-
Biochemical Engineering Journal 81 (2013) 15 23
Contents lists available at ScienceDirect
Biochemical Engineering Journal
jou rna l h om epage: www.elsev ier .c
Regular article
Biodies acisubcrit esfermen
Diniara S nc aDavid Ala Departament tro Pob Departament 8153c
Departament tnico
a r t i c l
Article history:Received 19 FeReceived in reAccepted 26
September 2013Available online 5 October 2013
Keywords:BiodieselHydroestericationLipasesBurkholderia
cSolid-state ferPacked-bed re
cation feedment
ethyl esters. The fermented solids were produced by cultivating
Burkholderia cepacia LTEB11 for 72 h ona 1:1 mixture, by mass, of
sugarcane bagasse and sunower seed meal. The esterication of fatty
acidsobtained from soybean soapstock acid oil was studied in the
packed-bed bioreactor, in a solvent-free sys-tem, with the best
results being a 92% conversion in 31 h, obtained at 50 C. When the
packed-bed reactorwas reused in successive 48-h esterication
reactions, conversions of over 84% of the fatty acids to esters
1. Introdu
Biodiesepetrodieseluse requiriin Brazil, w5% biodiese(methyl or
stocks usedare derivedever, these in biodieselcosts, it is to
increasePotential loresidual oil
Current vegetable o
CorresponE-mail add
1369-703X/$ http://dx.doi.oepaciamentationactor
were maintained for ve cycles at 50 C and for six cycles at 45
C. Unlike previous hydroestericationprocesses that have used
lipase-catalyzed hydrolysis followed by chemically-catalyzed
esterication, ourprocess 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 continuousmode.
2013 Published by Elsevier B.V.
ction
l is currently being produced as a substitute for, however, it
is not economically competitive, with itsng either subsidies or
government policies, such ashere all petrodiesel is currently
required to containl [1]. It is composed of esters of short-chain
alcoholsethyl alcohols) and long-chain fatty acids. The feed-
in most commercial biodiesel production processes from
triacylglycerols of edible vegetable oils [2]. How-oils are
relatively expensive, and since the feedstock
synthesis corresponds to 5085% of total productiondesirable to
use low-cost starting materials, in order
the commercial competitiveness of biodiesel [35].w-value
feedstocks include animal fat from sewage and
of domestic, industrial or commercial origin.industrial
processes for the production of biodiesel fromils use alkaline
transesterication, which gives high
ding author. Tel.: +55 41 33613470; fax: +55 41 33613006.ress:
[email protected] (N. Krieger).
yields (98%) in a short reaction time of about 1 h. However,
alka-line transesterication requires starting materials with low
levelsof moisture and free fatty acids and therefore is not
appropriate forthe production of biodiesel from low-value
feedstocks, which con-tain signicant amounts of free fatty acids or
water. Free fatty acidsreact with the alkaline catalyst, producing
soaps. This decreases thereaction yield and makes the separation of
products difcult. Toomuch water favors hydrolysis over
transesterication. Addition-ally, the alkaline catalyst
contaminates both the glycerol and thebiodiesel that are produced.
Its removal from the biodiesel requiresseveral washings with water.
This not only generates large amountsof wastewater, but also gives
rise to residual water in nal product[6,7].
Several strategies can be used to overcome problems caused bythe
presence of fatty acids in low-value feedstocks. It is possible
tocarry out the process using catalysis with an inorganic acid
[810],a heterogeneous catalyst [11,12] or lipases [4,1316], since
all cansimultaneously catalyze esterication and transesterication.
It isalso possible to use a two-step process. In the rst step, the
free fattyacids are converted to esters using an acid catalyst
(such as sulfuricacid), with the remaining acylglycerols in the
feedstock then beingconverted to biodiesel esters by alkaline
transesterication [17,18].
see front matter 2013 Published by Elsevier
B.V.rg/10.1016/j.bej.2013.09.017el production from soybean
soapstock ical water followed by lipase-catalyzedted solid in a
packed-bed reactor
oaresa, Andrei Ferreira Pintob, Alan Guilherme Goexander
Mitchell a, Nadia Kriegerb,
o de Bioqumica e Biologia Molecular, Universidade Federal do
Paran, Cx. P. 19046 Ceno de Qumica, Universidade Federal do Paran,
Cx. P. 19081 Centro Politcnico, Curitibao de Farmcia, Universidade
Federal do Paran, Av. Lothario Meissner, 3400, Jardim Bo
e i n f o
bruary 2013vised form 30 June 2013
a b s t r a c t
We investigated a new hydroesterifeedstocks: complete hydrolysis
of thea packed-bed reactor, containing a ferom/ locate /be j
d oil by hydrolysis interication using a
lvesc,
litcnico, Curitiba 81531-980, Paran, Brazil1-980, Paran, Brazil,
Curitiba, Paran, Brazil
strategy for the production of biodiesel from low-value oilstock
to fatty acids in subcritical water, followed by the use ofed solid
with lipase activity, to convert the fatty acids to their
-
16 D. Soares et al. / Biochemical Engineering Journal 81 (2013)
15 23
All of these processes transesterify the acylglycerols and
esterifythe free fatty acid, while avoiding the formation of soaps.
However,acid catalysts are highly corrosive to equipment, while
both hetero-geneous catalysts and lipases are expensive and
transestericationrates are rewater in thepromoting
Other stried out anwhich convglycerols into
produceacid-catalyzied the soand an acierols. This
aestericatioprocesses inbe separatewastewater
Recentlyoils has beeIn the rst sacids and glthe rst stepester
and wduction whis a hydrolytent of the fthe aqueouthan that ocess
[21,22]
Althoughydroesteriauthors haplant lipasework takes sis in
subcriuse lipases,estericatiobecause it iin Brazil. Inin the
formfermentatio
2. Materia
2.1. Raw m
Soybeancooking oilSoares e Ciawere of ana
2.2. Hydrol
Hydrolyin the pilot Ltda. (Pontaous hydrolyin a pressurmaterial
anof catalyst. top of the tothe bottom
were distilled and their compositions (Table 1) were
determinedby gas chromatography [25]. All free fatty acid
preparations con-tained less than 0.5% (w/w) water. Saponication
and acid valueswere determined according to the methods of the
American Oil
sts S].
icroo
epacim w
agarransfnd th0 h, as u
lid-s
fermf a m/w oool Mas per s
to os don
dry sded t wao Pved
ated mented sted
lessmenns.
rying
ee d ferm
in ash feted
ter). and imatn. In tced lC. Duined
y of tsh fete aneactinterf
fermeriv
s witashi
m anwereventlatively slow [7]. Also, if there is a signicant
amount of reaction medium then it will compete with the alcohol,the
hydrolysis of the acylglycerols.rategies have also been tried. Haas
et al. [8] rst car-
alkali-catalyzed saponication of soybean soapstock,erts both
free fatty acids and the fatty acids of acyl-to soaps. The soaps
were recovered and then acidied
free fatty acids, which were then esteried in aned process. On
the other hand, Wang et al. [19] acid-apstock, causing it to
separate into an aqueous phased oil phase containing free fatty
acids and acylglyc-cid oil phase was then subjected to an
acid-catalyzedn/transesterication process. However, both of
thesevolve homogeneous catalysis with acids, which mustd from the
product by successive washes, generating
contaminated with catalyst and reaction products., the
production of biodiesel by hydroesterication ofn proposed [2023].
The process involves two steps.tep, triacylglycerols are hydrolyzed
completely to fattyycerol. In the second step, the fatty acids
recovered from
are esteried with an alcohol to give the correspondingater. This
process is advantageous for biodiesel pro-en low-value feedstocks
are used. Since the rst stepsis step, the water content and the
free fatty acid con-eedstock do not interfere with nal yields.
Additionally,s glycerol produced in the hydrolysis step is more
purebtained in the alkali-catalyzed transesterication pro-.h both
the hydrolysis and esterication steps in acation process can be
carried out chemically, severalve investigated the potential of
using commercial ors to catalyze the hydrolysis step [2123]. The
presenta different approach. We produce fatty acids by
hydroly-tical water of several low-value feedstocks and we then
produced by Burkholderia cepacia LTEB11, to catalyzen with
ethanol. Ethanol was selected as the alcohols less toxic than
methanol and is abundantly available
order to minimize the costs of the lipases, we use them of dried
fermented solids, obtained by solid-staten.
ls and methods
aterials
oil, soybean soapstock acid oil, beef tallow and waste were
donated by the company Ubaldino Rodrigues. Ltda. (Ponta Grossa,
Paran, Brazil). All other reagentslytical grade.
ysis of fat feedstocks
sis of the different feedstocks (Table 1) was carried outplant
of the company Ubaldino Rodrigues Soares e Cia.
Grossa, Paran, Brazil). The process involves continu-sis of the
feedstocks in the presence of subcritical water,e tower at 60 atm
and 250 C [24]. In this process, fattyd water react in a
countercurrent ow in the absenceThe free fatty acids produced are
discharged from thewer and the water/glycerol mixture is discharged
from
of the tower. The free fatty acids from each feedstock
Chemi[26,27
2.3. M
B. cmediuto a LBwere task afor 81broth w
2.4. So
The(SSF) o(1:1, wde lcseed wsunowsievingSSF wamilledwas
adconten2000, Sautoclainoculthe ferfermenfermentent ofdry
ferreactio
2.5. D
Thrfrozen40 Cthe freintegradiamethe airapproxcolumfan-forat
30
determactivitthe fretriplica
In ravoid iity, thelipids dwasheeach w200 rpsolids the solociety
(AOCS Ofcial Method Cd 3-25 and Ca 5a-40)
rganism
a LTEB11 was maintained at 18 C in Luria Bertani (LB)ith 50%
(w/v) glycerol. A stock culture was transferred
plate and incubated for 48 h at 29 C. Isolated colonieserred to
30 mL of LB medium in a 250-mL Erlenmeyeren incubated on a rotary
shaker at 29 C and 200 rpmwhich represents mid-exponential phase.
This culturesed as inoculum for the solid-state fermentation.
tate fermentation
ented solid was obtained by solid-state fermentationixture of
sugarcane bagasse and sunower seed mealn a dry basis). Sugarcane
bagasse was donated by Usinaelhoramentos (Jussara, Paran, Brazil)
and sunowerurchased in the local market. Sugarcane bagasse andeed
were milled, separately, in a knife mill, followed bybtain
particles ranging between 0.85 and 2.36 mm. Thee in 2000-mL
Erlenmeyer asks, each containing 80 g ofubstrate. Phosphate buffer
solution (0.1 mol L1, pH 7.0)to obtain 75% moisture (w/w, wet
basis). The moistures determined in an infrared moisture balance
(Gehaka IVaulo, Brazil). Flasks were plugged with cotton wool
and
at 121 C for 20 min. After cooling, the substrates werewith 8 mL
of inoculum and incubated at 29 C. Duringtation, the hydrolytic and
esterication activities of theolids were determined every 24 h.
After incubation, thesolids were dried (see Section 2.5) to a
moisture con-
than 10% (w/w on a wet basis) and stored at 4 C. Theted solids
were then used directly in all esterication
and preparation of the fermented solid
ifferent drying processes were evaluated. In the rst,ented
solids were lyophilized for 24 h at 101 mbar and
lyophilizer (Jouan LP3, Virginia, USA). In the second,rmented
solids were dried in a column made with twopolyvinyl chloride tubes
(each 50 cm height and 4.3 cmThe lower tube was lled with activated
silica to drythe top contained 200 g of fresh fermented solids. Air
ately 25 C was blown at 20 L min1 into the bottom of thehe third,
200 g of fresh fermented solids was placed in aaboratory oven (Nova
tica 400-3ND, So Paulo, Brazil)ring the drying, the moisture
content of the solids was
with the infrared moisture balance, and the hydrolytiche dried
fermented solids was compared with that ofrmented solids. Values
are expressed as the means ofalyses the standard error of the
mean.
ons where n-hexane was used as the solvent, in order toerences
in the determination of the esterication activ-ented solids were
delipidated after drying to remove
ing from the fermentation. Delipidation involved threeh n-hexane
(10 mL per gram of fermented solids). Inng, the mixture was
agitated vigorously for 10 min atd 25 C. The solution was then
ltered and the retained
dried in a vacuum desiccator at room temperature. In-free
reaction system, the delipidation was unnecessary
-
D. Soares et al. / Biochemical Engineering Journal 81 (2013) 15
23 17
Table 1Characterization of free fatty acids from hydrolysis in
subcritical water.
Composition (% of free fatty acids)
FA-SO FA-SSAO FA-WCO FA-BT
Myristic Palmitic 16.5Palmitoleic Stearic 4.2Oleic 33.4Linoleic
44.2Others 1.7Average mol 277.3Acid value (m 195.5Saponicatio
199
Fatty acids fro -WCO
because thecompared t
2.6. Activity
The hydassessed by(67 mmol L
(2.5 mmol Lwas emulsithen for an a20 mL of emglass vesselthe
reactionpotentiomepH being mNaOH solutthe releaseconditions.
For estescrew-capp70 mmol L
alcohol mofermented sat 200 rpm ture were rthe Lowrylated by
conOne unit (Uproduced p
Both hyunits of acti
2.7. Stabilit
The stabmeasuring different sythe followinoleic acid aacid and
ethture). For thfermented srotary shaksolids wereto remove tthen
ltereture. The remethod usithe initial h
elimi
liminin thce an
oute (olamot xdualoleatcids f
lvent
pacl diad walumn
with presoun
e bull diarom r mal (coMHP
of tixturshedl. Throus nto e
by tiis.
the s-bedC14:0 C16:0 7.9C16:1 0.6 C18:0 4.8 C18:1 30.1 C18:2
52.6
4.0 ecular weight (g mol1) 278.6g KOH g1) 191.3 n value (mg KOH
g1) 196
m soybean oil (FA-SO), soybean soapstock acid oil (FA-SSAO),
waste cooking oil (FA
lipid content in the fermented solids was negligibleo the lipid
content of the reaction mixture.
measurement with the fermented solid
rolytic activity of the dry fermented solids was the titrimetric
method. The solution contained triolein1), gum arabic 3% (w/v),
CaCl2 (2 mmol L1), TrisHCl
1) and NaCl (150 mmol L1) in distilled water [28]. Ited with a
blender at high speed, initially for 5 min anddditional 1 min
immediately before use. For each assay,ulsion and 100 mg of
fermented solids were placed in a
maintained at 40 C. The free fatty acids released during were
titrated for 5 min in a Metrohm 718 STAT Titrinotric titrator
(Metrohm, Herisau, Switzerland) with theaintained at 7.0 through
the addition of a 0.05 mmol L1
ion. One unit (U) of hydrolytic activity was dened as of 1 mol
of fatty acid per minute, under the assay
rication activity, reactions were carried out in 12 mLed bottles
containing 10 mL of a mixture containing1 of fatty acid, 210 mmol
L1 of ethanol (i.e. an acid tolar ratio of 1:3) in n-hexane and 800
mg of delipidatedolids. These bottles were incubated on a rotary
shakerand 40 C. At xed intervals, 50 L samples of the mix-emoved
and analyzed for residual free fatty acids byTinsley method [29].
Ethyl ester production was calcu-sumption of free fatty acids from
the reaction mixture.) of esterication activity equals 1 mol of
ethyl esterer minute, under the assay conditions.drolytic and
esterication activity were expressed invity per gram of dry
fermented solid (U gdfs1).
y of the fermented solid
ility of the dry fermented solids was evaluated bythe hydrolytic
activity before and after incubation instems. The fermented solids
were incubated for 48 h in
2.8. Pr
Presolids presencarriedmixturstated 40 C. Afor resi(ethyl fatty
a
2.9. So
TheinternareceiveThe cosolids,lightlythis amthat thinternaacids
fa molaethanoner 45bottomtion mand waethanoanhydacids
iminedanalys
Forpackedg media: (a) ethanol, (b) n-hexane, (c) 70 mmol L1 ofnd
210 mmol L1 of ethanol in n-hexane and (d) oleicanol in a 1:3 molar
ratio (i.e. solvent-free reaction mix-ese tests, screw-capped
bottles containing 500 mg ofolids and 10 mL of reaction mixture
were incubated on aer at 200 rpm and 40 C. After incubation, the
fermented
washed four times, each time with 10 mL of n-hexane,he
substrates and reaction products. All samples wered and dried in a
vacuum desiccator at room tempera-sidual hydrolytic activity,
determined by the titrimetricng triolein as substrate, was
expressed as percentage ofydrolytic activity.
of 48 h. Thesolids and reach cycle, replaced wiexpressed
abatch.
2.10. GC an
Ethyl estCo., Kyoto, Jtor and a S2.1 5.922.0 30.51.5 5.9
4.4 10.0 35.6 45.3 32.6 0.8 1.8 1.6
274.3 269.6 190.9 199.3
198 203
) and beef tallow (FA-BT).
nary studies of biodiesel synthesis
ary tests were done to dene the amount of fermentede reaction
mixture and to compare the reaction in thed absence of n-hexane as
solvent. The reactions were
in 12 mL screw-capped bottles with 10 mL of reactioneic acid and
ethanol in a molar ratio of 1:3) and theunt of fermented solids,
with incubation at 200 rpm anded intervals, 50 L samples were
collected and analyzed
free fatty acids by the LowryTinsley method [29]. Estere)
production was calculated by consumption of freerom the reaction
mixture.
-free esterication in a packed-bed reactor
ked-bed reactor was made of a glass column (2.7 cmmeter and 21
cm high) with an external jacket thatter from a water bath at the
stated temperature (Fig. 1).
was packed with 12 g (on a dry basis) of fermented the solids
being poured in through a funnel and thensed with a glass rod.
Undertaking this procedure witht of fermented solids gave a bed
height of 16 cm, suchk density of the bed was 130 g L1. The
reservoir (4.4 cmmeter and 9 cm high) was loaded with 100 g of
fattysoybean soapstock acid oil (FA-SSAO) (on the basis ofss of 277
g, this corresponds to 361 mmol) and 50 g ofrresponding to 1089
mmol). A peristaltic pump (Sten-10, Florida, USA) pumped the
reaction mixture into thehe bed at a ow rate of 5 mL min1. Samples
of the reac-e were collected from the top of the packed-bed
reactor
with saturated NaCl solution to separate the excess ofe upper
organic fraction was collected and dried overNa2SO4 and
centrifuged. The conversion of the fattyster was monitored by
measuring the acid value deter-tration [27]. The content of ester
was determined by GC
tudy of the reutilization of the fermented solids in the
reactor, the reaction was repeated for eight cycles, each
experimental procedure, the amount of the fermentedeaction mixtures
used were as described above. Afterthe reaction mixture was
collected from the reactor andth a new mixture. Reaction yields
after each cycle weres fractions of the yield obtained at the end
of the rst
alysis
er content was determined using a GC-2010 (Shimadzuapan)
equipped with a hydrogen ame ionization detec-GE HT-5 capillary
column (0.32 mm internal diameter,
-
18 D. Soares et al. / Biochemical Engineering Journal 81 (2013)
15 23
Fig. 1. Schemvoir; (2) reactpacked with feling port; (7) w
25 m lengthwas dilutedheptadecanwith a splitand detecto120 C for
2tained for 3for 2 min. Pparison of tcontent (in of the inter
3. Results
3.1. Produc
B. cepaciarcane bagaand esteriover time. Tobtained at91.6 3.3
U96 h. Since cation reacremaining s
3.2. Stabilitfermented s
After festorage. Prlyophilizati
8
10
100
120
gdfs-
1 )
Hyd
roly
tic a
ctiv
ity (U
gdfs-
1 )
ctivitmentarepres
ce c, the ois
y of este
in thl mole to
hydophith dry dec
labod as e of 1atic representation of the packed-bed reactor
system. Key: (1) reser-ion mixture fed to reactor; (3) peristaltic
pump; (4) glass columnrmented solids; (5) reaction mixture removed
from reactor; (6) samp-ater jacket.
and 0.1 mm lm thickness). Prepared sample (56 mg) in 1 mL of an
internal standard solution of methyl
1
Fig. 2. Astate ferplotted
to redudryinghad a mactivituse inminedoriginapossib
Theafter lying wiactivitforcedselectetenancoate (1 mg mL ) in
n-heptane. Then 1 L was injected, ratio of 1:50, using N2 as the
carrier gas. The injectorr were set at 250 C. The oven program was
as follows:
min, heating at 10 C min1 to 180 C, 180 C main- min, heating at
5 C min1 to 230 C, 230 C maintainedeaks in the chromatograms were
identied by com-he retention times with a standard solution. The
estermass percent) was determined relative to the peak areanal
standard [25].
tion of fermented solids
a LTEB11 was cultivated on a 1:1 mixture (w/w) of sug-sse and
sunower seed meal, with both the hydrolyticcation activities of the
fermented solids being measuredhe highest esterication activity was
5.8 0.3 U gdfs1,
72 h (Fig. 2). At this time the hydrolytic activity was gdfs1,
although it did reach a slightly higher value atthe fermented
solids were intended for use in esteri-tions, a fermentation time
of 72 h was selected for thetudies.
y of the hydrolytic activity during drying of theolid
rmentation, the fermented solids must be dried foreviously in
our laboratory they have been dried byon [14,30], but this is an
expensive process. In order
activity in festable duriobtained wdata are av20% loss of
IRD43aIV [3around 33%zopus micro
3.3. Stabilit
The immin organic mpresent in tit in the var
The resiabove 95% reaction min-hexane, t100% after
immobilizevents has bsolvent mothereby facthe activitytion
mediaincubation rm that thstability in [33]. For ex0
2
4
6
0
20
40
60
80
120967248240
Este
rific
atio
n ac
tivity
(U
Time (h)y of the fermented solids from Burkholderia cepacia
LTEB11 in solid-tion. Key: () hydrolytic activity; () esterication
activity. Valuesent the mean of duplicate asks the standard error
of the mean.
osts, we evaluated alternative drying processes. Beforefresh
fermented solids, obtained at 72 h of fermentation,ture content of
72% (w/w, wet basis) with a hydrolytic83 5 U gdfs1. Although the
solids were intended forrication reactions, the hydrolytic activity
was deter-is experiment because the high water content of theist
solids used as the control meant that it was notundertake the assay
for esterication activity.rolytic activity of the fermented solids
was maintainedlization at 4 C for 24 h (90 4 U gdfs1) and after
dry-y air in a column at 25 C for 5 h (84 6 U gdfs1). Thereased to
37 5 U gdfs1 for the sample dried in a fan-ratory oven at 30 C for
8 h. Drying in the column wasthe drying method for the remaining
studies. The main-00% of activity with air drying suggests that the
lipolyticrmented solids obtained with B. cepacia LTEB11 is more
ng the drying step than that in the fermented solidsith
Rhizopus, the only other organism for which similarailable. Dry air
and lyophilization each caused aroundlipolytic activity for
fermented solids from Rhizopus sp.1], while oven drying and
lyophilization each caused
loss of lipolytic activity for fermented solids from Rhi-sporus
CPQBA 312-07 DRM [32].
y of the fermented solid in organic solvents
obilized lipase from B. cepacia is known for its stabilityedia
[3336]. However, since the stability of the lipasehe fermented
solids has not been studied, we evaluatedious solvents and reaction
mixtures used in this work.dual lipolytic activity of the fermented
solids remainedafter 8 h of incubation in ethanol and in
solvent-freexture (Fig. 3). In n-hexane and in reaction mixture
withhe activity initially increased slightly, remaining above8 h
incubation. The increase of the catalytic activity ofd lipases
after treatment with hydrophobic organic sol-een observed
previously and is attributed to residuallecules maintaining the
lipase in its open conformation,ilitating the access of the
substrates to the active site in
assay [35,3739]. In ethanol and in solvent-free reac-, the
residual activities remained above 80% after 24 hand above 72%
after 48 h incubation. These results con-e lipolytic activity of
the fermented solids has similarorganic media to that of
immobilized B. cepacia lipaseample, the residual lipolytic activity
of a lipase from
-
D. Soares et al. / Biochemical Engineering Journal 81 (2013) 15
23 19
100
120
140
y (%
)
Fig. 3. StabilitResidual activ210 mmol L1
oleic acid and ditions: 500 mand 200 rpm. Adetermined
byrepresent the
B. cepacia im120% after i
3.4. Ester p
High yietransesterilipase from[30,33,40]. ication ofessentially
immobilizesolids of Bwhether it wwhile main
In the so82% after 8with n-hexaconcentratifor the solvobtained
foof the solveratio of subneed for theprocessing
3.5. Effect o
The effecture (9, 12 mass of olerate of prodincrease in However,
a did not incrsolids madeof 12% ferm
3.6. Esterifatty acid so
We evalusing free fa
0
20
40
60
80
100
967248240
Subs
trat
e con
vers
ion
(%)
Time (h)
ffect of the fermented solids content on the esterication
reaction in afree system. Key: () 9%; () 12%; () 15% (mass of
fermented solids astage of the mass of oleic acid). Reaction
conditions: oleic acid 1890 mmol,5670 mmol, 40 C, 200 rpm. Values
plotted represent the mean of triplicate
the standard error of the mean.
btained through the hydrolysis of soybean oil, soybean soap-cid
oil, beef tallow and waste cooking oil (denoted as FA-SO,O, FA-BT
and FA-WCO, respectively). Esterication activityee fatty acids was
not directly related to the acyl chain length). Th
gdfstivitimons [41l EP-
reas7.0 U
the rroprlecter.
fect o
peracatio
LTE. cepO2-Pcia li0
20
40
60
80
483624120
Res
idua
l ac
tivit
Time (h)y of the fermented solids after incubation in different
systems. Key:ity in () ethanol; () n-hexane; () 70 mmol L1 of oleic
acid andof ethanol in n-hexane (i.e. reaction mixture containing
solvent); ()ethanol in a 1:3 molar ratio (i.e. solvent-free
reaction mixture). Con-g of dry fermented solids in 10 mL of
solution, incubated at 40 Cctivities were determined relative to an
initial activity of 84 U gdfs1
the titrimetric method, using triolein as the substrate. Values
plottedmean of triplicate analyses the standard error of the
mean.
mobilized on a macroporous resin ranged from 80 toncubation for
4 h in methanol, ethanol and acetone [36].
roduction in the presence and absence of n-hexane
lds of methyl or ethyl esters have been reported forcation and
esterication reactions catalyzed by the
B. cepacia LTEB11 in systems containing solventsIn previous
investigations of our group into the ester-
oleic acid with ethanol in n-heptane, we obtained95100% ester
yield in 13 h using B. cepacia LTEB11d on Accurel [33,40] and 94%
in 18 h with dry fermented. cepacia [30]. In the current work, we
investigatedould be possible to remove the solvent from the
system
taining acceptable conversion rates.lvent-free reaction mixture,
the conversion was only8 h, compared to a 92% conversion after 8 h
obtainedne as the solvent (Table 2). However, due to the higherons
within the reaction mixture, the ester productivityent-free system
was essentially twice as high as thatr the reaction with n-hexane.
Additional advantagesnt-free system are that, rstly, it uses a much
higherstrate to fermented solids and, secondly, it avoids the
recovery and recycling of the solvent, thereby lowering
Fig. 4. Esolvent-a percenethanol analyses
acids ostock aFA-SSAwith fr(Fig. 5(12.7 Ution
acPseudosystemAccure
TheSSAO (duringan appfore sereacto
3.7. Efreactor
Temestericepaciaoil by Phol (SiP. cepacosts.
f the amount of fermented solids on esterication
t of the amount of fermented solids in the reaction mix-and 15%
of fermented solids, expressed relative to theic acid) was
evaluated in the solvent-free system. Theuction of ethyl oleate
increased signicantly with anthe amount of fermented solids from 9
to 12% (Fig. 4).further increase in the fermented solids, from 12
to 15%,ease the reaction rate signicantly. Higher amounts of
it difcult to agitate the reaction mixture. The additionented
solids was used in the remaining studies.
cation activity of the fermented solids on differenturces
uated the esterication activity of the fermented solidstty acids
of different chain lengths, as well as free fatty
Fig. 5. EsteriConditions: 10210 mmol L1
represent the e highest activities were observed with palmitic
acid1) and caprylic acid (11.7 U gdfs1). Higher esterica-es with
palmitic acid have been obtained previously foras cepacia lipase
(LPS A001526) in AOT microemulsion] and B. cepacia lipase (LPS
AR01520) immobilized on100 [42].onably high esterication activity
obtained with FA-
gdfs1) is encouraging, since it is a byproduct generatedening of
soybean oil [8,4346]. Its low cost may make itiate feedstock for
biodiesel production [8]. It was there-d as the substrate for the
experiments in the packed-bed
f temperature on biodiesel synthesis in a packed-bed
tures from 40 to 60 C have been utilized for the trans-n of
soybean oil catalyzed by fermented solids from B.B11 [14],
transesterication of beef tallow and babassuacia lipase immobilized
in polysiloxane-polyvinyl alco-VA) [47], esterication of lauric
acid by an immobilizedpase from Amano [35] and regioselective
acylation of
FA-SOFA-SSAO
FA-BT15129630
C6:0FA -C8:0FA -
C12:0FA -C14:0FA -C16:0FA -C18:0FA -C18:1FA -C18:2FA -
FA-WCO
Esterification activity (U gdfs-1)
Free
fatty
aci
ds
cation activity of the fermented solids against different fatty
acids. mL of reaction mixture with n-hexane (70 mmol L1 of fatty
acid andof ethanol), 800 mg of fermented solids, 40 C, 200 rpm.
Values plottedmean of duplicate analyses the standard error of the
mean.
-
20 D. Soares et al. / Biochemical Engineering Journal 81 (2013)
15 23
Table 2Esterication reactions carried out in shake asks in the
presence and in the absence of solvent.
Solventa Solvent-free
Mass (mg) Molar concentration (mmol L1) Mass (mg) Amount
(mmol)
Reaction mixtureOleic acid 198 70 5760 20.4Ethanol 97 210 2813
61.2Fermented solids 500 500
Results
Ratio fermented solids/oleic acid (g/g) 2.5 0.09Results
Conversion in time taken 92% in 8 h 82% in 88 hProductivity 50
mg gdfs1 h1 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
estericationof FA-SSAO with ethanol in the packed-bed reactor. The
initial reac-tion rate (based on the conversions obtained at 12 h)
increased withincreasing temperature (Fig. 6). However, at 60 C the
conversionsfor reaction times above 24 h were lower than those
obtained atthe lower temperatures, probably due to a higher degree
of dena-turation. At
31 h; at the85% after 40
3.8. Operatpacked-bed
We usedcycles of 48cycle, the cthe value ofan attemptcycles, we
rcase the coduring six c
3.9. Compo
The propester compwork by es16.5% of ethand 44.2% o
Fig. 6. Effect o40 C; () 50 Cfatty acids fromacid to ethanomean
of dupli
obtained by de Sousa et al. [22] and Chen et al. [50], which
gavebiodiesel properties that met the Brazilian [51] and European
[52]standards, respectively.
4. Discussion
This work makes two contributions in the area of hydroester-n.
Firstly, this is the rst study of hydroesterication usingn sorst ss
be
twoof faeen his icondtionpackns hutiotentiep inck.
ntrib
of thlow-AO
onve
rsio
n (%
) 50 C, a 92% conversion of FA-SSAO was obtained at other
temperatures the conversions were still below
h of reaction.
ional stability of the fermented solids in the reactor
the same fermented solids in successive esterication h in the
packed-bed reactor at 50 C (Fig. 7). In the fthonversion remained
above 84% (which is above 90% of
92% conversion that was obtained in the rst cycle). In to keep
the original conversion for a larger number ofedid the
reutilization experiment at 45 C (Fig. 7). In thisnversion remained
above 90% of the initial conversionycles.
sition of the ethyl esters
erties of biodiesel are directly inuenced by the fattyosition
[49]. The ethyl ester that was obtained in thisterifying FA-SSAO
with ethanol contained, by mass,yl palmitate, 4.2% of ethyl
stearate, 33.4% of ethyl oleatef ethyl linoleate. This composition
is similar to those
60
80
100
sion
(%)
icatiosoybeais the step hamakescation have bacids, ttem.
Seproducout in reactiocontribthe potion stfeedsto
4.1. Co
Oneto use that SS0
20
40
483624120
Con
ver
Time (h)f temperature on esterication in the packed-bed reactor.
Key: (); () 60 C. Reaction conditions: 12 g of dry fermented
solids, 100 g of
soybean soapstock acid oil and 50 g of ethanol (i.e. molar ratio
of fattyl of 1:3), recirculation rate of 5 mL min1. Values plotted
represent thecate analyses the standard error of the mean.
Rel
ativ
e c
Fig. 7. Operatsame fermentcycles. Key: re12 g of dry ferm50 g of
ethano5 mL min1.apstock acid oil (SSAO) as the feedstock. Secondly,
thistudy of hydroesterication in which the esterication
en undertaken using enzymatic catalysis. The work also
contributions in the area of lipase-catalyzed esteri-tty acids.
Firstly, although lipolytic fermented solidsused previously to
catalyze the esterication of fattys the rst study that has done
this in a solvent-free sys-ly, lipase-catalyzed transesterication
reactions for the
of biodiesel in solvent-free systems have been carrieded-bed
bioreactors, but lipase-catalyzed estericationave not previously
been studied in this system. Thesens, which are discussed below,
combine to demonstrateal of using fermented solids to catalyze the
esterica-
a hydroesterication process that uses SSAO as the
utions in the area of hydroesterication
e strategies to reduce the cost of biodiesel production isvalue
feedstocks. In the current work we demonstratedis an adequate
feedstock for hydroesterication, with
5060708090100
5060708090
100110
conv
ersio
n (%
)010203040
010203040
87654321
Ori
gina
l
Cycles
ional stability of the fermented solids in the packed-bed
reactor. Theed solids were used to catalyze the esterication
reaction in 48-haction temperature of () 45 C and () 50 C. Reaction
conditions:ented solids, 100 g of fatty acids from soybean
soapstock acid oil and
l (i.e. molar ratio of fatty acid to ethanol of 1:3),
recirculation rate of
-
D. Soares et al. / Biochemical Engineering Journal 81 (2013) 15
23 21
Table 3Studies 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 sCatalyst (% w t
TemperatureSubstrates (r
t oil
Conversion i
Estericatio
Catalyst (% wTemperatureReaction mix
Conversion i
VEEG: vegetab lyst 1
the free fatsubcritical wSoapstock ivegetable othe originalthe
soybeanbreaking anthe acid oilacids, 28% t[8]. It sells f[43].
Our worfrom soybeterication.that hydroebiodiesel frothe
productthat have aone used w
Additionenzymatic estericatiohydrolysis/justify this smild
condiHowever, urequired, esused by Caalthough a to oil
volumconditions ucally involv(200 C) [21
The hydinverts thetial hydrolyis catalyzechemical-hThis
strategsoapstocks salts [43], sstrategy, thto these cowere not
fa[21] in theirwere of rela
ysis it to ceivtudieave iing ]. Altthe ms ope
CH/n comcatals relclessiblet-freesteri
the gions
pro-bedat r
by fel cone coougin btion
potethe step Enzymatic Enzymatic eight of oil) Thermomyces
lanuginos
(liquid lipase, 2.3%)VEEG from physic nu(10%)
& pressure 60 C & 1 atm 40 C & 1 atm atio in v:v)
Water:soybean oil (1:1) TrisHCl
0.1 mol L1:physic nu(9:1)
n time taken 89% in 48 h 98% in 2 h
n step Chemical Chemical
eight of fatty acid) Niobic acid (20%) Niobic acid (20%) &
pressure 200 C & 24 atm 200 C & 34 atm ture (molar ratio)
FA/methanol (1:3) FA/methanol (1:3)
n time taken 92% in 1 h 97% in 2 h
le enzyme extract from germinated seeds; FA: fatty acids from
hydrolysis; *Amber
ty acids that were obtained after its hydrolysis withater being
efciently converted into their ethyl esters.
s a byproduct from the alkaline neutralization step ofil rening
and represents about 6% of the volume of
crude vegetable oil [43]. In a typical industrial process,
soapstock is acidied with sulfuric acid for emulsiond then
separates into two phases, an aqueous phase and
phase. The acid oil contains, by weight, 59% free
fattyriacylglycerol, and around 5% di- and monoacylglycerolor
approximately half the cost of rened vegetable oils
k demonstrates, for the rst time, that acid oil derivedan
soapstock can be used as a feedstock for hydroes-
Despite the fact that previous authors have
recognizedsterication is a promising technology for producingm
low-value feedstocks, two of the previous studies ofion of
biodiesel esters in hydroesterication processesn enzymatic step
have used relatively pure oils, whileaste cooking oil (Table
3).ally, the three studies listed in Table 3 all involve anoil
hydrolysis step followed by a chemically catalyzedn step. This will
be referred to as the enzymatic-chemical-esterication (EH/CE)
strategy. The authorstrategy by pointing out that the hydrolysis
step involvestions of temperature and pressure (3060 C, 1 atm).nder
these conditions long reaction times are normallypecially at the
volumetric ratio of water to oil of 1:1valcanti-Oliveira et al.
[21] and Talukder et al. [23],98% conversion in 2 h has been
achieved with a wateretric ratio of 9:1 [22]. In addition, unlike
the mild
hydrolcatalysstep rethree sdate htion be[2123mode, batche
Ourtages ilipase-involve48-h cybe
possolventransecation,limitatcationpackedthan thalyzedoriginawith
th
Alththe maproduchas theuct of sed in the hydrolysis step, the
esterication step typi-es either high pressures (2434 atm) and
temperatures,22] or the use of solvents such as isooctane
[23].roesterication process studied in the present work
strategy of these previous studies, in that the ini-sis step is
a chemical step while the estericationd enzymatically. This will be
referred to as theydrolysis/enzymatic-esterication (CH/EE)
strategy.y has two advantages over the EH/CE strategy. Firstly,can
contain contaminants, such as sodium or potassiumulfur, phosphorous
and metal ions [53]. In the EH/CEe enzymes used in the initial
hydrolysis step are exposedntaminants and may be inactivated. These
problemsced by de Sousa et al. [22] and Cavalcanti-Oliveira et
al.
studies of the EH/CE strategy because the oils they usedtively
high quality. In contrast, in the CH/EE strategy the
air drying, tof the lipaslized lipaseCosts couldsunower
s[30,59].
4.2. Contrib
The currcontaining solvent-freemented soln-heptane can be
achiumetric proEnzymatic Subcritical waterCandida rugosa (0.05%)
Free of catalyst
30 C & 1 atm 250 C & 60 atmWaste cooking
oil:water(1:1)
Water:soybean soapstockacid oil (1:1)
100% in 10 h 95% in 1 h
Chemical Enzymatic
*Amberlyst 15 (100%) Fermented solid (12%)60 C & 1 atm 50 C
& 1 atmFA/methanol (4:1), inisooctane
FA/ethanol (1:3)
99% in 2 h 93% in 31 h
5: acidic styrene-divinylbenzene sulfonated ion-exchange
resin.
s carried out with subcritical water, such that there is
nosuffer inactivation, and the lipases in the estericatione a
contaminant-free fatty acid stream. Secondly, thes using the EH/CE
strategy that have been published tonvolved batch operation, with
the enzymatic prepara-used to catalyze the hydrolysis of a single
batch of oilhough we also used the packed-bed bioreactor in
batchaintenance of over 80% conversion in seven successivens up the
possibility of continuous operation.EE hydroesterication process
also has some advan-parison to lipase-catalyzed transesterication.
Firstly,
yzed transesterication in solvent-free media typicallyatively
long reaction times (Table 4). Although we used
for the esterication reaction, once optimized, it should to
reduce the reaction time to a few hours, even in
media, since esterication is typically much faster thancation
[13,14,54]. Secondly, in enzymatic transesteri-lycerol absorbs onto
the catalyst, causing mass transfer[5,5558], while this problem is
avoided in hydroesteri-cesses. This explains why the operational
stability of the
reactor for the esterication of FA-SSAO was highereported for
the transesterication of soybean oil cat-rmented solids from B.
cepacia LTEB11 [14], where theversion of 95% was maintained only
for three cycles,nversion decreasing to 62% after six cycles.h the
high cost of commercial lipases remains as one ofarriers against
their use in industrial processes for the
of biodiesel, the use of fermented solids in our processntial to
reduce lipase costs signicantly. The nal prod-olid-state
fermentation is simply subjected to a mild
hus avoiding the costs of recovery and immobilizatione that are
associated with the production of immobi-s that are produced by
submerged liquid fermentation.
be decreased further if it were possible to replace theeed meal
with a cheaper inducer of lipase production
utions in the area of esterication of fatty acids
ent work represents the rst time that fermented solidslipases
have been used to catalyze esterication in a
system. Previous studies of esterication with fer-ids have
involved the use of organic solvents such as[30,60] and n-hexane
[31]. Although high conversionseved in short times in the presence
of solvents, the vol-ductivity of such reactions is lower, due to
the much
-
22 D. Soares et al. / Biochemical Engineering Journal 81 (2013)
15 23
Table 4Studies of solvent-free biodiesel production by lipases
in packed-bed reactors.
Microorganism Support Reaction mixture Conversion in time taken
System Reference
Burkholderia cepacia LTEB11 Fermented solid FA-SSAO + ethanol
93% in 31 h Batch This workBurkholderiaRhizopus oryCandida
antaCandida antaCandida anta ethanPseudomonaaRecombina ethan
a Aspergillus suppo
lower substhen it is esthis extra st
Althougbiodiesel uhave all invrepresents
solvent-freePacked-bedavoiding thagitated reawith the sa(w/w), 74
hthis convers
5. Conclus
The currthe costs ohydroesteristock acid othe second sfree
systemcontains lip80% can be involving szation of thlaboratory.
Acknowled
This resselho NacioBrazilian gotechnologyreira Pinto,research
scSoares, for tplant for th
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gements
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alves, Andrei Fer-
David Mitchell and Nadia Krieger also thank CNPq forholarships.
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Biodiesel production from soybean soapstock acid oil by
hydrolysis in subcritical water followed by lipase-catalyzed
ester...1 Introduction2 Materials and methods2.1 Raw materials2.2
Hydrolysis of fat feedstocks2.3 Microorganism2.4 Solid-state
fermentation2.5 Drying and preparation of the fermented solid2.6
Activity measurement with the fermented solid2.7 Stability of the
fermented solid2.8 Preliminary studies of biodiesel synthesis2.9
Solvent-free esterification in a packed-bed reactor2.10 GC
analysis
3 Results3.1 Production of fermented solids3.2 Stability of the
hydrolytic activity during drying of the fermented solid3.3
Stability of the fermented solid in organic solvents3.4 Ester
production in the presence and absence of n-hexane3.5 Effect of the
amount of fermented solids on esterification3.6 Esterification
activity of the fermented solids on different fatty acid sources3.7
Effect of temperature on biodiesel synthesis in a packed-bed
reactor3.8 Operational stability of the fermented solids in the
packed-bed reactor3.9 Composition of the ethyl esters
4 Discussion4.1 Contributions in the area of
hydroesterification4.2 Contributions in the area of esterification
of fatty acids
5 ConclusionsAcknowledgementsReferences