Sustainable co-production of 1,3-PDO, ethanol and H2 from glycerol via dark fermentation by Citrobacter telavivensis T1.2D-1 isolated from the deep subsurface

The increasing production of biodiesel has led to a surplus of glycerol, a polluting by-product in need of valorization. In this study, we demonstrate that Citrobacter telavivensis T1.2D-1, an extremophile bacterium isolated from the Iberian Pyrite Belt, effectively converts glycerol into valuable compounds via dark anaerobic fermentation. Genomic and bioinformatic analyses confirmed the presence of the dha and pdu operons, responsible for 1,3-propanediol (1,3-PDO) synthesis, and the hyc operon and fdhF gene involved in hydrogen (H 2 ) production. Batch fermentations revealed that optimal yields of both H 2 (0.94 mol . mol-glycerol -1 ) and 1,3-PDO (0.66 mol‧mol-glycerol -1 ) were achieved at 25 °C using 2 g L -1 of supplied glycerol. Optimum yield of ethanol (1 mol‧mol-glycerol -1 ) was achieved using 12.5 g L -1 of supplied glycerol. Interestingly, 1,3-PDO and H 2 production inversely correlated with ethanol formation, suggesting metabolic competition. Antibiotic sensitivity profiling revealed susceptibility to multiple antibiotics, supporting future genetic engineering efforts. We suggest opperating with reactors at low concentrations to produce 1,3-PDO and H 2 with high yields, and at medium concentrations to generate ethanol. Our findings support C. telavivensis T1.2D-1 as a promising venue for the sustainable biotechnological production of biohydrogen and bio-based 1,3-PDO from glycerol, offering a dual solution to both energy demands and industrial waste management.