ORI GI NAL PAPER

Synthesis of SBA-15 from low cost silica precursor obtained

from sugarcane leaf ash and its application as a support matrix
for lipase in biodiesel production
A. Arumugam

V. Ponnusami
Received: 29 January 2013 / Accepted: 16 May 2013 / Published online: 21 May 2013
Springer Science+Business Media New York 2013
Abstract Ordered mesoporous silica material was synthe-
sized froma low-cost precursor, sugarcane leaf ash, was used
as a support matrix for lipase for the production of biodiesel.
The mesoporous samples were characterized using Fourier
transforminfra red spectroscopy. The surface topography and
morphology of the mesoporous materials were studied using
scanning electron microscope. The pore diameter, pore vol-
ume, Brunauer Emmett and Teller surface area of the meso-
porous material were determined by N
2
gas adsorption
technique. Different pore size Santa Barbara Acid-15 (SBA-
15) samples were synthesized and their lipase immobilization
capacity and specic enzyme activity of immobilization
lipase were determined and compared. Lipase from Candida
Antarctica immobilized on SBA-15 (C) had shown maximum
percentage immobilization and specic enzyme activity. The
immobilized lipase mesoporous matrix was used for biodiesel
production from crude non-edible Calophyllum inophyllum
oil. The percentage yield of fatty acid methyl ester, 97.6 %
was obtained under optimized conditions: 100 mg of lipase
immobilized on SBA-15, 6:1 methanol to oil molar ratio, the
reaction of 2 g C. inophyllum oil with methanol.
Keywords Sugarcane leaf ash Biodiesel SBA-15
Methanolysis Lipase immobilization Calophyllum
inophyllum oil
1 Introduction
Santa Barbara Acid-15 nds application in various elds
including chemical catalysis, medical imaging, drug
delivery, and chromatography. It is characterized by high
surface area and pore volume. Well ordered mesoporous
silica had been identied as a potential host for protein
immobilization [1]. SBA-15 possesses hexagonally ordered
tuneable and uniform mesopores. SBA-15 had been proven
to be very promising material for size selective adsorption
of large biomolecules as its pore diameters are in the range
required for the biomolecules. Tailoring the pore sizes of
mesoporous silicas for specic application can be achieved
either by using surfactants with various chain lengths,
manipulation of processing parameters or by addition of 1,
3, 5-trimethylbenzene (TMB) amines as swelling agents [2].
Template assisted synthesis of SBA-15 where Pluron-
ics P123 (poly(ethylene oxide)-poly(propylene oxide)-
poly(ethylene oxide)) is employed as structure directing
agent has the advantage of rendering a uniform and denite
pore size; and the inner walls are strengthened by using a
template; that is essential for post synthesis modication of
the particle [3]. SBA-15 is a combined micro- and meso-
porous material with hexagonally ordered tuneable uniform
mesoporous (414 nm). The micropores in the walls of the
SBA-15 mesoporous originate from the polyethyleneoxide
blocks (PEO) in the triblock copolymers that are directed to
the aqueous solution, whereas the polypropyleneoxide
blocks (PPO) are more hydrophobic and give rise to the
internal structure of the mesoporous. Hydrochloric acid
was used to maintain the pH well below the isoelectric
point of silica (pI = 2) to ensure the precipitation of the
precursor [4].
Several silica precursors like tetra-alkoxysilane, fumed
silica, water glass and sodium silicate for SBA-15 synthesis
had been reported in the recent literatures [5]. However,
these silica precursors are expensive and/or highly toxic.
Therefore, development of low cost eco-friendly silica
precursors is essential to bring down the cost of SBA-15.
A. Arumugam (&) V. Ponnusami
School of Chemical and Biotechnology, Sastra University,
Thirumalaisamudram, Thanjavur, India
e-mail: aruchemxl@scbt.sastra.edu
1 3
J Sol-Gel Sci Technol (2013) 67:244250
DOI 10.1007/s10971-013-3070-1
In this work, we have employed sugar leaf ash as a low cost
silica precursor for the synthesis of SBA-15.
Performance of the SBA-15 developed from sugarcane
leaf ash had been tested for a specic applicationsupport
for lipase enzyme production in this work. Lipase immo-
bilised on SBA-15 was then employed in the production of
biodiesel from Calophyllum inophyllum oil. C. inophyllum
is native from East Africa, through India and Southeast Asia
to the Philippines, Taiwan, and the Marianas to name a few.
The chemical characteristics of the C. inophyllum oil ME
(methyl esters) meet the requirement of diesel engine [6].
Enzymatic transesterication of vegetable oils is preferred
over chemical transesterication owing to its inherent
advantages like (1) no formation of soaps, mono and dia-
cylglycerols, (2) employs mild reaction conditions (3) avoid
unwanted pre-treatments which is uneconomical [7].
Thus, main objectives of the present work were (1) To
synthesis SBA-I5 from a low cost silica source obtained
from sugarcane leaf ash (2) Immobilisation of lipase on the
synthesized SBA-15 and (3) Transesterication of crude C.
inophyllum linn oil, a non-edible oil using immobilized
lipase.
2 Materials and methods
2.1 Materials
The sugarcane leaf ash was obtained from nearby sugar
industry. Pluronic P123, (a triblock co-polymer Polyethylene
glycol-polypropylene glycol-polyethylene glycol, PEO
20
P-
PO
70
PEO
20
), was used as a template. Potassium dihydrogen
phosphate, Gum Arabic, Sodium potassium tartrate, dipo-
tassium hydrogen phosphate, Sodium Carbonate, Ethanol,
Acetone, Copper Sulphate Pentahydrate, and Sodium Potas-
sium Tartarate were purchased from Himedia Laboratories
Pvt. Ltd. Folins Ciocalteau Reagent, Trimethyl benzene,
Hydrochloric acid fromMerck Chemicals Pvt. Ltd., were also
used. Lipase enzyme from Candida Antarctica was pur-
chased fromQualigens Fine Chemicals Pvt. Ltd and Himedia
Laboratories Pvt. Ltd. respectively. C. inophyllum oil was
obtained from local farm. The average molecular weight of
the oil was calculated to be 869 g/mol from the chemical
composition of the oil given in the Table 1.
2.2 Preparation of mesoporous silica support
Template assisted solgel method was employed for the
synthesis of SBA-15 using Pluronic P123 as a structure
directing agent [8]. Sugarcane leaf ash has been used as
silica source for the replacement of other precursors due to
its low cost and high SiO
2
content. Table 2 gives the X-ray
uorescence (XRF) analysis of sugarcane leaf ash. Si, Ca,
Mg, Al, Na, K and Fe were the major components present
of the ash. 1 g of nely ground ash and sodium hydroxide
in the weight ratio of 1:1.2 were fused at a temperature of
450 C for 1 h. The fused mass was resuspended in water
in the ratio of 1:4 (by weight) and then sonicated to dis-
perse the particles completely. The solution was then l-
tered repeatedly until a colorless ltrate was obtained [9].
An acidied solution prepared by dissolving 10 g of
concentrated HCl in 43.2 g of demineralized water and 5 g
P123 triblock copolymer was added and stirred for 1 h at
35 C until a homogeneous solution was obtained. The
pH of the solution was maintained well below pH 2
(iso-electric point of silica) below which silica exists as
SiO
2
H
?
. TMB was in 1:1 ratio with P123 triblock
copolymer, as a swelling agent for increasing the pore size.
12 g of sodium silicate obtained from sugar cane leaf ash
was dissolved in 5 ml of 1 N sodium hydroxide solution.
The surfactant solution and the solution containing sodium
silicate were well mixed and maintained at 35 C for 24 h
and transferred to a Teon bottle and aged for 72 h at either
100 C or 120 C to get SBA-15 depending upon the pore
size required. The solid products (SBA-15) were ltered
and washed repeatedly with de-ionized water, room dried
for 24 h and then calcined for 8 h at 550 C in air.
2.3 Characterization
Mesoporous silica was characterized by Fourier transform
infrared spectroscopy (spectrum 100, Perkin Elmer, USA).
Table 1 Fatty acid composition of calophyllum inophyllum oil (%)
Fatty acid Molecular formula wt%
Palmitic acid C16:1 16.25
Stearic acid C18:0 14.75
Oleic acid C18:1 38.12
Linoleic acid C18:2 29.34
Linolenic acid C18:3 0.20
Table 2 XRF results for ele-
ments in their oxide form of
sugarcane leaf ash
Formula Concentration
(wt%)
SiO
2
80.14
CaO 6.06
MgO 5.02
K
2
O 3.09
SO
3
2.25
Al
2
O
3
0.89
Cl 0.67
Fe
2
O
3
0.51
Na
2
O 0.28
MnO 0.17
J Sol-Gel Sci Technol (2013) 67:244250 245
1 3
Microscopic observation of the mesoporous material was
done using scanning electron microscope (6701 F, JEOL,
Japan). The transmission electron microscopy (CM 20,
PHILIPS) was performed to analyse the microstructure of
the sample. Methyl esters formed were examined by gas
chromatographymass spectroscopy (CLARUS 500, Perk-
inElmer, USA).
2.4 Immobilization and Lipase activity estimation
Two gram of mesoporous silica was added to 10 ml of
phosphate buffer (pH 7.5) solution containing pure C.
Antarctica lipase (20 mg). The mixture was then stirred at
37 C for 24 h [10]. After equilibration with lipase solu-
tion, the silica hosts were separated by centrifugation and
used for further studies. The supernatant was then ltered
and the activity of immobilized enzyme was determined
[11]. Lipase loading was calculated by determining the
protein content (as lipase is the only protein present) in the
enzyme solution before and after loading using Lowrys
method [12]. Lipase loading is the amount of lipase bound
to the support matrix. S1(2 % Sodium Carbonate in 0.1 N
NaOH), S2(1 % Sodium potassium tartrate in water),
S3(0.5 % Copper (II) Sulphate, Pentahydrate in water),
S4(49 ml of S1, 0.5 ml of S2, 0.5 ml of S3), S5-(Phenol
Reagent-1 part FolinPhenol : 1part water). 1 ml of
sample was taken in a test tube, and 2 ml of solution S4
was added. And the solution was allowed to stand for
10 min at room temperature. 0.3 ml of FolinCiocalteau
reagent was added and allowed to stand for 30 min at room
temperature and the absorbance was noted at 580 nm.
Enzyme activity was determined using olive oil emul-
sion method and expressed as LU/g (One lipase unit of
activity is equivalent to the amount of enzyme required to
liberate 1 lmol free fatty acid (FFA) per minute). A 50 ml
conical ask was taken and the following reagents were
pipette out: Phosphate buffer45 mL, Olive Oil [10 %
v/v]5 mL, Gum Arabic [4.5 % w/v]2.25 g. The con-
tents were mixed by swirling for 15 min at 35 C and
immobilized lipase enzyme was added. The mixture was
kept for incubation for 15 min following which the
quenching agent (95 % Ethanol & Acetone [1:1]) was
added. The contents were mixed and two drops of phe-
nolphthalein indicator was added. The resulting solution
was titrated with 50 mM NaOH [13].
2.5 Enzymatic transesterication
The reaction was carried out by mixing 2 g of C. ino-
phyllum linn oil, 3 % v/v of water and 1.83 g of methanol
(alcohol: oil molar ratio 6:1), in 6 mL screw-capped vials.
The reactions were initiated by adding 100 mg of immo-
bilized lipase to the mixture and carried out at 30 C for
8 h. Reaction vials were shaken in a horizontal shaking
water bath at 100 oscillations/min [14].
2.6 Analysis of methyl ester
The reaction mixture containing methyl esters was analyzed
by gas chromatographymass spectroscopy. The apparatus
was equipped with a HP5 column 30 m long and an
internal diameter of 0.25 mm. The column was heated to
280 C at the rate of 10 C/min. For GCMS quantication,
5 ll of reaction mixture and 300 ll of 1.4lmol/l methyl
esters of heptadecanoic acid (internal standard) and hexane
as solvent were used. 1.0 ll of the reaction mixture was
injected into the column for analysis [15].
3 Results and discussion
3.1 Characterization of mesoporous silica
Scanning electron microscopy was used primarily to study
surface topography and morphology of solid materials.
Fig. 1ac shows that SBA-15 consists of aggregates of
uniform rope-like particles [16]. The rod-like particles
(0.51.0 lm long and 0.40.8 lm in diameter) are
agglomerated into larger bres 3 lm in length. Fig. 1d
shows the TEM image of SBA-15 and conrms the ordered
hexagonal mesoporous channels of silica [17].
Figure 2 shows FTIR spectrum of SBA-15. The peak
observed at 3,451 cm
-1
in the FTIR conrms the presence
of surface silonal group (SiOH) on SBA-15 and the band
at 1,629 cm
-1
in the spectrum of SBA-15 is attributed to
the adsorbed water [18]. Peak observed at 1,087 cm
-1
conrms the presence of SiOSi group in samples [19].
The volume of pores, average pore diameter and surface
area of mesoporous material were determined by N
2
gas
adsorption technique at195.79 C. Brunauer-Emmett-
Teller (BET) analysis provides specic area of materials
using nitrogen multilayer adsorption. Barrett-Joyner-Hal-
enda model (BJH) is used to determine pore size distribu-
tion by assuming cylindrical pore geometry [20]. The pore
size distributions displayed in Fig. 3 were determined by
the BJH method. From the Table 3 the surface area of SBA-
15 decreased by 22 % and pore diameter increased from
4.12 and 7.16 nm with increase in aging temperature from
100 and 130 C. Increase in aging temperature reduces the
hydrophilicity of the ethylene oxide chains which goes into
the hydrophobic core of the micelle and expands it, thereby
pushing out the silica walls and increase the pore diameter.
TMB was added as swelling agent [21]. Addition of TMB
has increased pore size from 7.16 to 14.24 nm and
decreased the surface area by 12 %. For the SBA-15 (C),
BET surface area was determined to be 589 m
2
g
-1
,
246 J Sol-Gel Sci Technol (2013) 67:244250
1 3
average pore volume was estimated to be 2.89 mL g
-1
with
an average pore diameter of 14.24 nm [22].
3.2 Immobilization of lipase
Immobilization of lipases was done on SBA-15 with dif-
ferent pore size and surface area. From the Fig. 4 is could
be seen that SBA-15 (A) and SBA-15 (B) had shown
similar percentage enzyme loading. Maximum enzyme
loading was obtained with SBA-15 (C). Higher percentage
enzyme loading in SBA-15 (C) might be due to larger pore
size which offers lesser mass transfer resistance for enzyme
diffusion [23]. Similarly enzyme activity was least in the
case of SBA-15 (A), and maximum in case of SBA-15 (C).
The major variable that inuenced the enzyme loading rate
and specic enzyme activity was pore diameter. As SBA-
15 (A) pore size was in the range of size of lipase
(a) SBA-15 (A) (b) SBA-15 (B)
(c) SBA-15 (C) (d) SBA-15 (C)
Fig. 1 SEM image of SBA-15
(a,b and c) and TEM images of
SBA-15
400 800 1200 1600 2000 2400 2800 3200 3600 4000
%

T
r
a
n
s
m
i
t
t
a
n
c
e

(
A
r
b
u
t
a
r
a
r
y

s
c
a
l
e
)
Wavenumber (1/cm)
S
i
-
O
H

p
e
a
k


3
4
5
1

c
m
-
1
S
i
-
O
-
S
i

s
t
r
e
t
c
h

1
0
8
7

c
m
-
1
H
-
O
-
H
s
t
r
e
a
t
c
h


1
6
3
8

c
m
-
1
Fig. 2 FTIR spectra of SBA-15
0
0.2
0.4
0.6
0.8
1
1.2
1 10 100 1000
P
o
r
e

V
o
l
u
m
e

(
c
m

/
g

n
m
)
Pore Diameter (nm)
SBA-15 (A)
SBA-15 (B)
SBA-15 (C)
Fig. 3 BJH desorption dV/dD pore volume
J Sol-Gel Sci Technol (2013) 67:244250 247
1 3
molecules (diameter of 5 nm and length of 4 nm) in the
aqueous medium transport of enzymes was hindered and
the resistance to mass transfer was high [24]. This was
possible reason for low immobilisation and specic
enzyme activity with SBA-15 (A). With larger pore
diameters the resistance to pore diffusion decrease and
hence the percentage immobilization and specic activity
also increase. However, the increase in immobilisation and
specic enzyme activity was not proportional to increase in
pore diameter. When the pore diameter becomes too large,
molecules may freely diffuse through the pore and also
may leave the pore freely [25]. SBA-15 (A) showed a
lower percentage immobilization and specic enzyme
activity of immobilized enzyme due to low pore diameter
even though the surface area was the maximum. The
activity of the immobilized lipase prepared was determined
to be 11,667 U/g. Hence lipase immobilized on SBA-15
(C) was used for further studies [26].
3.3 Effect of methanol to oil ratio
Biodiesel was produced with various methanol to oil ratio.
The Fig. 5 shows that maximum yield of FAME (fatty acid
methyl ester) was obtained when the methanol to oil ratio
was 6:1 [27]. The results indicated that the FAME yield
initially increased with increasing methanol to oil ratio
from 2 to 4 and reaches its maximum at 6 in this study.
However, a further increase in methanol to oil ratio
decreased FAME yield. It was in agreement with the earlier
observations that excessive methanol concentrations lead
to lipase enzyme inactivation [28]. One way ANOVA was
performed using Minitab 15. The F and p values were
40.44 and 0.002 respectively. Critical F value for 3, 4
degrees of freedom (F
3, 4
) at 95 % condence level is
6.591. As the calculated F value was greater than F
table
the
methanol to oil ratio has a signicant inuence on the
percentage yield of FAME. This was also conrmed from
the low p value obtained (p \0.05).
3.4 Reusability
The results for reusability of the SBA-15 are shown in
Fig. 6. The percentage yield of FAME decreased by 8.9 %
after eight recycles. The reusability of the immobilized
lipase was important in cutting down the enzymes
requirement and hence cost of biodiesel production. Thus,
it can be used as suitable matrix for immobilisation of
lipase to be employed in large scale methanolysis [29]. In
Fig. 6 loss of activity of immobilized lipase and loss of
enzyme due to leaching are shown along with FAME yield
Table 3 Synthesis conditions
for SBA-15 samples, Surface
area, pore volume and pore
diameter of the mesoporous
materials
Samples Post synthesis
treatment C
TMB/P123 Specic surface
area (m
2
g
-1
)
Pore volume
(mL g
-1
)
Pore diameter
(nm)
SBA-15 (A) 100 860 0.938 4.12
SBA-15 (B) 120 670 1.256 7.16
SBA-15 (C) 120 1:1 589 2.89 14.24
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40
45
50
SBA-15 (A) SBA-15 (B) SBA-15(C)
S
p
e
c
i
f
i
c


e
n
z
y
m
e

a
c
t
i
v
i
t
y

(
U
/
m
g
)

%


I
m
m
o
b
i
l
i
z
a
t
i
o
n
SBA-15
% Immobilization
Specific enzyme
activity (U/mg)
Fig. 4 Enzyme loading and specic enzyme activity of lipase SBA-
15
50
55
60
65
70
75
80
85
90
95
100
0 1 2 3 4 5 6 7 8 9
%

Y
i
e
l
d


o
f


F
A
M
E
Methnol/oil ratio (molar)
Fig. 5 Effect of Methanol to oil molar ratio on immobilized lipase
catalyzed methanolysis of C. inophyllum oil for Temperature 35 C
and reaction time of 8 h
248 J Sol-Gel Sci Technol (2013) 67:244250
1 3
for eight cycles. From the Fig. 6 it is evident that the
gradual decrease in FAME yield was attributed to both loss
of activity of immobilized lipase and loss of enzyme due to
leaching [30].
4 Conclusion
Mesoporous SBA-15 had been successfully synthesised
from water glass from sugarcane leaf ash, a low cost silica
precursor, it was employed for the production of Biodiesel.
The variable pore size and structure of the supports were
strongly inuence the enzyme loading and specic enzyme
activity. High enzyme loading and specic enzyme activity
of lipase on the mesoporous support matrix was observed
which can prove to be efcient in industrial scale biodiesel
production. Biodiesel was produced from renewable and
nonedible feedstock C. inophyllum oil using immobi-
lized lipase on SBA-15 as a support matrix. The optimum
conditions for the methanolysis: methanol to oil molar ratio
-6:1. The reusability studies of immobilized lipase have
shown that the activity of enzyme toward methanolysis was
retained for eight cycles.
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