The micromorphology of Trichoderma reesei analyzed in cultivations on lactose and solid lignocellulosic substrate, and its relationship with cellulase production

Vera Novy, Maximilian Schmid, Manuel Eibinger, Zdenek Petrasek, Bernd Nidetzky

Trichoderma reesei is the principal producer of cellulolytic enzymes. Because of the strong influence on the enzyme production, the morphology of the filamentous fungi is a key parameter for process optimization. For cost-effective production of cellulolytic enzymes, the cultivation of T. reesei is performed on lignocellulosic waste streams. These insoluble substrates prevent the application of the conventional light microscopy for the analysis of fungal morphology. Here, we present a novel method for the micromorphological analysis based on confocal laser-scanning microscopy (CLSM) and the computer-aided image analysis. This method enabled the quantification of the dimensions of the single cell (intercalary length and cell width) and the degree of branching in cultivations on the industrially relevant substrates wheat straw and lactose. The micromorphology of two T. reesei strains, QM9414 and a carbon catabolite derepressed cre1 knockout mutant (Δcre1), was analyzed in dependence of substrate, inoculation method, and agitation velocity. Trichoderma reesei strain Δcre1 formed shorter cells (10.09 µm) on average and developed more ramified mycelia (0.36 branches/cell) than strain QM9414 (12.03 µm, 0.22 branches/cell). Cultivated on wheat straw, the average cell length of QM9414 (10.87 µm) and Δcre1 (9.74 µm) was 10 and 21 % shorter as compared to reference cultivations on lactose. When inoculation was done with spores as compared to hyphal biomass, cell lengths of QM9414 (10.97 µm) and Δcre1 (9.10 µm) were on average about 20 % shorter. Strain performance was evaluated in protein concentration and total cellulase activity, which varied between 0.69 and 2.31 FPU/mL for Δcre1 and between 0.84 and 1.64 FPU/mL for QM9414. The cell length exhibited slightly negative correlation with the protein (regression coefficient -0.04 g/(L µm), R (2) 0.33) and the cellulase (-0.30 FPU/(mL µm), R (2) 0.53) production. The dimensions of the single cell of T. reesei were dependent on strain background, substrate used and process conditions applied. Micromorphological changes were correlated semi-quantitatively with the efficiency of enzyme production. In providing a process analytical tool for enzyme production by T. reesei on lignocellulosic substrate, this study has relevance for the characterization and optimization of a critical step in the overall saccharification process.