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 C min1 to 180 C, 180 C maintained for 3 min, heating at 5 C min1 to 230 C, 230 C maintained
for 2 min. Peaks in the chromatograms were identied 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 [25].

to reduce costs, we evaluated alternative drying processes. Before

drying, the fresh fermented solids, obtained at 72 h of fermentation,
had a moisture content of 72% (w/w, wet basis) with a hydrolytic
activity of 83 5 U gdfs1 . Although the solids were intended for
use in esterication reactions, the hydrolytic activity was determined in this experiment because the high water content of the
original moist solids used as the control meant that it was not
possible to undertake the assay for esterication activity.
The hydrolytic activity of the fermented solids was maintained
after lyophilization at 4 C for 24 h (90 4 U gdfs1 ) and after drying with dry air in a column at 25 C for 5 h (84 6 U gdfs1 ). The
activity decreased to 37 5 U gdfs1 for the sample dried in a fanforced laboratory oven at 30 C for 8 h. Drying in the column was
selected as the drying method for the remaining studies. The maintenance of 100% of activity with air drying suggests that the lipolytic
activity in fermented solids obtained with B. cepacia LTEB11 is more
stable during the drying step than that in the fermented solids
obtained with Rhizopus, the only other organism for which similar
data are available. Dry air and lyophilization each caused around
20% loss of lipolytic activity for fermented solids from Rhizopus sp.
IRD43aIV [31], while oven drying and lyophilization each caused
around 33% loss of lipolytic activity for fermented solids from Rhizopus microsporus CPQBA 312-07 DRM [32].
3.3. Stability of the fermented solid in organic solvents

3. Results
3.1. Production of fermented solids
B. cepacia LTEB11 was cultivated on a 1:1 mixture (w/w) of sugarcane bagasse and sunower seed meal, with both the hydrolytic
and esterication activities of the fermented solids being measured
over time. The highest esterication activity was 5.8 0.3 U gdfs1 ,
obtained at 72 h (Fig. 2). At this time the hydrolytic activity was
91.6 3.3 U gdfs1 , although it did reach a slightly higher value at
96 h. Since the fermented solids were intended for use in esterication reactions, a fermentation time of 72 h was selected for the
remaining studies.
3.2. Stability of the hydrolytic activity during drying of the
fermented solid
After fermentation, the fermented solids must be dried for
storage. Previously in our laboratory they have been dried by
lyophilization [14,30], but this is an expensive process. In order

The immobilized lipase from B. cepacia is known for its stability

in organic media [3336]. However, since the stability of the lipase
present in the fermented solids has not been studied, we evaluated
it in the various solvents and reaction mixtures used in this work.
The residual lipolytic activity of the fermented solids remained
above 95% after 8 h of incubation in ethanol and in solvent-free
reaction mixture (Fig. 3). In n-hexane and in reaction mixture with
n-hexane, the activity initially increased slightly, remaining above
100% after 8 h incubation. The increase of the catalytic activity of
immobilized lipases after treatment with hydrophobic organic solvents has been observed previously and is attributed to residual
solvent molecules maintaining the lipase in its open conformation,
thereby facilitating the access of the substrates to the active site in
the activity assay [35,3739]. In ethanol and in solvent-free reaction media, the residual activities remained above 80% after 24 h
incubation and above 72% after 48 h incubation. These results conrm that the lipolytic activity of the fermented solids has similar
stability in organic media to that of immobilized B. cepacia lipase
[33]. For example, the residual lipolytic activity of a lipase from

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

Substrate conversion (%)

Residual activity (%)

140
120
100
80
60
40
20

19

100
80
60
40
20
0

0
0

12

24

36

24

48

48

Time (h)
Fig. 3. Stability of the fermented solids after incubation in different systems. Key:
Residual activity in () ethanol; () n-hexane; () 70 mmol L1 of oleic acid and
210 mmol L1 of ethanol in n-hexane (i.e. reaction mixture containing solvent); ()
oleic acid and ethanol in a 1:3 molar ratio (i.e. solvent-free reaction mixture). Conditions: 500 mg of dry fermented solids in 10 mL of solution, incubated at 40 C
and 200 rpm. Activities were determined relative to an initial activity of 84 U gdfs1
determined by the titrimetric method, using triolein as the substrate. Values plotted
represent the mean of triplicate analyses the standard error of the mean.

B. cepacia immobilized on a macroporous resin ranged from 80 to

120% after incubation for 4 h in methanol, ethanol and acetone [36].
3.4. Ester production in the presence and absence of n-hexane
High yields of methyl or ethyl esters have been reported for
transesterication and esterication reactions catalyzed by the
lipase from B. cepacia LTEB11 in systems containing solvents
[30,33,40]. In previous investigations of our group into the esterication of oleic acid with ethanol in n-heptane, we obtained
essentially 95100% ester yield in 13 h using B. cepacia LTEB11
immobilized on Accurel [33,40] and 94% in 18 h with dry fermented
solids of B. cepacia [30]. In the current work, we investigated
whether it would be possible to remove the solvent from the system
while maintaining acceptable conversion rates.
In the solvent-free reaction mixture, the conversion was only
82% after 88 h, compared to a 92% conversion after 8 h obtained
with n-hexane as the solvent (Table 2). However, due to the higher
concentrations within the reaction mixture, the ester productivity
for the solvent-free system was essentially twice as high as that
obtained for the reaction with n-hexane. Additional advantages
of the solvent-free system are that, rstly, it uses a much higher
ratio of substrate to fermented solids and, secondly, it avoids the
need for the recovery and recycling of the solvent, thereby lowering
processing costs.

3.6. Esterication activity of the fermented solids on different

fatty acid sources
We evaluated the esterication activity of the fermented solids
using free fatty acids of different chain lengths, as well as free fatty

96

Fig. 4. Effect of the fermented solids content on the esterication reaction in a

solvent-free system. Key: () 9%; () 12%; () 15% (mass of fermented solids as
a percentage of the mass of oleic acid). Reaction conditions: oleic acid 1890 mmol,
ethanol 5670 mmol, 40 C, 200 rpm. Values plotted represent the mean of triplicate
analyses the standard error of the mean.

acids obtained through the hydrolysis of soybean oil, soybean soapstock acid oil, beef tallow and waste cooking oil (denoted as FA-SO,
FA-SSAO, FA-BT and FA-WCO, respectively). Esterication activity
with free fatty acids was not directly related to the acyl chain length
(Fig. 5). The highest activities were observed with palmitic acid
(12.7 U gdfs1 ) and caprylic acid (11.7 U gdfs1 ). Higher esterication activities with palmitic acid have been obtained previously for
Pseudomonas cepacia lipase (LPS A001526) in AOT microemulsion
systems [41] and B. cepacia lipase (LPS AR01520) immobilized on
Accurel EP-100 [42].
The reasonably high esterication activity obtained with FASSAO (7.0 U gdfs1 ) is encouraging, since it is a byproduct generated
during the rening of soybean oil [8,4346]. Its low cost may make it
an appropriate feedstock for biodiesel production [8]. It was therefore selected as the substrate for the experiments in the packed-bed
reactor.
3.7. Effect of temperature on biodiesel synthesis in a packed-bed
reactor
Temperatures from 40 to 60 C have been utilized for the transesterication of soybean oil catalyzed by fermented solids from B.
cepacia LTEB11 [14], transesterication of beef tallow and babassu
oil by P. cepacia lipase immobilized in polysiloxane-polyvinyl alcohol (SiO2 -PVA) [47], esterication of lauric acid by an immobilized
P. cepacia lipase from Amano [35] and regioselective acylation of

Free fatty acids

3.5. Effect of the amount of fermented solids on esterication

The effect of the amount of fermented solids in the reaction mixture (9, 12 and 15% of fermented solids, expressed relative to the
mass of oleic acid) was evaluated in the solvent-free system. The
rate of production of ethyl oleate increased signicantly with an
increase in the amount of fermented solids from 9 to 12% (Fig. 4).
However, a further increase in the fermented solids, from 12 to 15%,
did not increase the reaction rate signicantly. Higher amounts of
solids made it difcult to agitate the reaction mixture. The addition
of 12% fermented solids was used in the remaining studies.

72

Time (h)

FA-BT
FA-SSAO
FA-SO
FA-WCO
FA - C18:2
FA - C18:1
FA - C18:0
FA - C16:0
FA - C14:0
FA - C12:0
FA - C8:0
FA - C6:0
0

12

15

Esterification activity (U gdfs-1)

Fig. 5. Esterication activity of the fermented solids against different fatty acids.
Conditions: 10 mL of reaction mixture with n-hexane (70 mmol L1 of fatty acid and
210 mmol L1 of ethanol), 800 mg of fermented solids, 40 C, 200 rpm. Values plotted
represent the mean of duplicate analyses the standard error of the mean.

20

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

Table 2
Esterication reactions carried out in shake asks in the presence and in the absence of solvent.
Solventa

Reaction mixture
Oleic acid
Ethanol
Fermented solids
Results
Ratio fermented solids/oleic acid (g/g)
Results
Conversion in time taken
Productivity

Solvent-free

Mass (mg)

Molar concentration (mmol L1 )

Mass (mg)

Amount (mmol)

198
97
500

70
210

5760
2813
500

20.4
61.2

2.5

0.09
92% in 8 h
50 mg gdfs1 h1

82% in 88 h
118 mg gdfs1 h1

Reactions were done in shake asks with 10 mL of reaction mixture (molar ratio of oleic acid: ethanol of 1:3), at 40 C and 200 rpm.
a
Sufcient n-hexane was added to give a total volume of 10 mL.

andrographolide by immobilized B. cepacia lipase from Amano [48].

This temperature range was therefore tested for the esterication
of FA-SSAO with ethanol in the packed-bed reactor. The initial reaction rate (based on the conversions obtained at 12 h) increased with
increasing temperature (Fig. 6). However, at 60 C the conversions
for reaction times above 24 h were lower than those obtained at
the lower temperatures, probably due to a higher degree of denaturation. At 50 C, a 92% conversion of FA-SSAO was obtained at
31 h; at the other temperatures the conversions were still below
85% after 40 h of reaction.
3.8. Operational stability of the fermented solids in the
packed-bed reactor
We used the same fermented solids in successive esterication
cycles of 48 h in the packed-bed reactor at 50 C (Fig. 7). In the fth
cycle, the conversion remained above 84% (which is above 90% of
the value of 92% conversion that was obtained in the rst cycle). In
an attempt to keep the original conversion for a larger number of
cycles, we redid the reutilization experiment at 45 C (Fig. 7). In this
case the conversion remained above 90% of the initial conversion
during six cycles.

obtained by de Sousa et al. [22] and Chen et al. [50], which gave
biodiesel properties that met the Brazilian [51] and European [52]
standards, respectively.
4. Discussion
This work makes two contributions in the area of hydroesterication. Firstly, this is the rst study of hydroesterication using
soybean soapstock acid oil (SSAO) as the feedstock. Secondly, this
is the rst study of hydroesterication in which the esterication
step has been undertaken using enzymatic catalysis. The work also
makes two contributions in the area of lipase-catalyzed esterication of fatty acids. Firstly, although lipolytic fermented solids
have been used previously to catalyze the esterication of fatty
acids, this is the rst study that has done this in a solvent-free system. Secondly, lipase-catalyzed transesterication reactions for the
production of biodiesel in solvent-free systems have been carried
out in packed-bed bioreactors, but lipase-catalyzed esterication
reactions have not previously been studied in this system. These
contributions, which are discussed below, combine to demonstrate
the potential of using fermented solids to catalyze the esterication step in a hydroesterication process that uses SSAO as the
feedstock.

3.9. Composition of the ethyl esters

4.1. Contributions in the area of hydroesterication

Relative conversion (%)

Conversion (%)

100

One of the strategies to reduce the cost of biodiesel production is

to use low-value feedstocks. In the current work we demonstrated
that SSAO is an adequate feedstock for hydroesterication, with

80
60
40
20
0

110
100
90
80
70
60
50
40
30
20
10
0

100
90
80
70
60
50
40
30
20
10
0
1

12

24

36

48

Time (h)
Fig. 6. Effect of temperature on esterication in the packed-bed reactor. Key: ()
40 C; () 50 C; () 60 C. Reaction conditions: 12 g of dry fermented solids, 100 g of
fatty acids from soybean soapstock acid oil and 50 g of ethanol (i.e. molar ratio of fatty
acid to ethanol of 1:3), recirculation rate of 5 mL min1 . Values plotted represent the
mean of duplicate analyses the standard error of the mean.

Original conversion (%)

The properties of biodiesel are directly inuenced by the fatty

ester composition [49]. The ethyl ester that was obtained in this
work by esterifying FA-SSAO with ethanol contained, by mass,
16.5% of ethyl palmitate, 4.2% of ethyl stearate, 33.4% of ethyl oleate
and 44.2% of ethyl linoleate. This composition is similar to those

Cycles
Fig. 7. Operational stability of the fermented solids in the packed-bed reactor. The
same fermented solids were used to catalyze the esterication reaction in 48-h
cycles. Key: reaction temperature of () 45 C and () 50 C. Reaction conditions:
12 g of dry fermented solids, 100 g of fatty acids from soybean soapstock acid oil and
50 g of ethanol (i.e. molar ratio of fatty acid to ethanol of 1:3), recirculation rate of
5 mL min1 .

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

21

Table 3
Studies of biodiesel production by hydroesterication with an enzymatic step.
Reference

Cavalcanti-Oliveira et al.
[21]

de Sousa et al. [22]

Talukder et al. [23]

This work

Hydrolysis step
Catalyst (% weight of oil)

Subcritical water
Free of catalyst

30 C & 1 atm
Waste cooking oil:water
(1:1)

250 C & 60 atm

Water:soybean soapstock
acid oil (1:1)

Conversion in time taken

89% in 48 h

Enzymatic
VEEG from physic nut
(10%)
40 C & 1 atm
TrisHCl
0.1 mol L1 :physic nut oil
(9:1)
98% in 2 h

Enzymatic
Candida rugosa (0.05%)

Temperature & pressure

Substrates (ratio in v:v)

Enzymatic
Thermomyces lanuginos
(liquid lipase, 2.3%)
60 C & 1 atm
Water:soybean oil (1:1)

100% in 10 h

95% in 1 h

Esterication step

Chemical

Chemical

Chemical

Enzymatic

Catalyst (% weight of fatty acid)

Temperature & pressure
Reaction mixture (molar ratio)

Niobic acid (20%)

200 C & 24 atm
FA/methanol (1:3)

Niobic acid (20%)

200 C & 34 atm
FA/methanol (1:3)

Fermented solid (12%)

50 C & 1 atm
FA/ethanol (1:3)

Conversion in time taken

92% in 1 h

97% in 2 h

*Amberlyst 15 (100%)
60 C & 1 atm
FA/methanol (4:1), in
isooctane
99% in 2 h

93% in 31 h

VEEG: vegetable enzyme extract from germinated seeds; FA: fatty acids from hydrolysis; *Amberlyst 15: acidic styrene-divinylbenzene sulfonated ion-exchange resin.

the free fatty acids that were obtained after its hydrolysis with
subcritical water being efciently converted into their ethyl esters.
Soapstock is a byproduct from the alkaline neutralization step of
vegetable oil rening and represents about 6% of the volume of
the original crude vegetable oil [43]. In a typical industrial process,
the soybean soapstock is acidied with sulfuric acid for emulsion
breaking and then separates into two phases, an aqueous phase and
the acid oil phase. The acid oil contains, by weight, 59% free fatty
acids, 28% triacylglycerol, and around 5% di- and monoacylglycerol
[8]. It sells for approximately half the cost of rened vegetable oils
[43].
Our work demonstrates, for the rst time, that acid oil derived
from soybean soapstock can be used as a feedstock for hydroesterication. Despite the fact that previous authors have recognized
that hydroesterication is a promising technology for producing
biodiesel from low-value feedstocks, two of the previous studies of
the production of biodiesel esters in hydroesterication processes
that have an enzymatic step have used relatively pure oils, while
one used waste cooking oil (Table 3).
Additionally, the three studies listed in Table 3 all involve an
enzymatic oil hydrolysis step followed by a chemically catalyzed
esterication step. This will be referred to as the enzymatichydrolysis/chemical-esterication (EH/CE) strategy. The authors
justify this strategy by pointing out that the hydrolysis step involves
mild conditions of temperature and pressure (3060 C, 1 atm).
However, under these conditions long reaction times are normally
required, especially at the volumetric ratio of water to oil of 1:1
used by Cavalcanti-Oliveira et al. [21] and Talukder et al. [23],
although a 98% conversion in 2 h has been achieved with a water
to oil volumetric ratio of 9:1 [22]. In addition, unlike the mild
conditions used in the hydrolysis step, the esterication step typically involves either high pressures (2434 atm) and temperatures
(200 C) [21,22] or the use of solvents such as isooctane [23].
The hydroesterication process studied in the present work
inverts the strategy of these previous studies, in that the initial hydrolysis step is a chemical step while the esterication
is catalyzed enzymatically. This will be referred to as the
chemical-hydrolysis/enzymatic-esterication (CH/EE) strategy.
This strategy has two advantages over the EH/CE strategy. Firstly,
soapstocks can contain contaminants, such as sodium or potassium
salts [43], sulfur, phosphorous and metal ions [53]. In the EH/CE
strategy, the enzymes used in the initial hydrolysis step are exposed
to these contaminants and may be inactivated. These problems
were not faced by de Sousa et al. [22] and Cavalcanti-Oliveira et al.
[21] in their studies of the EH/CE strategy because the oils they used
were of relatively high quality. In contrast, in the CH/EE strategy the

hydrolysis is carried out with subcritical water, such that there is no

catalyst to suffer inactivation, and the lipases in the esterication
step receive a contaminant-free fatty acid stream. Secondly, the
three studies using the EH/CE strategy that have been published to
date have involved batch operation, with the enzymatic preparation being used to catalyze the hydrolysis of a single batch of oil
[2123]. Although we also used the packed-bed bioreactor in batch
mode, the maintenance of over 80% conversion in seven successive
batches opens up the possibility of continuous operation.
Our CH/EE hydroesterication process also has some advantages in comparison to lipase-catalyzed transesterication. Firstly,
lipase-catalyzed transesterication in solvent-free media typically
involves relatively long reaction times (Table 4). Although we used
48-h cycles for the esterication reaction, once optimized, it should
be possible to reduce the reaction time to a few hours, even in
solvent-free media, since esterication is typically much faster than
transesterication [13,14,54]. Secondly, in enzymatic transesterication, the glycerol absorbs onto the catalyst, causing mass transfer
limitations [5,5558], while this problem is avoided in hydroesterication processes. This explains why the operational stability of the
packed-bed reactor for the esterication of FA-SSAO was higher
than that reported for the transesterication of soybean oil catalyzed by fermented solids from B. cepacia LTEB11 [14], where the
original conversion of 95% was maintained only for three cycles,
with the conversion decreasing to 62% after six cycles.
Although the high cost of commercial lipases remains as one of
the main barriers against their use in industrial processes for the
production of biodiesel, the use of fermented solids in our process
has the potential to reduce lipase costs signicantly. The nal product of the solid-state fermentation is simply subjected to a mild
air drying, thus avoiding the costs of recovery and immobilization
of the lipase that are associated with the production of immobilized lipases that are produced by submerged liquid fermentation.
Costs could be decreased further if it were possible to replace the
sunower seed meal with a cheaper inducer of lipase production
[30,59].
4.2. Contributions in the area of esterication of fatty acids
The current work represents the rst time that fermented solids
containing lipases have been used to catalyze esterication in a
solvent-free system. Previous studies of esterication with fermented solids have involved the use of organic solvents such as
n-heptane [30,60] and n-hexane [31]. Although high conversions
can be achieved in short times in the presence of solvents, the volumetric productivity of such reactions is lower, due to the much

22

D. Soares et al. / Biochemical Engineering Journal 81 (2013) 1523

Table 4
Studies of solvent-free biodiesel production by lipases in packed-bed reactors.
Microorganism

Support

Reaction mixture

Conversion in time taken

System

Reference

Burkholderia cepacia LTEB11

Burkholderia cepacia LTEB11
Rhizopus oryzae
Candida antarctica Novozyme 435
Candida antarctica Novozyme 435
Candida antarctica Novozyme 435
Pseudomonas cepacia
a
Recombinant Aspergillus oryzae

Fermented solid
Fermented solid
Polyurethane foam
Acrylic resin
Acrylic resin
Acrylic resin
Fe3 O4 nanoparticle
Polyurethane foam

FA-SSAO + ethanol
Soybean oil + ethanol
Soybean oil + methanol
Rapeseed oil + methanol
Waste oil + methanol
Rapeseed and soybean oils + methanol
Soybean oil + methanol
Rapeseed and soybean oils + methanol

93% in 31 h
95% in 46 h
91% in 50 h
99% in 72 h
90%, time not mentioned
9699%, time not mentioned
>88%, 192 h residence time
96% in 14 h

Batch
Batch
Batch
Batch
Continuous
Continuous
Continuous
Batch

This work
[14]
[13]
[54]
[55]
[56]
[57]
[58]

Aspergillus oryzae expressing Fusarium heterosporum lipase was immobilized within biomass support particles (BSPs), made of reticulated polyurethane foam.

lower substrate concentrations. In addition, if solvents are used,

then it is essential to recover them at the end of the process, and
this extra step increases process costs.
Although packed-bed reactors have been used to produce
biodiesel using lipases in solvent-free systems, previous studies
have all involved transesterication (Table 4). The present study
represents the rst time that lipase-catalyzed esterication in a
solvent-free system has been carried out in a packed-bed reactor.
Packed-bed reactors allow high catalyst to substrate ratios, while
avoiding the mixing problems when the same ratios are used in
agitated reactors. For example, in the current work, at 40 C and
with the same ratio of fermented solid to fatty acids, namely 12%
(w/w), 74 h were required for 84% conversion in shake asks, while
this conversion was reached in only 43 h in the packed-bed reactor.
5. Conclusions
The current work has proposed a potential new route to lower
the costs of production of biodiesel using lipases, namely the
hydroesterication of low-value feedstocks, such as soybean soapstock acid oil. The rst step involves hydrolysis in subcritical water;
the second step involves esterication of the fatty acids in a solventfree system, using a reactor packed with a fermented solid that
contains lipases produced by B. cepacia. Conversion to ester of over
80% can be obtained in each of seven cycles of 48 h. Further studies
involving scale-up of the packed-bed bioreactor and characterization of the biodiesel are currently under investigation in our
laboratory.
Acknowledgements
This research was supported by a grant from CNPq (Conselho Nacional de Desenvolvimento Cientco e Tecnolgico), a
Brazilian government agency for the advancement of science and
technology. Diniara Soares, Alan Guilherme Goncalves, Andrei Ferreira Pinto, David Mitchell and Nadia Krieger also thank CNPq for
research scholarships. We are also grateful to Ubaldino Rodrigues
Soares, for the supply of the fatty feedstocks and the use of the pilot
plant for the subcritical water hydrolysis step.
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