Komagataeibacter spp. has been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently, studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose-producing bacterium capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from the biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in a minimal medium containing glucose (3.3 ± 0.3 g/L), pure glycerol (2.2 ± 0.1 g/L) and crude glycerol (2.1 ± 0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from the central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in a minimal medium supplemented with acetate, BC production was not observed. However, in the presence of readily utilizable substrates, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.