Metal additives for boosting hydrogen production in anaerobic fermentation: Focus on the change of gene expression and cost analysis

Dark fermentation has emerged as a promising strategy for sustainable hydrogen production. However, the low hydrogen yield produced by anaerobic microbes continues to be a challenge. Although metal ion additives have been proposed to enhance hydrogen production, their entire impacts and underlying mechanisms are poorly understood. The present study aimed to examine the effects of Fe2+, Ni2+, Mg2+, and Mo6+ on dark fermentative hydrogen production, as well as their influences on microbial community analysis and gene expression in a batch system. The experiments were conducted under anaerobic conditions at 37 degrees C, utilizing 100 mM glucose as a substrate and an initial pH of 6.8. The results showed that Mo6+ at 0.015 mg-Mo6+/L significantly enhanced the hydrogen yield (2.21-fold increment) by upregulating nitrogenase gene expression (2.19-fold increment). Fe2+ at 50 mg-Fe2+/L was the second most effective metal ion, with a 1.81-fold increase in hydrogen yield and upre-gulation of ferredoxin (1.83-fold increment) and pyruvate formate-lyase gene (3.36-fold increment) expression. Mg2+ at 70 mg-Mg2+/L and Ni2+ at 25 mg-Ni2+/L also increased hydrogen yield by 1.51-fold and 1.31-fold, respectively, with upregulation of glyceraldehyde-3-phosphate dehydrogenase (1.02-fold and 1.81-fold in-crements, respectively) and ATP synthase (1.46-fold and 1.10-fold increments, respectively) expressions for both metal additives, pyruvate formate-lyase (2.77-fold increment) and pyruvate kinase (1.44-fold increment) gene expression for Mg2+. Regarding the mixed metal approach, it was observed that although certain combinations of mixed metals, such as Mg2++Mo6+, resulted in an enhancement of hydrogen yield by a factor of 1.37-fold, the increment was found to be lower than that of an individual metal. Therefore, the results of this study suggest that using a single metal would be more effective. Economic potential calculations further revealed that Mo6+ exhibited the highest economic feasibility, with the lowest expense of 2.02x10-5 $-metal/m3-H2, accounting for 0.002% of the average total production cost. This was followed by Mg2+, Fe2+, and Ni2+. Overall, our findings provide mechanistic insights into the use of metal ion additives for clean hydrogen production and offer a foundation for optimising their economic feasibility in large-scale hydrogen production.