Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2019 / In an effort to align the current research with the country’s biofuel strategy, the aim of the study was to utilize banana pseudostem in the production of fungal cellulolytic enzymes and bioethanol through fermentation of the banana pseudostem hydrolysate. The selection of microorganisms was based on the ability of the fungi to grow on agar containing Avicel (microcrystalline cellulose) followed by assaying for cellulases in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from screening process showed positive endoglucanase activity on carboxymethyl cellulose – Congo Red agar plate. The six fungal isolates selected based on high cellulase activity belonged to Trichoderma and Aspergillus genera. In submerged fermentation (SmF), the maximum cellulase and endoglucanase production under optimal conditions by all fungal isolates was achieved in media with an initial of pH 6.5 at 30 °C. Under these conditions, the total cellulase activity was 9.79 filter paper units (FPU)/mL and endoglucanase activity 45.2 U/mL for Trichoderma longibrachiatum LMLUL 14-1 and total cellulase activity of 7.7 FPU/mL and endoglucanase activity of 32.7 U/mL for Trichoderma harzianum LMLUL 13-5. These cellulase activities were higher than in the crude enzymes system for all Aspergillus fumigatus. The production conditions for maximum β-glucosidase varied amongst the Aspergillus spp. For example, Aspergillus fumigatus LMLUL 13-4 had produced higher β-glucosidase activity in a medium with an initial pH of 6.5 and at an incubation temperature of 30 °C whereas A. fumigatus LMLUL 13-1 had produced higher β-glucosidase activity at an initial pH of 7.0 and at 35 °C. Solid state fermentation (SSF) to produce cellulase enzymes system was influenced by temperature, nature of the substrate (i.e. moisture, modification) and culturing technique/strategy (i.e. monoculture versus co-culture). Higher cellulase enzymes system was produced under the conditions of 30 °C, 75% moisture content of untreated (native) BPS and pH 6.5. All the fungi investigated, produced thermotolerant and acidophilic cellulase and endoglucanase, whilst β-glucosidase is both acidophilic and alkaliphilic. The cellulase enzymes complex of T. harzianum LMLBP07 13-5 is most stable, followed by A. fumigatus LMLPS 13-4 and the least stable cellulase enzymes complex was for T. longibrachiatum LMLULSA 14-1. For the pretreatment of BPS, the material was first subjected to three different pretreatment conditions; namely alkaline (3% NaOH), acid (5% H2SO4) and hot water (autoclave method) pretreatment to remove lignin and loosen the cellulose structure. After the pretreatments, alkaline method exposed more cellulose than other pretreatments methods. The alkaline pretreated BPS contained 52.3% cellulose, 10.8% hemicellulose and 8.7% lignin, which is 2.3-fold more cellulose and 0.48-fold less hemicellulose as well as 0.6-fold less lignin to the native BPS. The enzymatic saccharification of the alkaline pretreated BPS at different substrate loadings at 50 °C for 76 hours by an individual crude cellulase enzymes system from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 cultures were used at a final concentration of 10 FPU/g. Saccharification released maximum glucose of 43.5 g/L and 20.1 g/L form alkaline pretreated BPS by crude cellulase enzymes from T. longibrachiatum LMLSAUL 14-1 and T. harzianum LMLBP07 13-5 measured at the highest solid loading. The production of bioethanol was carried out in separate hydrolysis and fermentation (SHF). Fermentation of nutrient supplemented BPS hydrolysate with an initial pH of 5.0 by S. cerevisiae UL01 occurred at 30 °C for 48 hours. The maximum ethanol concentration obtained after fermentation was 17.6 g/L corresponding to ethanol yield of 60% of the maximum theoretical yield. In conclusion, banana pseudostem is a suitable alternative substrate for the production of second-generation bioethanol. / National Research Foundation (NRF) and Vlaamse Interuniversitaire Raad (VLIR- UOS)