Fe2O3-Based Microbial Hybrids for Enhancing Dark Fermentation Hydrogen Production: Performance and Mechanistic Insights
Dark fermentation offers a sustainable route for hydrogen production, yet its yield is often limited by inefficient electron transfer and low microbial metabolic activity. This study engineered a mixed microbial biohybrid system incorporating Fe2O3 nanoparticles to overcome these bottlenecks. At an optimal Fe2O3 concentration of 300 mg/L (S300), the hydrogen yield reached 2.94 mol H2 per mol glucose, equivalent to 73.5% of the theoretical maximum and 1.59 times higher than the control (S0). Mechanistic analyses revealed that Fe2O3 nanoparticles stimulated microbial metabolism, as evidenced by a 4.09-fold increase in ATP content and a 1.30-fold rise in total protein concentration. Hydrogenase and dehydrogenase activities were enhanced by 24.62% and 63.11%, respectively, while electron transfer system activity increased by 3.44-fold, accompanied by a significant reduction in charge transfer resistance. Notably, the gradual release of Fe2+ ions from Fe2O3 reduction by dissimilatory iron-reducing bacteria (DIRB) was identified as a key factor in stimulating enzyme activity and electron transfer. Microbial community analysis showed that the relative abundance of Clostridium, a key hydrogen-producing genus, increased by 9.75 percentage points to 42.60% in S300. This study demonstrates that Fe2O3-based biohybrids offer a promising strategy to enhance dark fermentation hydrogen production, providing both performance improvements and mechanistic insights into nanomaterial-microbe synergies.