2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 64 -
conventional technique varying one factor at a time while
keeping the other factors constant.
The enzymatic hydrolysis reaction was carried out at
room temperature in an orbital shaker (150 rpm) and
incubated with different amounts of leaf waste (0-40 g/L)
for 0-32 h of incubation. Moreover, the effect of cellulase
concentrations (0.13, 0.17, 0.22 and 0.27 mg/ml) was also
studied. Cellulase from Sigma (E.C. 3.2.1.4) derived
from
Aspergillus niger
was utilized throughout this study.
D. Fermentation studies Microorganism and cultivation
medium
Flocculation yeast strain
Saccharomyces cerevisiae
TISTR 5048 (Culture collection, Thailand Institute of
Scientific and Technological Research, Thailand) was
used in all the cultivation experiments. The strain was
maintained on yeast malt peptone (YMP) agar plates
made of yeast extract 10 g/L, soy peptone 20 g/L and
agar 20 g/L with D-glucose 20 g/L as an additional
carbon source [21].
E. Starter preparation and cultivation conditions
Starter cultures were prepared by inoculation of 24 h
cultures from agar slant into 100 mL nutrient broth (NB)
and incubated at 35
o
C on a shaker (200 rpm) for 48 h.
The starter culture (10%) of
S. cerevisiae
(contained 1.5 x
10
4
viable cells/mL) was inoculated into NB and
cultivated on a shaker (200 rpm) for 96 h.
F. Simultaneous saccharification and fermentation (SSF)
A steam-autoclaved (121
o
C, 1 atm for 15 min)
suspension of leaf waste in 0.05 M Tris-HCl buffer, pH
4.5 was used as SSF medium. SSF experiments were
started by inoculation with yeast starter and addition of
filter-sterile enzyme solution under optimum condition
obtained above.
G. Separate hydrolysis and fermentation (SHF)
A filter-steriled hydrolyzate produced from leaf waste
under optimal condition was used as fermentation
medium with no supplement addition. A control
fermentation was also run, on YMP medium
supplemented with reagent-grade of glucose (Merck,
Gemany) at the same concentrations present in the sludge
hydrolyzate used for the SHF process.
The production of ethanol was operated under a 5-L
fermentor (MD300, Eyela) containing 3-L of culture
medium with three six bladed Rushton turbine impellers
(40 mm dia.)
and equipped with
pH, DO, antifoam and
temperature probes which were connected to the
controller
(
B. E. Marubishi). Controlling parameters such
as agitation speed, aeration rate, pH set point were
selected to give desired culture conditions.
H. Analytical method
Ethanol and sugar were measured. Triplicate samples
of ethanol were analyzed by HPLC (Waters, USA) which
was equipped with a reflective index (RI) detector an
Aminex HPX-87H column (Bio-Rad, USA). Ethanol
identification was performed with at 60
o
C with eluent of
5 mM sulfuric acid as mobile phase at a flow rate of 0.4
mL·min [21].
Reducing sugar content in the hydrolyzate was
determined quantitatively by using Nelson Somogyi
method as outlined [22]. After mixing the samples with
the assay reagent, the absorbance was measured at 520
nm against the appropriate blank solution by using
spectrophotometer. The amount of reducing sugar
released was calorimetrically determined. A calibration
curve was obtained using D-glucose as standard.
IV. R
ESULT AND
D
ISCUSSION
A. Characteristics of
Acacia auriculiformis Cunn.’s
leaves
The basic structure of leaf waste consists of three basic
polymers: cellulose (C
6
H
10
O
5
)
x
, hemicellulose and lignin
[C
9
H
10
O
3
.(OCH
3
)
0.9-1.7
]
n
[17, 23]. Two of the main
polymers of the biomass should be provided as precursor
and broken down into fermentable sugars in order to be
converted into ethanol or other valuable products [24].
The effect of pretreated on cellulose content was also
determined. Samples were pretreated with various
reagents including HCl, NaOH and (NH
4
)
2
SO
4
at
concentration of 2% (w/v) for 24 with/without stream
autoclave (121
o
C, 15 min). The biochemical
characteristic of leaf waste was shown in Table 1.
TABLE I
B
IOCHEMICAL COMPOSITIONS OF LEAF WASTE UNDER VARIOUS
PRETREATMENT REAGENTS WITH
/
WITHOUT STREAM AUTOCLAVE
Treatments
Autoclave Cellulose
(%)
Holocellulose
(%)
Lignin
(%)
Before
Leaf waste
48.9±7.6
55.7±1.0
44.3±0.6
After
2% HCl
–
49.1±0.6
58.7±1.3
41.3±0.3
+
85.9±0.2
86.7±3.1
13.3±0.6
2% NaOH
–
49.5±0.8
58.5±2.4
41.5±0.9
+
88.4±1.2
88.7±1.6
11.3±2.1
2%
(NH
4
)
2
SO
4
–
53.6±1.5
69.8±1.2
30.2±1.2
+
93.1±0.4
93.6±0.7
6.4±1.2
Different pretreatments were applied to increase the
degradation activity of the cellulase from
A. niger
. Steam
explosion with 2% of (NH
4
)
2
SO
4
was the best
pretreatment that caused significant increased cellulose
(93.1±0.4%) followed by NaOH (88.4±1.2%) and HCl
(85.9±0.2%). However, pretreatment with reagent
without stream autoclave did not negatively affect on
cellulose content.
Steam explosion is the most commonly used method
for pretreatment of lignocellulosic materials [25]. In this
method, biomass is treated with high-pressure saturated
steam and then the pressure is swiftly reduced, which
makes the materials undergo an explosive decompression.
Steam explosion is typically initiated at a temperature of
160-260
o
C (corresponding pressure 0.69
–
4.83 MPa) for
several seconds to a few minutes before the material is
exposed to atmospheric pressure. The process causes
hemicellulose degradation and lignin transformation due
to high temperature, thus increasing the potential of
cellulose hydrolysis [17, 25]. Ninety percent efficiency of
