Research PapersThe influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30
Introduction
The ability of filamentous fungi to secrete large amounts of proteins has motivated their extensive use for the production of industrial enzymes. However, the morphology, mechanisms of cell growth, and product formation are not well known. Fungal fermentation is widely recognized as a complex process with several problems, some of which are related to the diversity of fungal morphologies under cultivation [1].
In submerged cultures, the morphology of filamentous microorganisms usually varies between the pelleted and the dispersed forms, depending on the respective culture conditions and the genotype of the strain of organism used [2]. The dispersed form consists of branched and unbranched hyphae (freely dispersed) and aggregates (clumps); pellets are highly entangled, dense masses of hyphae which can range in size from several hundred micrometers to several millimeters. The general structure of fungal pellets is a core of such densely packed hyphae, surrounded to a greater or lesser extent by a more annular dispersed or “hairy” region, containing radially growing hyphae [3]. Free filamentous mycelia lead to a highly viscous and pseudoplastic fermentation broth that reduces the gas-liquid mass transfer and the homogeneity of the medium content in fermenters. Pellets have probably also internal mass transfer limitations. The fungal morphology determines the medium rheology and cell growth as well.
For most fungal fermentations, productivity is dependent on an optimal morphology. To achieve and control the optimal morphology, the relation between operational conditions and fungal morphology must be known [4].
Cellulases are one of the most extensively investigated multicomponent enzyme systems because of their ability to decompose the cellulosic biomass into glucose, which in turn can be converted to other valuable chemicals and energy [5].
Complete cellulase enzyme systems can be produced by a large diversity of microorganisms. Among the best characterized and most widely studied of these systems are the inducible cellulases of the filamentous fungus Trichoderma reesei [6]. Cellulases produced by this filamentous fungus have a high industrial interest. They are widely used in the food and feed industries and recently also in the textile and pulp and paper industries [7]. Its cellulase system consists of three general classes of enzymes: cellobiohydrolases (EC 3.2.1.91), endo-1,4-β-D-glucanases (EC 3.2.1.4) and 1,4-β-D-glucosidase (EC 3.2.1.21).
However, there is a limited knowledgement on the effect of the culture conditions on the morphology of T. reesei and the possible relations between morphology, growth and enzyme production [8].
In this work, the morphology of the T. reesei Rut C-30 during submerged cultivations is examined. The relation between the microscopic morphology and the cellulase production is also studied. The T. reesei Rut C-30 is a mutant strain which produces cellulolytic enzymes more efficiently than a wild-type T. reesei, and cellulase expression is not repressed by glucose to the same extent as in some other strains [9].
Section snippets
Strain, media, and growth conditions
T. reesei Rut C-30 strain was a gift from Dr. M. Pentillä (VTT, Finland). Fungal spores from a stock, kept at −80°C in 20% (v/v) glycerol, were used to inoculate potato dextrose agar (Difco) slants. Five days after inoculation, the spores were harvested and washed with sterilized water and the spore concentration was determined in a counting chamber after appropriate dilution.
A preculture in a 250 ml Erlenmeyer flask containing 50 ml of medium was prepared. This medium was inoculated with 50 μl
Morphology evaluation
Five different media were used (Table 1), referred as medium 1 to 5. Medium 1 and 2 have the same composition as previously defined in Section 2, only medium 2 has half of the metal ions concentration. Medium 3 has the same composition as medium 1, but it was supplemented with Tween 80. Medium 4 has the same composition as medium 3, but it was supplemented with yeast extract, and peptone. Medium 5 has the same composition of medium 4, however Solka floc cellulose was used as inducer instead of
Conclusions
The influence of inoculum size and media composition on the T. reesei Rut C-30 morphology and enzyme activity were studied. The inoculum size and media composition seems to influence morphogenesis (development of morphology) and product (cellulase) formation.
At low inoculum size pellet growth is dominant and the majority of the pellets were large. With the increase in inoculum, concentration pulp growth became prevalent. Composition of fermentation medium was very important on the morphology.
Acknowledgements
We thank Dr M. Pentillä from VTT, Finland for their kind gift of T. reesei Rut C-30 strain.
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