Cellulase Yield Research Articles

A hypercellulolytic mutant of Fusarium oxysporum

Multiple mutagenesis of Fusarium oxysporum DSM 841 resulted in enhanced yields of cellulases. The hypercellulolytic mutant (NTG-19) secretes high levels of extracellular cellulases on different cellulosic substrates. Addition of surfactant, Tween-80, further increased enzyme secretion by about 30%. The results on hydrolysis of wheat straw by parent strain, DSM 841 and mutant NTG-19 cellulases also revealed a significant improvement in the hydrolytic potential of the cellulolytic enzymes from the mutant NTG-19.

Enhanced production of cellulases byPenicillium citrinum in solid state fermentation of cellulosic residue

Penicillium citrinum, using rice husks in a solid state fermentation, produced maximum cellulase yields (37 Units/g) after 12 days with a cellulose utilization of more than 70%. Enzyme yields were three times higher than in shake-flask cultures.

Regulation of Cellulase Synthesis in Acidothermus cellulolyticus

AbstractThe regulation of cellulase synthesis by induction and catabolite repression in the thermophilic, aerobic bacterium Acidothermus cellulolyticus was studied in batch fermentations. Various compounds, such as L‐sorbose, cyclic AMP (cAMP), L‐glucose, 2‐deoxyglucose (2‐DG), glucose 1‐phosphate (G‐1‐P), sophorose, salicin, sugar alcohols, and isopropyl thioglucoside (IPTGlu), were added along with Solka Floe to improve extracellular cellulase formation by the culture. When cAMP was added exogenously to A. cellulolyticus cultures in the concentration range of 0.01‐0.2 g/L, cAMP did not affect cell growth; however, cellulase yields were increased with increasing levels of cAMP. The enzyme production rates with the different levels of cAMP addition during Solka‐Floc fermentations were identical. L‐Sorbose, L‐glucose, 2‐DG, G‐1‐P, sophorose, IPTGlu, and sugar alcohols enhanced cellulase activity produced in the medium, but the starting time and the time required to reach the maximum enzyme activity were different in each condition. All these substances may function as moderators of cellulase synthesis. From the experimental results, only cellobiose, xylose, sophorose, and unknown soluble derivatives from cellulose were considered as inducers. In a possible regulatory mechanism of cellulase synthesis, the repressor, inducer, cAMP, and moderator may be all involved in controlling the rate and the yield of enzyme production.

Production of protein and cellulase byAspergillus niger in solid state culture

Single-cell protein (SCP) and cellulase production byAspergillus niger AS-101, grown on alkalitreated corn cobs, was studied under various cultural conditions. The maximum yields of SCP and cellulase, under solid-state fermentation were obtained when the culture was incubated at pH 5.5 for 18 d at 30°C with 12% substrate.

Production of amylase and cellulase: Degradation of starch and carboxymethylcellulose by extracellular enzymes from four fungal species

Four fungal species, Aspergillus terreus (SA 562), Fusarium moniliforme (SA 1190), Aspergillus carneus (SA 1326) and Phoma sorghina (SA 1397) have been screened for the production of cellulase (EC 3.2.1.4) and amylase (EC 3.2.1.1). All the species grew profusely on culture plates containing 1% ground millet, starch or carboxymethylcellulose (CMC) as the carbon source. Maximum growth was observed within about 96 h. When grown in submerged liquid culture, stationary or shaken at 20 strokes min −1, the species produced a good yield of extracellular cellulase and amylase. Differences in yield of enzymes for stationary and shaking growth were negligible. A preliminary study of the action of the crude enzymes on CMC, starch, ground millet (Pannisetum typhoidees) and acha (Digitaria exilis) is reported.

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.

Enhanced cellobiohydrolase production from Aspergillus ustus and Trichoderma harzianum

The mutants Aspergillus ustus M35 and Trichoderma harzianum M5 were selected for their ability to grow faster on crystalline cellulose as sole carbon source and to excrete higher cellulase yields, particularly exo-cellobiohydrolase (1,4-β- d-glucan cellobiohydrolase EC 3.2.1.91), than the wild types. At optimum pH and temperature of enzyme activity the respective yields of exo-cellobiohydrolase, endo-1,4-β- d-glucanase [1,4-(1,3;1,4)-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β- d-glucosidase (β- d-glucoside glucohydrolase, EC 3.2.1.21) were 1.0, 69.0 and 19.2 U ml −1 culture fluid for A. ustus M35 and 1.0, 33.5 and 2.5 U ml −1 culture fluid for T. harzianum M5. The exo-cellobiohydrolase yields were among, if not the highest reported in the literature.

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.

Production of Cellulase byTrichoderma reesei on Waste Cellophane

Utilization of waste cellophane for production of extracellular cellulose-degrading enzymes byTrichoderma reesei was studied on laboratory-scale experiments using liquid media containing varnished or non-varnished cellophane disintegrated physically to different degrees. Enzyme activities were directly proportional to the degree of cellophane disintegration,i.e. its specific surface area. Hydrolytic activities of cellulase on 1 % milled cellophane reached approximately 50 % yield of the activities acquired on 1 % microcrystalline cellulose. Addition of Xylocel, concentrated prehydrolyzate of beech wood, added to the media in amounts corresponding to 0.2 to 1.0 % concentration of reducing groups, did not improve the yield of cellulase on milled cellophane. However, β-D-xylanase activity was increased several times due to the presence of lower xylooligosaccharides serving as specific β-D-xylanase inducers. No differences in occurrence of multiple forms of endo-l,4-β-D-glueanases and endo-l,4-β-D-xylanases and their isoelectric points were observed.

Cellulase production by trichoderma harzianum in static and mixed solid‐state fermentation reactors under nonaseptic conditions

Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8., 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 h. The extension from static column cultivation to stirred tank reactor of 65 L capacity gave similar yields of cellulase.

Comparisons between cellulase production by Aspergillus fumigatus in agitated vessels and in an air‐lift fermentor

Aspergillus fumigatus was cultured in disc-turbine-agitated vessels and in an air-lift fermentor. In the agitated vessels the yield of cellulase was reduced when the agitation rate was increased, although extracellular protein levels rose. The enzyme complex itself was shown to be exceptionally stable under conditions similar to those in the agitated vessels, so probably shear damage to the mycelium had occurred, liberating intracellular contents. These appeared to contain an inhibitor that could be removed by fabricated inorganic protein absorbents, such as kieselguhr and alumina. However, the inhibitor was not likely to be protease, since only relatively low levels could be detected and its identity has not been established. The use of an air-lift fermentor avoided the shear effects due to use of the disc turbine agitator in the conventional fermentors, and yields of enzyme were then found to increase by about 20%, maximum yields being obtained at maximum K(L)a values.

Regulation of cellulase biosynthesis and secretion in fungi.

The cellulase complex of the mesophilic fungus Trichoderma reesei is capable of efficiently hydrolysing crystalline cellulose to glucose and provides a potential route to the conversion and recycling of renewable cellulosic biomass. To date, however, the high cost of these enzymes has hampered their large-scale industrial application. This high cost is principally due to the low yield and low specific activity of enzymes produced by the available microbial strains. In recent years a number of improved mutant strains have been isolated, many of which produce as much as 20mg of extracellular protein/ml with substantial increases in yields of cellulase (for a review see Montenecourt, 1983). However, only one strain, RL-P37, exhibits an increase in specific activity (Sheir-Neiss & Montenecourt, 1984). In spite of the improvements that have been made in strain development of T. reesei, major new advances cannot be anticipated until the mechanisms controlling the synthesis and secretion of cellulases are fully understood.

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), beta-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, beta-glucosidase, and xylanases.

Selection of Hypercellulolytic Mutants of Trichoderma reesei Based on Resistance to Nystatin

Nystatin-resistant mutants of Trichoderma reesei were isolated, and several derived from the wild strain showed increased production of cellulase. Thus, the concept of gaining greater enzyme release through interference with membrane function was confirmed. Certain nystatin-resistant mutants derived from the hypercellulolytic strain RUT-C30 also showed increased cellulase yields, but these strains proved unstable and reverted to giving basal enzyme yields.

Pilot scale production of cellulase by Trichoderma reesei (Rut c-30)

A 150-litre pilot scale fermentation using Trichoderma reesei (RUT C-30) for the production of cellulase is described in which a cellulase yield of 12.0 IU.ml−1 (filter paper activity) and productivity of 75 IU.L−1h−1 were achieved.

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- d-glucosidase [β- d-glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater .

Optimization of Clostridium thermocellum growth on cellulose and pretreated wood substrates

Two strains of Clostridium thermocellum ATCC 27405 and NRCC 2688 demonstrated similar product yields and cellulase activities when grown on solka floc. A sequential culture of C. thermocellum and Zymomonas anaerobia supplemented with cellobiase could produce 1.8 mg/ml of ethanol when grwon on 1% solka floc. Different media were evaluated for their ability to enhance the product and cellulase yields of C. thermocellum grown on cellulose substrates. Ethanol and reducing sugar values of 1.5 and 3.8 mg/ml respectively and an endoglucanase activity of 3 IU/ml were obtained after growth of Clostridium thermocellum in a modified medium containing 1% solka floc. Three different pretreated wood fractions were assessed as substrates for growth. A steam exploded wood fraction gave comparable values to those obtained after growth on solka floc. Sequential cultures of C. thermocellum and Zymomonas anaerobia grown on a 1% steam exploded wood fraction could produce 1.6 mg/ml ethanol after 3 days growth.

Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling

AbstractCultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio = 8.5) produced 4.5 IU/ml celulase 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr). In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml, and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.

Secretion of cellulase and beta-glucosidase by Trichoderma viride ITCC-1433 in submerged culture on different substrates.

Trichoderma viride ITCC-1433 produces high yields of cellulase and especially beta-glucosidase when grown in submerged culture on different carbon sources. Cellulase synthesis was strongly repressed in the presence of glucose and only a low constitutive activity of beta-glucosidase and carboxymethylcellulase, but no Avicelase, could be demonstrated when culturing T. viride on glucose. With carboxymethylcellulose (CMC) as a substrate the secretion of enzyme as well as growth depended on the degree of substitution, but in general CMC cannot be regarded either as a powerful inducer or as a carbon source. With insoluble cellulose, maximum enzyme production and activities were obtained using an alkali-treated cellulose powder. On this substrate the excretion of soluble protein into the culture broth increased and the protein concentration corresponded to cellulolytic activities.

Cellulolytic and physiological properties of Clostridium thermocellum.

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, α-cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 μmoles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 μmole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.