BackgroundGlycerol, a by-product of the biodiesel industry, has garnered attention as a substrate for sustainable bioprocessing. This study investigates the metabolic adaptability of a newly isolated strain, Klebsiella variicola JYP01, focusing on glycerol utilization under both aerobic and anaerobic conditions, as well as the potential of this strain as a proficient producer of 1,3-propanediol (PDO) in sustainable bioprocessing. The research explores the potential of K. variicola JYP01 as the efficient PDO producers with the emphasis on the integration of zero-valent iron (ZVI) to enhance energy production and PDO yield. MethodsIsolation and characterization of the JYP01 strain, assessing its growth and PDO production under both aerobic and anaerobic conditions, and incorporating ZVI (as an electron donor) to identify the metabolic fluxes. Several analyses, including 16S rRNA analysis, high-performance liquid chromatography, and bio-electrochemical system reactors, were employed to elucidate metabolic strategies and the bioconversion efficiency of glycerol to PDO of JYP01 particularly with ZVI. Significant findingsThe integration of ZVI into the bioconversion process significantly enhanced PDO production, leading to 171 % increase from 44.0 mM to 75.2 mM. This demonstrates the distinct ability of the K. variicola JYP01 strain to utilize ZVI as an extra reducing energy in an anaerobic environment. The findings underscore this strain's metabolic capacity, its suitability for glycerol conversion on a large scale, and the novel strategy of using ZVI to elevate PDO production, advancing sustainable bioprocessing techniques.