3.2.1 Cellulase
3.2.2 Screening of cellulase-producing microorganisms
3.2.3 Strain improvement for cellulase production
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.
A cellulosic enzyme system consists of three major components: endo-ß-glucanase (EC 3.2.1.4), exo-ß-glucanase (EC 3.2.1.91) and ß-glucosidase (EC 3.2.1.21). The mode of action of each of these being:
(1) Endo-p-glucanase, 1,4-ß-D-glucan glucanohydrolase, CMCase, Cx: "random" scission of cellulose chains yielding glucose and cello-oligo saccharides.(2) Exo-P-glucanase, 1,4-ß - D-glucan cellobiohydrolase, Avicelase, C1: exo-attack on the non-reducing end of cellulase with cellobiose as the primary structure.
(3) ß-glucosidase, cellobiase: hydrolysis of cellobiose to glucose.
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.
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. Fungi are the main cellulase-producing microorganisms, though a few bacteria and actinomycetes have also been recently reported to yield cellulase activity (Table 3-1). Microorganisms of the genera Trichoderma and Aspergillus are thought to be cellulase producers, and crude enzymes produced by these microorganisms are commercially available for agricultural use. Microorganisms of the genus Trichoderma produce relatively large quantities of endo-ß-glucanase and exo-ß-glucanase, but only low levels of ß-glucosidase, while those of the genus Aspergillus produce relatively large quantities of endo-ß-glucanase and ß-glucosidase with low levels of exo-ß- glucanase production.
In order to obtain a high titer cellulase-producing strain, various cellulase-producing strains obtained from Japanese and overseas research institutions were screened in our laboratory. On the basis of this screening, Trichoderma reesei QM-9414, isolated by the U.S. Army Natick Laboratory, was selected as the best cellulase-producing strain and was subjected to mono-colony isolation in order to obtain strain KY-746, which was used as a parent strain. A plate screening system which enables the isolation and evaluation of mutants was initially investigated, which led to the development of methodology for producing high titer cellulase (Section 3.2.3.1). Fungal strains of the genus Trichoderma, when cultured on an agar medium, are generally difficult to isolate, owing to the lateral spreading of hyphae over the surface. Hyphal growth was controlled by the addition of Triton X-1007 and L-sorbose, an inhibitor of P-l,3-glucan biosynthesis, to the medium (Fig. 3-3), thus enabling a large number of strains to be screened. The genealogy of the representative strains created in our laboratory is presented in Fig. 3-4. Large numbers of mutant strains exhibiting a diversity of characteristics were obtained by UV and nitrosoguanidine (NTG) treatment and various other techniques. A strain free from catabolite repression for example, was induced using a culture medium containing glycerol and glucose, while a high titer strain was induced by addition of L-sorbose to the medium. In addition, a strain of elevated (3-glucosidase activity was isolated from medium containing 4-methyl-umbelliferyl-ß-D-glucoside.
Figure 3.2 - Schematic representation of sequential stages in cellulolysis
Table 3-1 Representative Cellulase-producing Microorganisms
|
|
Microorganism |
|
Microorganism |
|
Fungi |
Acremonium cellulolyticus |
Bacteria
|
Clostridium thermocellum |
|
Aspergillus acculeatus |
Ruminococcus albus |
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|
Aspergillus fumigatus |
Streptomyces sp. |
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|
Aspergillus niger |
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|
Fusarium solani |
|||
|
Irpex lacteus |
|||
|
Penicillium funmiculosum |
|||
|
Phanerochaete |
Actinomycetes
|
Streptomyces sp. |
|
|
chrysosporium |
Thermoactinomyces sp. |
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|
Schizophyllum commune |
Thermomonospora curvata
|
||
|
Sclerotium rolfsii |
|||
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Sporotrichum cellulophilum |
|||
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Talaromyces emersonii |
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Thielavia terrestris |
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|
Trichoderma koningii |
|||
|
Trichoderma reesei |
|||
|
Trichoderma viride |
3.2.3.1 Development of a process for high-titer cellulase production
3.2.3.2 Cellulase production at low cost
3.2.3.3 Potential for mass production of cellulase
The commercial use of cellulases is dependent on the following:
i) high titer and good enzymatic activity
ii) low production cost, and
iii) feasible mass production.
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 can also be induced by elaborately controlling culture conditions.
Figure 3.3 - Clearing zones formed by T. reesei mutants on Walseth's Cellulose Agar Plates
CMCase, Avicelase, and FPU activities exhibited a pH optimum of approximately 4, while the pH optimum of P-glucosidase was between pH 5 and 6. pH adjustment was therefore investigated with the objective of uniformly inducing all three activities. Culturing at pH 4 for 2 days, followed by a shift to either pH 5 or 6 on the third day, resulted in a marked increase in the p-glucosidase activity of PC-3-7, but no beneficial effect was observed in the case of CDU-11 and of KDG-3, which was already of high [3-glucosidase activity (Table 3-4). On the basis of these findings, a scale-up experiment was conducted in a 1-kL tank using the semi-batch culturing method with 10% Avicel as a carbon source and soybean curd as a nitrogen source. The pH was controlled and shifted as described above. Results are shown in Fig. 3-6. With a shortened culture period of 12 days, elevated activities of 530 U/ml and 8 U/ml were obtained for CMCase and p-glucosidase respectively, with an elevated protein content of 40 mg/ml in the culture broth. The soluble protein content of the culture broth was 53 mg/ml, which was considered favorable.
Figure 3.4 - Genealogy of artificial mutants of Trichoderma reesei
Table 3-2 Cellulase Productivity of T. reesei Mutants in Batch Culture
|
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 | ||||
Table 3-3 Cellulase Productivity of T. reesei Mutants in Semi-Batch Culture
|
Strain |
Protein (mg/ml) |
CMCase (U/ml) |
ß-Glucosidase (U/ml) |
Avicelase (U/ml) |
FPU (U/ml) |
|
KDG-3 |
36.7 |
448 |
7.6 |
27.6 |
23.2 |
|
PC-3-7 |
37.8 |
418 |
7.0 |
28.7 |
33.5 |
|
CDU-11 |
41.4 |
451 |
17.6 |
31.2 |
31.6 |
|
pH: 4.0 (adjusted with ammonia) | |||||
Table 3-4 Cellulase Productivity of T. reesei Mutants Using the pH Shifting System
|
Strain |
Protein (mg/ml) |
CMCase (U/ml) |
ß-Glucosidase (U/ml) |
Avicelase (U/ml) |
FPU (U/ml) |
|
KDG-3 |
22.8 |
332 |
8.7 |
20.1 |
19.5 |
|
PC-3-7 |
24.0 |
345 |
12.3 |
23.6 |
22.5 |
|
CDU-11 |
23.6 |
328 |
17.6 |
22.0 |
19.1 |
|
pH: Initial: 4.0; 5.0 after 48 hours | |||||
Cellulase is an inducible enzyme. Sophorose, a decomposition product of crystalline cellulose, is thought to be an indispensable inducer of cellulase activity. Although cellulase culture has been investigated with the use of Avicel and other crystalline celluloses as carbon sources, cheaper carbon and nitrogen sources are desired in order to lower production costs.
Figure 3.5 - Effect of initial substrate concentration on cellulase production by batch fermentation
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.
Figure 3.6 - Time course of cellulase production by T. reesei KDG-3 in a 1kL tank
Table 3-5 Cellulase Production From Cheese Whey
|
CMCase [U/ml] |
545 |
|
FPU [U/ml] |
32 |
|
(3-Glucosidase [U/ml] |
23 |
|
Avicelase [U/ml] |
42 |
|
Protein [mg/ml] |
45 |
|
Carbon source: 10% Cheese whey and 1% Avicel | |
Figure 3.7 -Effect of alkali concentration of the production of cellulase from biomass
Cellulase fermentation was conducted with high-titer enzyme-producing strain CDU-11 under semi-batch conditions in a 4-L fermentor, using inexpensive carbon and nitrogen sources and the pH-shift system. This resulted in the production of CMCase activity exceeding 600 U/ml and (3-glucosidase activity exceeding 30 U/ml. Such activity levels could feasibly be practically applied to the development of scaled-up processes.
