Abstract: Glycerol, generated in large amounts during the production of biodiesel, present an excellent opportunity to convert it into bioethanol. A native, nonpathogenic bacterial strain such as Escherichia coli (E. coli), able to ferment glycerol to useful products under anaerobic condition is very desirable. The objective was to develop mutant strains of E. coli (ATCC 11505) that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant bioethanol yield, was assessed. A glycerol-utilizing mutant E. coli strain for production of bioethanol using low power microwave have been developed (frequency 2.45 GHz and irradiation time 5 min pulse irradiation). In the present work, the mutant E. coli were permeabilized and then cells were immobilization on glutaraldehyde cross-linked chitosan for bioethanol production from glycerol. The fermentation runs was carried out with glycerol as substrate in 2.0 L as worked volume containing 100 g of immobilized cells coated with chitosan cross linking with glutaraldehyde. The Scanning Electron Microscopy (SEM) images confirmed that the bacteria cells were entrapped in the porous network of cross-linked chitosan beads. The results showed that the chitosan beads-cross-linked with glutaraldehyde immobilized with permeabilized mutant E. coli showed great potential for use during the bioethanol production, which boosted up the production 2.9-folds comparing with the reactor with free cells. Particularly, the research confirmed the effectiveness of cells immobilization in chitosan beads reflected by the high yield of bioethanol (96.7 g L-1 compared of those using free cells) and the ability to utilize glycerol in high glycerol containing medium.