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 °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 upregulation 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 increments, 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.

暗发酵已成为可持续制氢的一种有前途的策略。然而，厌氧微生物产生的低氢气产量仍然是一个挑战。尽管已经提出金属离子添加剂来提高氢气产量，但人们对它们的整体影响和潜在机制知之甚少。本研究旨在研究 Fe ²⁺、Ni ²⁺、Mg ²⁺和 Mo ⁶⁺对暗发酵产氢的影响，以及它们对批处理系统中微生物群落分析和基因表达的影响。实验在 37°C 的厌氧条件下进行，使用 100 mM 葡萄糖作为底物，初始 pH 值为 6.8。结果表明，Mo ⁶⁺在 0.015 mg-Mo ⁶⁺ /L 时，通过上调固氮酶基因表达（2.19 倍增量）显着提高氢产量（2.21 倍增量）。50 mg- Fe ²⁺ /L 的 Fe 2+ 是第二个最有效的金属离子，氢产量增加 1.81 倍，铁氧还蛋白（增加 1.83 倍）和丙酮酸甲酸裂解酶基因（增加 3.36 倍）^增加） 表达。Mg ²⁺为 70 mg-Mg ²⁺ /L 和 Ni ²⁺为 25 mg-Ni ²⁺/L 还分别使氢产量增加了 1.51 倍和 1.31 倍，并上调了甘油醛-3-磷酸脱氢酶（分别增加了 1.02 倍和 1.81 倍）和 ATP 合酶（分别增加了 1.46 倍和 1.10 倍） ，分别）两种金属添加剂、丙酮酸甲酸裂解酶（2.77 倍增量）和丙酮酸激酶（1.44 倍增量）Mg ²⁺基因表达的表达。关于混合金属方法，观察到虽然混合金属的某些组合，例如 Mg ²⁺ +Mo ⁶⁺, 导致氢产率提高 1.37 倍，发现增量低于单个金属。因此，这项研究的结果表明，使用单一金属会更有效。经济潜力计算进一步表明，Mo ⁶⁺表现出最高的经济可行性，成本最低，为2.02x10 ^-5 $-metal/m ³ -H ₂，占平均总生产成本的0.002%。其次是 Mg ²⁺、Fe ²⁺和 Ni ²⁺. 总体而言，我们的研究结果为使用金属离子添加剂进行清洁制氢提供了机理见解，并为优化其在大规模制氢中的经济可行性奠定了基础。