2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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pretreated with 80% phosphoric acid in 20 sec microwave
irradiation, whereas a pretreatment with 80% phosphoric
acid in 5 sec microwave irradiation resulted in a glucose
yield of about 60% (Fig. 2). Application of lower
phosphoric acid concentrations in the pretreatment
procedures resulted in lower glucose yields (around 40%
to 50%) in subsequent enzymatic cellulose conversion
(Fig. 2). When applying a scheme of microwave
irradiation of 4 rounds of 5 sec irradiation and 5 sec of no
irradiation, when higher concentrations of phosphoric
acid were applied (80% and 40%), the resulting glucose
yields in the subsequent enzymatic hydrolysis were
significantly lower as in microwave experiments in which
20 sec in a constant incubation was applied (Fig. 2). In
contrast, for the lowest applied phosphoric acid
concentration (20%), no differences in the yields were
seen regardless of the time of microwaving applied
(Fig. 2).
0%
20%
40%
60%
80%
100%
Buffer,no enzyme
Buffer +enzymes
80% 5 sec
80% 20 sec
80% 20 sec*
40% 5 sec
40% 20 sec
40% 20 sec*
20% 5 sec
20% 20 sec
20% 20 sec*
Conversion of cellulose to glucose [%]
Fig. 2. Enzymatic conversion of cellulose to glucose of
A. grandis
wood
particles pretreated prior to enzymatic hydrolysis for 5 or 20 sec in a
microwave (Intellowave microwave, LG Co., Ltd, Germany) at a rating
power of 1000 W, an out power of 700 W, and a working voltage of 230
V and 50 Hz, with 8 ml of various concentrations of phosphoric acid.
Each 100 mg of left cellulosic pellets were incubated with 4 U ml
-1
of
cellulase and 4 U ml
-1
xylanase and 4 U ml
-1
laccase in 50 mM sodium
acetate buffer, pH 5.0 for 24 hrs at 37
o
C under constant shaking. Three
different samples per pretreatment and three controls with 100 mg
original
A. grandis
wood particles were treated in parallel and average
values of conversion of cellulose to glucose the experimentally
determined amount in the respective samples 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). * Incubation for 5 sec, stop for
5 sec, repeated for 4 cycles; total 20 sec.
Another interesting result from the experiments
presented in Fig. 2 is, that reasonable yields of glucose
were obtained both with application of 40% and of 20%
phosphoric acid in the microwave pretreatments but that
overall there were no or only marginal differences in the
glucose yields. Thus, a combination of very low amounts
of phosphoric acid with laccase gives the ecological
favorable situation.
As indicated already from the conclusion from the
results presented in Fig. 2, wood extractives are one of
the possible drawbacks of enzymatic hydrolysis processes
since the functions of the enzymes might be inhibited by
the wood extractives. Water extractives were subjected to
a GC-MS analysis in order to identify their individual
compounds (Fig. 3). The different compounds were
present in the chromatograms of water extractives with an
identification quality higher than 95%: 4-Hydroxy-3-
methoxy cinnamic acid (ferulic acid), 4-Hydroxy
cinnamic acid (
p
-coumaric acid) and 3-Methoxy-4-
hydroxybenzoic acid (vanillic acid) were obtained highest
amount in concentration of 17.82, 16.28 and 14.43 μg in
1 g dry wood, respectively (Fig. 3; filled arrows).
Fig. 3
.
GC-MS analysis of compounds in water extractives from
A. grandis
wood particles. Filled arrows mark compounds obtained
highest amount in concentration (μg) in 1 g dry wood
.
This section therefore investigates the influences of
wood extractives in cellulase hydrolysis carried out in
50 mM sodium acetate buffer, pH 5.0 (Fig. 4). A
commercial carboxymethylcellulose (CMC) preparation
(product No. 21901, Fluka) was used as a substrate in
cellulase hydrolysis at a concentration of 5 mg in 5 ml
50 mM sodium acetate buffer pH 5.0 with either
0.008 U ml
-1
cellulase (Cellulase “Onozuka R
-
10” from
Trichoderma viride
, SERVA Electrophoresis GmbH,
Heidelberg, Germany) or 0.008 U ml
-1
cellulase and
0.008 U ml
-1
laccase (Novozyme, EEC No. 420-150-4)
added to the samples for a 2 and a half hour incubation at
37
o
C. Water extractives were used to simulate the
influence of extractives on enzymatic cellulose
hydrolysis. Moreover, addition of laccase was also used
to possibly improve cellulase hydrolysis under such
circumstances.
Fig. 4A shows that water extractives negatively
influenced hydrolysis of 5 mg CMC in 50 mM sodium
acetate buffer, pH 5.0 by cellulase over the whole time of
incubation observed, although the kinetic curve of
production of glucose (PG) by CMC hydrolysis through
cellulase increased linearly according to the time of
incubation (T), (Fig. 4A). At the end of the experiment,
the glucose yield was 13 μg ml
-1
and thus only half as
much as achieved with the enzyme without addition of
wood extractives (24 μg ml
-1
; Fig. 4A). However, when
adding in addition laccase to the samples, the negative
effect by the wood extractives was counteracted and a
nearly identical kinetic curve for CMC hydrolysis was
obtained than for CMC+cellulase alone (compare Fig. 4A
and 4B).
14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
ß-
Sit
est
er
ol,
T
M
S
Ca
m
pe
ste
rol
,
T
M
S
de
hy
dr
oa
bie
tic
aci
d
Ol
eic
aci
d,
T
M
S
est
er
D-
Gl
uci
tol,
T
M
S
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00
22.00 23.00
24.00
25.00
26.00
27.00
50000
150000
250000
3500
450000
55000
650000
750000
50
950000
1050000
115
Retention time
Abundance
ß-Sitosterol
Campesterol
2,6-Dibromo-4-nitrophenol
Dehydroabietic acid
Octadecanoic acid
Oleic acid
4-Hydroxy-3-methoxy cinnamic
Hexadecanoic acid
D-Glucitol
4-Methoxy cinnamic acid
3-Methoxy-4-hydroxybenzoic acid
4-Hydroxy
cinnamic acid
a
b
c
c
d e d de de
f f
