2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 65 -
enzymatic hydrolysis has been achieved in 24 h for
poplar chips pretreated by steam explosion, compared to
only 15% hydrolysis of untreated chips [26].
B. Optimization for enzymatic hydrolysis using leaf
waste
The effect of waste concentration, enzyme
concentration and incubation time was studied.
C. Effect of different amount of leaf waste
An initial increasing trend of sugar formation was
observed when more of substrate was degrade with fixed
enzyme concentration (0.17 mg/mL). The highest
reducing sugars concentration at 6.7 g/L was obtained
from the optimal leaf waste at an amount of 30 g/L. In
detail, the concentration of sugars was found to increase
with increasing leaf waste from 2
–
30 g. Leaf waste in
the range of 2
–
30 mg gave sugar amount at 0.36
–
6.70
g/L. High amounts of leaf waste tested (40 g) gave lower
sugars at 1.3 g/L (Table 2).
T
ABLE II
E
FFECT OF DIFFERENT AMOUNT OF LEAF WASTE ON SUGAR PRODUCTION
Leaf waste (g)
Sugar production (g/L)
0
0
2
0.36±0.2
3
0.39±0.1
4
0.61±0.1
5
0.74±0.3
10
0.82±0.1
20
3.57±0.2
30
6.70±0.1
40
1.30±0.3
D. Effect of cellulase concentration and incubation time
The effect of cellulase concentrations (0.13, 0.17, 0.22
and 0.27 mg/ml) and incubation time was also studied.
Cellulase from
Aspergillus niger
(Sigma) was used
throughout this study. The highest sugar production
6.70±0.3 g/L was obtained from 0.17 mg/mL of cellulase
after 16 h of incubation time. Increase of enzyme
concentration gave a decrease in sugar production (Fig.
1).
Cellulose can also be hydrolysed enzymically under
milder reaction conditions. A cellulosic enzyme
(cellulase) system consists of three major components:
endo-
β
-glucanase (EC 3.2.1.4), cellobiohydrolase (EC
3.2.1.91) and β
-glucosidase (EC 3.2.1.21). Endo-
β
-
glucanases randomly hydrolyse the internal glycosidic
bond to decrease the length of the cellulose chains.
Cellobiohydrolases are exo- or endo-processive enzymes
that split off cellobiose. Cellobiose is subsequently
hydrolysed by β
-glucosidases to glucose [27].
Although a large number of microorganisms are
capable of degrading cellulose, only a few of these
microorganisms produce significant quantities of cell-free
enzymes capable of completely hydrolysing crystalline
cellulose
in vitro.
Microorganism of the genera
Aspergillus
is thought to be cellulase producers, and a
crude enzyme produced by this microorganism is
commercially
available
for
agricultural
use.
Microorganisms of the genus
Aspergillus
produce
relatively large quantities of endo-
β
-
glucanase and β
-
glucosidase with low levels of exo-
β
-glucanase
production [27].
Fig. 1. Effect of cellulase concentration and incubation
time on sugar production from leaf waste (30 g/L). The
concentration of enzyme was varied at 0.13 mg/ml (●),
0.17 mg/mL (■), 0.22 mg/mL (♦) and 0.27 mg/mL (▲).
E. Identification of wastepaper hydrolyzate composition
Cellulose, major component of paper can be converted
enzymatically to sugars (e.g., glucose, cellobiose,
cellotriose xylose and L-arabinose, etc.) and subsequently
fermented to valuable products. Therefore, sugar content
obtained from leaf waste hydrolyzate was identified by
HPLC analysis. Glucose was found to be a major end
product (6.7 g/L) in enzymatic hydrolyzate. The present
of glucose suggests the complete action of enzyme during
lignocellulose hydrolysis (Fig. 2).
F. Ethanol production using leaf waste as raw substrate
Hydrolysate obtained from leaf waste under optimal
condition was used as fermentation medium, with no
nutritional supplementation, for the separate hydrolysis
and fermentation (SHF) and sequential saccharification
and fermentation (SSF) process, to produce ethanol with
S. cerevisiae
TISTR 5048
.
The fermentation of the hydrolyzate (containing
glucose at 6.7 g/L) was compared with that of YMP
medium supplemented with reagent-grade glucose at
these same concentration.
The time course profiles obtianed for both
fermentation experiments are represented in Fig. 3. The
ethanol production obtained in the SSF process was 1.18
g/L followed by SHF (1.06 g/L) after 36 h of cultivation,
coresponding to an ethanol volumetric production rate of
0.03 g ethanol/L·h. However, the highest ethanol
concentration was obtained from YMP medium at 2.90
g/L (Fig 3). Ethanol was produced along with cell growth
in the course of the SSF process. The highest cell mass
was also obtained after 36 h of cultivation time. Yeast
cells readily consumed glucose to produce ethanol and
total consumption occurred within less than 35 h of
incubation [28].
