AimsA low yield of bacterial cellulose (BC) always results from an excessive accumulation of organic acids. Screening and the selection of bacterial mutants with a low accumulation of organic acids is an efficient approach for improving BC production. Methods and ResultsIn combination with the proton suicide method (medium containing NaBr-NaBrO3), diethyl sulphate chemical mutagenesis coupled with Co-60- irradiation treatment were performed for the screening and selection of desired mutant lines with a high yield of BC. Two high-yield strains, Br-3 and Co-5, as well as a low-yield strain, Br-12, were obtained. Amplified fragment length polymorphism (AFLP) was applied to explore the differences between the mutant lines and the wild type. For the Br-12 line, three specific fragments were verified, corresponding to TonB-dependent transport (TBDT), exopolysaccharides output protein (PePr) and an unknown gene. For Co-5, two specific fragments were matched, acsD and UDP-galactose-4-epimerase. In addition, metabolic analysis for the mutant lines indicated that BC production may be limited by excessive accumulation of organic acids in the fermentation. The limitation would be resolved by the cross-talk of genes involved in BC biosynthesis. ConclusionsReduced organic acid by-products from glucose in bypasses were found to be responsible for the high-yield BC synthesis in Komagataeibacter hansenii mutant strains. Significance and Impact of the StudyThe metabolic process was varied by mutagenesis-induced gene disruption of the metabolic products. A new idea was provided for the targeted screening and characterization of mutants in the future.