# Cellulases Production by a _Trichoderma_ sp. Using Food Manufacturing Wastes
## 3 Results and Discussion  
A fungal strain was isolated from olive stones kept at room temperature. After successive passages onto PDA plates, a homogenous fluffy-type mycelium of white color was observed which turned green after 5 days of cultivation (Figure 1a). Examination of samples of the lactophenol-cotton-blue-stained mycelium under optical microscopy showed the presence of segmented hyphae, tree-like reproductive structure, and conidia showing round to ovoidal form. Similar features are found in _Trichoderma_ species (Figure 1b,c) [33]. PCR amplification using ITS-1 and ITS-4 primers and the mycelium DNA produced a fragment of around 570 bp (Figure 2a). This fragment was cloned in pGEM-T vector and used for transformation of _Escherichia coli_ competent cells. After selection of colonies containing the recombinant plasmid, positive colonies were inoculated in LB medium for plasmid purification. Plasmids containing the PCR product were further sequenced using the T7 and SP6 vector primers. The amplified ITS1-5.88-ITS2 sequence from strain FG displayed 100% homology to the same region of several strains of _Trichoderma asperellum_ associated with plants worldwide. Also, this region is 99% identical to that of _T. asperellum_ UC 1 (Genbank ID: MF774876) isolated in Malaysia, which is reported to be a high cellulase and xylanase producer [34]. A phylogenetic tree was constructed using these data and related sequences extracted from the NCBI database, as shown in Figure 2b. This figure shows that strain FG sequence was located in a clade together with other _Trichoderma_ species. These data suggest that strain FG belongs to _Trichoderma_ sppTo evaluate the behavior of the _Trichoderma_ sp. on FMWs, the fungus was grown on both solid and liquid Mandel medium supplemented with 0.5% FMWs. For comparison purposes, glucose was added to a fourth set of liquid cultures and plates. On these plates, _Trichoderma_ sp. growth was observed on the three FMWs, however the highest radius value at 72 h was observed in OMSW and GP plates (4.3 cm) followed by glucose (4.1 cm) and TP (4.0 cm) (Figure 3) plates. The samples were incubated in 100 mL liquid cultures at 30 \({}^{\circ}\)C, and the total biomass (wet weight) obtained after 5 d of culture was 17.4 g for glucose, 11.0 g for OMSW, 9.8 g for G,P and 8.4 g for TP. In addition, the final protein concentrations in these samples were 0.6, 0.7, 0.3, and 0.4 mg/mL, respectively. The visual inspection of the FMW cultures on the fifth day revealed incomplete degradation; however, OMSW seemed to be the most preferable carbon source among the three FMWs. Figure 1: **Morphology of strain FG. ** (**a**) Mycelium from strain FG grown on a potato dextrose agar (PDA) plate for 5 d; (**b**) A sample taken from a mycelium grown on a PDA plate and stained with lactophenol cotton blue. This sample was observed under an optical microscope using 40x magnification. Bar indicates 20 μm. Tree-like structures containing conidia can be observed (black arrow); (**c**) Microscopic observation of spores by an optical microscope using 100x magnification. Bar indicates 5 μm. Figure 2: **Molecular analysis of strain FG. ** (**a**) Gel electrophoresis of strain FG DNA. Lane 1: Molecular marker 100 bp; lane 2: genomic DNA from strain FG extracted with the hexadecyltrimethylammonium bromide (CTAB) method; lane 3: PCR product using ITS primers and genomic DNA as a template. Arrow indicates the PCR product of the expected size (\(\sim\)570 bp); (**b**) Neighbor-joining analysis with the Kimura-2-parameter model after aligning ITS1-5-8-ITS2 sequences with Clustal X in MEGA7. Bootstrap analyses were performed for 10,000 replicates. The ITS1-5-8-ITS2 sequence from _Fusarium solani_ was used as outgroup. Asterisk indicates fungal strains characterized as high cellulases producers [18; 34; 35; 36; 37; 38]. Fungal growth was evidenced when the fungal strain was inoculated on plates containing LB medium with 1% carboxymethyl cellulose stained with Congo red solution. Initially, the plates were fully stained red.