<P><B>Abstract</B></P> <P>Bacterial cellulose (BC) has received considerable attention as an environment-friendly, biodegradable nanomaterial. In this study, the strain <I>Komagataeibacter</I> sp. nov. CGMCC 17276, which showed rapid cell growth and high BC-production ability, was isolated and classified into a novel species in the <I>Komagataeibacter</I> genus. Four BC synthase operons were annotated using whole-genome analysis, partially explaining the high BC yield of strain CGMCC 17276. Operons <I>bcs Ⅱ</I> and <I>bcs Ⅲ</I> showed high transcriptional levels under static and agitated culture conditions, indicating their importance in BC synthesis. Of the eight suitable carbon sources identified by whole-genome analysis, the highest BC production was achieved using glycerol as a single carbon source. Finally, waste glycerol was successfully used as an eco-friendly and sustainable strategy for BC production. This study provides valuable insights into the mechanism of BC synthesis, genetic structure of BC-producing strains, and industrialization of BC production using an eco-friendly and low-cost strategy.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Strain CGMCC 17276 is a high-yield BC-producing novel species of <I>Komagataeibacter</I>. </LI> <LI> bcs II and bcs III operon showed high transcriptional expression under static and agitated conditions. </LI> <LI> The highest yield of BC production reached using glycerol as single carbon source. </LI> <LI> Waste glycerol was successfully used as a sustainable strategy for BC production. </LI> </UL> </P>