2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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The purpose for this is on the development of approaches
and technologies with a significant promise in reducing
production cost and in caring for environmentally
friendly procedures in order to make lignocellulosic
ethanol economically competitive with the first
generation biofuel production. This knowledge is thus
urgently needed and also knowledge from timely research
in how to integrate enzymatic hydrolysis of material such
as wood into already existing basic fermentation
processes in order to adjust the available biotechnology
for biofuel production to the specific challenges meet
when transforming cellulose from wood e.g. into ethanol
[2, 3, 4].
II. M
ATERIAL AND
M
ETHOD
Wood
Wood particles of
A. grandis
were prepared in
cooperation with the work group of Prof. Kharazipour
from the Division of Molecular Wood Biology and
Technology and Technical Mycology, Büsgen-Institute,
Göttingen, Germany, by chipping wood log into wood
shaving (length about 1 cm, width about 1 cm and height
about 2 cm) in a drum chipper (Klöckner Trommelhacker
KTH 120 x 400 H2WT, Klöckner Wood Technology
GmbH, Hirtscheid, Germany), flaking these with a knife
ring flaker (Condux HS 350, Condux Maschinenbau
GmbH. and. Co. KG, Hanau, Germany) into smaller
particles and then sieving the wood particles
(60-40 mesh), following the methods presented in [21].
Wood extractives
Wood particles (10 g) was extracted with a Soxhlet
apparatus in 450 ml boiling water for 6 hrs, following
TAPPI Test Method T 204 om-88 (1988). The water
extractives were concentrated and evaporated to dryness
using a rotatory evaporator (Heidolph, W105/38, V 220,
and Hz 50) at 60
o
C. Wood extractives were dissolved in
dimethyl sulfoxide (DMSO) in a concentration of 1 ml
DMSO per 1 g extracted wood. Wood extractives were
analyzed by GC-MS (Gas Chromatography Mass
Spectrometry, Agilent technologies, 6890N, network GC
system, USA) to define individual extractive constituents
in the solution. The extractives in 0.5 ml DMSO were
concentrated to dryness by speed-vacuum-centrifugation
(Concentrator 5301, Eppendorf, Wesseling-Berzdorf,
Germany) at 45
o
C. Dry samples were dissolved in
50 μl pyridine (PIERCE Biotechnology, Rockford, USA)
and 50 μl bis-N,O-trimethylsilyl trifluoroacetamide
(BSTFA: PIERCE Biotechnology, Rockford, USA) for
derivatization in order to increase the volatile phase of
compounds and give more thermal stability to the
samples. The extractives were directly dried and
resuspended in 300 μl toluene to be injected into the
GC-MS (capillary column, Agilent 122-5532).
Compounds were identified by comparing resulting data
with standard references in the data program NIST
(National Institute of Standards and Technology,
Maryland, USA). Concentration of specific compounds
were determined by injecting mixtures of specific
compounds (2-methoxyphenol, 4-hydroxybenzaldehyde,
1,4-dihydroxybenzene, 3,5-di-tertbut-4-hydroxy-toluene,
3-methoxy-4-hydroxybenzaldehyde,
4-hydroxybenzoic
acid, 3-methoxy-4-hydroxybenzoic acid, 4-hydroxy
cinnamic acid, and 4-hydroxy-3-methoxy cinnamic acid)
in defined amounts (0.025 μg each, 0.05 μg each, 0.1 μg
each, 0.25 μg each, 0.5 μg each, 2.5 μg each, and 5.0 μg
each) into the GC-MS and creating for comparison a
standard curve of abundances for each tested compounds.
Chemical and physico-chemical pretreatment
Chemical and physico-chemical pretreatments
followed with modifications the procedure presented by
[9]. Wood particles were treated with phosphoric acid
(8 ml per 1 g wood in 25 ml glass beaker), then incubated
in a hot water bath at 50
o
C or incubated with microwave
irradiation (Intellowave microwave, LG Co., Ltd,
Germany). For irradiation of 1 g wood particles in 8 ml
phosphoric acid in a 25 ml glass beaker, a rating power
1000 W, an out power 700 W, and working voltage
230 V and 50 Hz was used.
Chemicals and enzymes
All chemicals were reagent grade and purchased from
Sigma-Aldrich (St. Louis, MO), unless otherwise noted.
A commercial cellulase enzyme (Cellulase “Onozuka R
-
10” from
Trichoderma viride
, SERVA Electrophoresis
GmbH,
Heidelberg,
Germany),
xylanase
from
Trichoderma viride
(Fluka),
β
-glucosidase from almonds
and recombinant laccase from
Aspergillus
sp.
(Novozyme, EEC No. 420-150-4) and pure laccase V
(as purified by [22]) from
C. cinerea
Okayama 7 were
used for enzymatic hydrolysis.
Enzymatic cellulose hydrolysis
All enzymatic hydrolysis experiments were carried
out at 37
o
C in 5 ml of 50 mM sodium acetate buffer, pH
5.0 for 100 mg substrate in 50 ml Falcon tubes
(SARSTEDT, Nümbrecht, Germany) on a standing
shaker (Uniform, Infors AG, Bottmingen, Switzerland)
applying 200 rpm for shaking. 500 μl samples were
periodically removed and centrifuged with 16,000 g for
5 min. The production of sugar was determined by using
a glucose detection GAGO-20 assay, Sigma-Aldrich,
Steinheim, Germany through measurements in a
spectrophotometer (Spectra max340PC, Molecular
Devices, California, USA). In another set of experiments,
the influences of wood extractives and specific phenolic
compounds on cellulose hydrolysis were determined by
analyzing a kinetic curve between production of glucose
and incubation time. The experiment was carried out by
using 5 mg carboxymethylcellulose (CMC) as a substrate
in a 50 ml Falcon tube on a standing shaker 200 rpm,
37
o
C in 5 ml of 50 mM sodium acetate buffer, pH 5.0.
500 μl samples were periodically collected after 60, 90
and 120 min of incubation, and boiled for 5 min to stop
the enzymatic reaction, and centrifuged for 16,000 g for
