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3.2.1 Cellulase
3.2.2 Screening of cellulase-producing microorganisms
3.2.3 Strain improvement for cellulase production
3.2.1 Cellulase
Successful utilization of cellulosic materials as renewable carbon sources is dependent
on the development of economically feasible process technologies for cellulase
production, and for the enzymatic hydrolysis of cellulosic materials to low molecular
weight products such as hexoses and pentoses. Spano et al. (4) showed that cellulase
production, was the most expensive step during ethanol production from cellulosic
biomass, in that it accounted for approximately 40% of the total cost. Significant cost
reduction is required in order to enhance the commercial viability of cellulase production
technology.
Reese et al. 1950 (5) proposed that exo-ß-glucanase causes a disruption in cellulose
hydrogen bonding, followed by hydrolysis of the accessible cellulose with endo-
ß-gucanase. Although cellulase isolation techniques have not been fully developed, the
hypothesis depicted in Fig. 3-2 is now accepted. According to this hypothesis, in a
synergistic sequence of events, endo-ß-glucanase acts randomly on the cellulose chain,
while exo-ß-glucanase acts on exposed chain ends by splitting off cellobiose or glucose.
Cellobiose is subsequently hydrolysed by p-glucosidase to glucose. This hypothesis is
however the opposite of that proposed by Reese et al. (5), and indicates that three,
rather that two enzymes are essential for the decomposition of cellulosic biomass.
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Microorganism Microorganism
Acremonium cellulolyticus Clostridium thermocellum
Aspergillus acculeatus Ruminococcus albus
Fungi Aspergillus fumigatus Bacteria Streptomyces sp.
Aspergillus niger
Fusarium solani
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Irpex lacteus
Penicillium funmiculosum
Phanerochaete Streptomyces sp.
Sclerotium rolfsii
Sporotrichum cellulophilum
Talaromyces emersonii
Thielavia terrestris
Trichoderma koningii
Trichoderma reesei
Trichoderma viride
Representative mutants derived from KY-746 were batch cultured in a 5-L fermentor.
Considerable enhancement of titer was observed in four strains: KDG-3, PC-3-7,
PCD-10, and CDU-11 (Table 3-2). An evaluation of carbon source utilization by these
microorganisms, suggested that the production of high-titer cellulases necessitated an
increase in the concentration of Avicel, the carbon source. A somewhat lower than
expected maximum cellulase titer was however obtained at Avicel concentrations greater
than 6% in batch culture (Fig. 3-5). This relatively lower titer, may be attributed either to
adsorption of the cellulase produced on to the Avicel, or rate limitation of aeration due to
the high viscosity of the culture medium. In order to overcome this, semi-batch culturing
was investigated, where it was found that an initial Avicel concentration of 6%, followed
by subsequent addition of 4% Avicel together with other components after a few days,
resulted in relatively higher titer cellulase than that obtained in the batch process (Table
3-3). Titer improvement by the semi-batch process was however dependent on the types
of microorganisms used. While the titer of KY-746 was relatively unaffected by the semi-
batch process, various degrees of improvement in titer were observed for KDG-3,
PC-3-7, PCD-10, and CDU-11. This semi-batch process was further utilized in order to
optimize the stirring rate, aeration volume, feed change, seed volume, and the seed
culture period. T. reesei generally exhibits poor (3-glucosidase activity. Strain CDU-11
was therefore created in order to enhance p-glucosidase activity. P-glucosidase activity
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Mutant CMCase (U/ml) FPU (U/ml) ß-Glucosidase (U/ml) Extracellular protein (mg/ml)
QM-941 4 60 6.1 3.2 10
KY-746 88 7.2 3.5 11
K-14 106 12 1.1 17
KDR-27 150 18 2.9 20
KDG-3 324 19.5 7.7 23
PC-3-7 345 21 6.7 24
PCD-10 385 22.4 6.4 28
CDU-11 330 19 17.4 23
5-L jar fermentor, 6% Avicel, 28/C, 7 days
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Table 3-4 Cellulase Productivity of T. reesei Mutants Using the pH Shifting System
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An evaluation of the use of cheese whey, bagasse, and rice straw, as potential
substrates for cellulase production, confirmed that PC-3-7 was capable of fermenting
these substrates and efficiently producing cellulase enzymes. PC-3-7 was created as a
strain, capable of partially producing cellulase enzymes. Table 3-5 shows an example of
cellulase production using cheese whey as the carbon source. Flask-scale production of
cellulase using bagasse and rice straw, the main raw materials used for fuel alcohol
production, is shown in Fig. 3-7. Alkali pre-treatment of biomass was observed to
enhance cellulase production. This seems to suggest that cellulase production is directly
proportional to the crystallinity of the biomass from which it is produced, i.e. the higher
the crystallinity, the better the yield of cellulase.
The use of soybean curd, a low-cost nitrogen source, which is applicable as a substitute
for expensive yeast extract and polypeptone, was investigated with cellulase
fermentation residues (mainly waste cells). Results obtained, revealed that addition of
5% of the fermentation residue induced cellulase activity similar to that induced by the
use of either yeast extract or polypeptone.
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