# Cellulases Production by a _Trichoderma_ sp. Using Food Manufacturing Wastes
## 3 Results and Discussion
Once the cellulose was degraded after 5 d of incubation, the red color disappeared leaving a pale-colored halo, which was indicative of the presence of cellulases released into the culture medium (Figure 4a). This preliminary evidence prompted us to search for cellulases when the fungi were grown in FMWs. Culture supernatants prepared from FMWs liquid cultures after 5 d of fungal growth were evaluated for their total cellulase activity (i.e., endo-\(\beta\)-1,4-D-glucanase, 1,4-\(\beta\)-D-glucan  
Figure 4: **Cellulolytic activities of culture supernatants from the Trichoderma=sp. grown on different food manufacturing wastes (FMWs). ** (**a**) The Trichoderma=sp. was grown on a Petri plate containing carboxymethyl cellulose and stained with Congo red solution. The white halo is indicative of the presence of cellulases released into the culture medium. The arrow indicates the lack of staining due to the activity of cellulases secreted by the fungus (**b**) Filter paper activity (FPase) from culture supernatants (**c**) \(\beta\)-glucosidase activity from culture supernatants. For b and c, the culture supernatants were extracted on the fifth day of growth in Mandel medium supplemented with glucose (black bar), OMSW (gray bar), TP (striped bar), and GP (white bar). Activities are expressed in mg of total protein. For FPase, the activity, expressed in U/mL, was 2.6, 63.2, 33.0, and 112.0, respectively. For \(\beta\)-glucosidase, it was 0.2, 1.7, 1.6, and 1.4 U/mL, respectively. Figure 3: **Radial growth of the _Trichoderma_ sp. on FMWs plates. **_Trichoderma_ sp. was grown on Mandels medium supplemented with glucose (black bar), olive-mill solid waste (OMSW, gray bar), tomato pomace (TP, striped bar), and grape pomace (GP, white bar) at 0.5% as the sole carbon source. Error bars indicate the standard deviations (SDs) based on three different replicated experimental values. Numbers on the top of each bar indicate the average radius corresponding to mycelial growth. cellobiohydrolase, and \(\beta\)-glucosidase) and \(\beta\)-glucosidase activities through filter paper and pNPG assays, respectively. Filter paper specific activity was clearly higher in the FMW culture supernatants in comparison to the glucose supernatant, with the GP culture supernatant having the highest value (Figure 4b). The same trend was observed for \(\beta\)-glucosidase specific activities, although the TP supernatant had the highest value (Figure 4c). To confirm the presence of cellulases, proteins were extracted from the culture supernatants of the _Trichoderma_ sp. grown on glucose and FMWs on the fifth day. Proteins were separated by SDS-PAGE (denaturant electrophoresis) and analyzed by mass spectrometry. This qualitative analysis identified 336, 219, 244, and 210 proteins in the supernatant media from cells grown in glucose, OMSW, GP, and TP, respectively (results not shown). The identity of the proteins in each fraction is found in Table 1, and a detailed description is given in Supplementary Material (ST1-ST8). In total, 24 distinct cellulases were found in 4 four different residues. Endo-\(\beta\)-1,4-D-glucanase,1,4-\(\beta\)-D-glucan cellobiohydrolase and \(\beta\)-glucosidase were found in the culture supernatants of FMWs, whereas the glucose supernatants did not contain any exo- or endo-glucanases. The ability of species belonging to genus _Trichoderma_ to grow in complex substrates has been previously reported. Using a synthetic medium, containing cellulose as the sole carbon source, [PERSON] et al. identified few cellulases in the culture supernatant of _Trichoderma harzianum_[36]. In contrast, after growth of _T. harzianum_ using the same medium supplemented with sugarcane bagasse, [PERSON] et al. found that this complex substrate induced a higher secretion of cellulolytic and xylanolytic activities when compared with other substrates such as glucose [37]. In a comparative study of the extracellular proteins produced by _T. asperellum_ S4F8 and _T. reesei_ Rut C30 after solid-state fermentation on the same substrate, [PERSON] et al.