Fe-0 and Ni-0 nanoparticles (NPs) of certain size were synthesized and added to the hydrogen production system from cotton stalk hydrolysate using Klebsiella sp. WL1316. Fe-0 and Ni-0 NPs with a size of 50 nm at all concentrations effectively improve hydrogen production during mid to late fermentation stages; particularly, the highest daily hydrogen production obtained following treatment with 50 nm Fe-0 NPs at 30 mg/L fermented for 96 h significantly increased by 61% comparing to the control treatment. The reducing sugar consumption in cotton stalk hydrolysate and Delta OD600 could be improved to some extent by Fe-0 and Ni-0 NPs supplementation. Addition of Fe-0 or Ni-0 NPs of 50 nm at a concentration of 30 mg/L resulted in enhanced cumulative hydrogen production with improvement of hydrogen yield reached higher than 20%, and the values of Y(H-2/S) were all higher than 90 mLig substrate, reflecting good hydrogen production and substrate consumption. The analysis of the main soluble metabolites profile revealed that supplementation with Fe-0 and Ni-0 NPs of suitable size and concentration may decrease the metabolic flux in the competitive branch of hydrogen production and increase the metabolic flux of the key node that leads to hydrogen generation, thus promoting biohydrogen synthesis. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.