Nickel-Iron Layered Double Hydroxide Enhances Hydrogen Production by Clostridium Fermentation
Dark fermentative biohydrogen production technology faces challenges for large-scale application due to low substrate conversion efficiency, inhibition by metabolic by-products, and limited hydrogen production rates. This study constructed a biohybrid system based on nickel-iron layered double hydroxide (NiFe-LDH) and Clostridium pasteurianum DSM 525 (denoted as NiFe-LDH@C. pasteurianum DSM 525). We systematically investigated the effects of the Ni/Fe molar ratio and NiFe-LDH addition concentration on the hydrogen production performance of the system. The results demonstrated that at a Ni/Fe molar ratio of 1:3 and a NiFe-LDH concentration of 20 mg/L, the cumulative hydrogen production reached 4.20 mmol, representing a 1.68-fold increase compared to the control group. The maximum hydrogen production rate increased to 0.54 mmol/h, which was 2.35 times that of the control. Mechanistic studies revealed that NiFe-LDH, through direct contact with the bacterial cells, significantly downregulated the expression of genes related to flagellar assembly while concurrently upregulating genes involved in biofilm synthesis. This shift prompted the bacteria to redirect ATP/NADH energy towards constructing a biofilm structure capable of buffering external pH fluctuations. Simultaneously, released iron (Fe) upregulated hydrogenase gene expression, accelerating hydrogen production to consume intracellular protons and thereby alleviating acid stress. Enhanced expression of DNA repair genes also safeguarded genomic stability. This contact-dependent regulation significantly improved the acid tolerance of the bacteria and activated glycolysis, short-chain fatty acid metabolism, and lactate utilization pathways. Ultimately, this led to a substantial enhancement in both the hydrogen production rate and total yield. This study provides a theoretical basis for developing efficient microbe-material hybrid systems for hydrogen production and broadens the application horizon of LDH materials in the field of bioenergy.