Pillai, Santhosh Kumar KuttanBux, FaizalKiambi, Sammy LewisMthethwa, Nonsikelelo Precios2019-05-292019-05-292019709135http://hdl.handle.net/10321/3217Submitted in fulfillment of the requirements of the degree of Masters in Applied Sciences: Biotechnology, Durban University of Technology, Durban, South Africa, 2019.Aquatic weeds are well known for their fast growth rate and high carbohydrate content that can be easily hydrolysed into fermentable sugars. This study was aimed at the utilization of an indigenous aquatic weed, Pistia stratiotes for biohydrogen production through the dark fermentation process. Characterization of the biomass, effect of pre–treatment methods on biomass hydrolysis, effect of reactor operational conditions and type of inoculum on enhancing hydrogen production potential of P. stratiotes was assessed. Physical and chemical pre–treatments were employed on P. stratiotes biomass to increase digestibility and to achieve high conversion rates of fermentable sugars. The highest sugar yield of 139± 0.8 mg/g was obtained when the oven dried biomass was subjected to H2SO4 (2.5%) pre– treatment followed by autoclaving at 121°C for 30 min. Biohydrogen production under different operational conditions was thereafter optimized using One–factor–at–a–time (OFAT) batch experiments in 120 mL serum bottles. A maximum hydrogen yield (HY) of 2.46 ± 0.14 mol-H2/mol-glucose (3.51 ± 0.20 mg-H2/g-dry weight) and 2.75 ± 0.07 mL h-1 hydrogen production rate was observed under optimized conditions (pH 5.5, Temp 35°C, S/X: 1.0 g-COD/g-VSS and HRT 8 h). The organic mass balance (92 – 96%) and electron– equivalent balance (92 – 98%) further indicated the reliability of the obtained fermentation data. Assessment of microbial activity was achieved using molecular techniques such as quantitative polymerase chain reaction (qPCR) targeting both 16s rRNA (of Clostridium spp., Bacillus spp., and Enterobacter spp.) and the functional hydrogenase gene (hydA). The highest gene activity of hydrogenase was noted at pH of 5.5 with 2.53×104 copies/ng-DNA compared to low pH: 4.5 (6.95 × 103 copies/ng-DNA) and high pH: 8.5 (7.77×103 copies/ng- DNA). A similar trend was also observed for the species containing a highly active hydrogenase (i.e. Clostridium spp., Bacillus spp., and Enterobacter spp.). During the optimum reactor conditions, three hydrogen producing bacterial strains Bacillus cereus and Enterobacter cloacae were successfully isolated. These isolates were used as inoculums for the pure culture studies and achieved HYs of 2.2, 1.10 and 1.97 mol-H2/mol-glucose respectively under optimized fermentation conditions. However, the thermally treated mixed culture displayed a marginally higher HY (2.46 mol-H2/mol-glucose) compared to the pure culture used alone. Furthermore, the cost estimation indicated a potential and economically feasible for biotransformation of P. stratiotes to hydrogen energy. In conclusion, the results from this study has revealed the potential of employing P. stratiotes biomass for biohydrogen production. The results also indicated the importance of employing suitable pre–treatment methods, operating conditions as well as inoculum types for enhanced hydrogen production using P. stratiotes.184 penHydrogen--BiotechnologyWater lettuceFermentationBiomass energyHydrogen as fuelThesishttps://doi.org/10.51415/10321/3217