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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Cellulase production; Lactose; Lignocellulose; Morphology; Trichoderma reesei; Wheat straw.

Fig. 1

CLSM images of wheat straw cultivations by T. reesei strains QM9414 (A1, A2) and Δcre1 (B1, B2). Precultures were inoculated with 10⁵ spores/mL (A1, B1) or with a piece of overgrown agar (A2, B2). All cultivations were incubated at 190 rpm

Fig. 2

CLSM images of lactose cultivations by T. reesei strains QM9414 (A1, A2) and Δcre1 (B1, B2). Precultures were inoculated with 10⁵ spores/mL (A1, B1) or with a piece of overgrown agar (A2, B2). All cultivations were incubated at 190 rpm

Fig. 3

Influence of the inoculation method (A1, A2) and the agitation velocity (B1, B2) on cell length. Cell length was analyzed in wheat straw (A1, B1) and lactose (A2, B2) cultivations. A1, A2 Precultures were inoculated with a piece of overgrown agar (black bars) or 10⁵ spores/mL (grey bars). B1, B2 Agitation velocity was 150 rpm (black bars) or 190 rpm (grey bars) in an orbital incubator shaker. Cultivations were accomplished with T. reesei strain Δcre1 (“cre”) or QM9414 (“QM”). Incubation was at 150 or 190 rpm, as indicated. The addition of Tween80 is marked with “Tw”. Data were taken from Table 1, where bars indicate the mean length and error bars show the spread. Data sets with 1, 2, or 3 stars mark significance in a 95, 99, and 99.9 % confidence level, respectively

Fig. 4

Correlation between cell length and total cellulase activity in wheat straw cultivations (A) and total protein concentration in wheat straw and lactose cultivations (B). A Depicted are cell length (grey bars) and total cellulase activity (empty circles). Cultivations were accomplished with T. reesei strain Δcre1 (“cre”) or QM9414 (“QM”). Inoculation was with a piece of overgrown agar (“agar”) or 10⁵ spores/mL (“spores”). Incubation was at 150 and 190 rpm, as indicated. The addition of Tween80 is marked with “Tw”. B Depicted are cell length and the corresponding protein concentration in the supernatant of all experimental setups. Data are taken from Table 1

Fig. 5

Correlation between cell length and cell width. Data are taken from Table 1 and include all experimental setups