2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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5 min. The production of sugar was measured by using
the glucose oxidase/peroxidase method of the GAGO-20
assay, Sigma-Aldrich.
Sugar assay
Production of sugar was quantified by using a glucose
(GO) assay kit, GAGO-20 (Sigma) following the
suppliers directions. An adapted methodology was carried
out using a 96-microplate format. Per individual test,
40 μl of centrifuged, diluted sample was mixed with
80 μl glucose reagent and incubated at 37
o
C on a standing
shaker 200 rpm for 30 min. Reactions were stopped at 30-
60 seconds by adding 80 μl of 12 N sulphuric acid
(H
2
SO
4
). Absorbance at 540 nm was measured in a
microplate spectrophotometer. In order to convert
absorbance values to glucose concentration, measured
values were interpolated using a glucose calibration curve
which kept a linear relationship between 37.04 and
1.37 μg ml
-1
.
Statistical analysis
Group values for all parameters in the tests were
compared by analysis of variance (ANOVA) tests using
the Fisher’s least significant difference (LSD) and
Duncan’s test procedure for multiple comparison (SPSS
8.0 for Windows; USA). Relations among the values of
samples were compared for each factor to the controls
kept under the same conditions than the samples of
interest.
III. R
ESULT AND
D
ISCUSSION
The effects of different combinations of enzymes in
cellulose hydrolysis by using each time 100 mg of
cellulosic pellets obtained in an up-scaled pretreatment
process in which in 1 hr incubation 5 g of
A. grandis
wood were incubated in 40 ml of 85% phosphoric acid in
a 100 ml beaker kept at 50
o
C in a hot water bath.
Each 100 mg of the cellulosic pellet were hydrolyzed
either by a single enzyme or by combinations of enzymes
in 50 mM sodium acetate buffer, pH 5.0 at 37
o
C for one
day. In a comparative analysis, single enzyme hydrolysis
was followed up, thereby using 4 U ml
-1
of either
cellulase, xylanase, laccase, laccase+mediator (mediator:
HBT; 1-
hydroxybenzotriazole; 1 mM) or β
-glucosidase,
respectively (Fig. 1). Of these, only cellulase and
xylanase hydrolysis obtained about 60% glucose yields,
whereas laccase, laccase+HBT and β
-glucosidase
obtained glucose yields lower than 10% (Fig. 1) and no
glucose yield was obtained by un-hydrolyzed controls.
C
ombinations of enzymes (laccase and/or β
-glucosidase
together with cellulase and xylanase) in contrast were in
all instances more successful in yielding glucose from
cellulose degradation (up to 100% glucose yield) but the
combination of only cellulase and xylanase (Fig. 1). This
combination of only two enzymes reached just about 60%
glucose yield, similarly to that what was achieved with
the two enzymes in individual digestion (Fig. 1).
Interesting to note is that regardless of whether
laccase, laccase with a mediator (HBT),
β
-glucosidase or
combinations of these were added, the yield of glucose
was always optimal (Fig. 1). The laccase applied were
supplied from Novo, Denmark (Novozyme, laccase EEC
No. 420-150-4). To elucidate possible contaminations,
0.12 U of the laccase solved in 30 μl of 50 mM sodium
acetate buffer, pH 5.0 were used in final total volumes of
1 ml of 100 mM sodium lactate buffer, pH 5.0 to
determine cellulase activity with 4.0% (w/v) CMC and
xylanase activity with 1.5% (w/v) with xylan form oat
spelts. To determine
β
-glucosidase activity with 1 mM
p
-nitro phenol (pNP-Glc), 2 U of laccase solved in 500 μl
of 100 mM sodium acetate buffer, pH 5.0 were tested for
foreign enzymatic activities. In no case, such enzymatic
activities were detected as contaminations in the
preparations. Accordingly, if these types of enzymes are
at all present, they should occur in very minor amounts in
the solutions of 4 U ml
-1
of laccase applied in the
A. grandis
wood pretreatments.
Fig. 1. Conversion of cellulose to glucose in cellulosic pellets of
A.
grandis
wood particles pretreated prior to enzymatic hydrolysis with 8
ml of 85% phosphoric acid at 50
o
C for one hour in a hot water bath, and
washed with water. Each time 100 mg of left cellulosic pellet were
incubated with enzymes in 50 mM sodium acetate buffer, pH 5.0 for
24 hrs at 37
o
C under constant shaking. Per different treatment, three
samples were incubated in parallel and average values and standard
deviations were calculated. The different superscripts on the chart
indicate values of glucose yields that differ significantly (
p
<0.05)
between treatments as based on analysis of variance (ANOVA).
Effects of laccase were further tested on
A. grandis
wood that had undergone phosphoric acid-microwave
pretreatments by five or 20 sec irradiation in a volume of
8 ml phosphoric acid at the different concentrations of
80%, 40% and 20% acid, respectively. Each time for
enzymatic hydrolysis, 100 mg of resulting cellulosic
pellets were hydrolyzed by a combination of 4 U ml
-1
cellulase, 4 U ml
-1
xylanase and 4 U ml
-1
laccase in
50 mM sodium acetate buffer, pH 5.0 for 24 hrs. The
glucose yields after enzymatic hydrolysis obtained were
highest with about 100% when wood particles were
0%
20%
40%
60%
80%
100%
No enzyme
Cellulase
Xylanase
Laccase
Laccase+HBT
Beta-Glucosidase
Cellulase+xylanase
Cellulase+xylanase+laccase
Cellulase+xylanase+Beta-glucosidase
Cellulase+xylanase+Beta-glucosidase+laccase
Cellulase+xylanase+laccase+HBT
Cellulase+xylanase+Beta-glucosidase+laccase+HBT
Conversion of cellulose to glucose [%]
a
a a a
a
b
b b
c c
d
