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The microbial mediated production of bio-ethanol from the rhizome of Imperata cylindrica (spear grass) was carried out in this research work. Proximate analysis of the rhizome was done to determine the protein, carbohydrate, moisture, ash, lipid, and fibre contents. Sugar profile analysis was carried out using Gas Chromatography (GC). The sugars obtained from the rhizome include ribose, xylose, arabinose, rhamnose, fructose, glucose, maltose, lactose, and sucrose. Ethanol yield increased simultaneously from day 5 to day 20 for all reactors in six different experimental design i.e, Saccharomyces cerevisiae alone; Serratia marcescens alone, Aspergillus flavus + Saccharomyces cerevisiae; Apergillus flavus + Saccharomyces cerevisiae + Serratia marcescens; Aspergillus flavus + Serratia marcescens including the control (containing no microorganism). The highest ethanol yield was achieved at pH 7 and temperature range between 280C to 380C. The reducing sugar decreased from 0.37 to 0.03 g/mol as the period of incubation increased while cell density increased from 0.1 to 0.68 nm as the period of incubation increased. Reactor containing co-cultures of Saccharomyces cerevisiae, Serratia marcescens and Aspergillus flavus in combination produced the highest ethanol yield at day 20 while the reactor containing only Serratia sp produced the lowest ethanol yield. This study has demonstrated that the use of Saccharomyces cerevisiae, Serratia marcescens and Aspergillus flavus co-cultures is more effective in bio-ethanol production using I. cylindrica (spear grass) compared to individual cultures.