Low-temperature lignosulfonate-based biochar promotes electron transfer and boosts dark fermentative hydrogen production by Klebsiella

Traditional biochar production via high-temperature thermochemical processes is energy-intensive and often destroys redox-active functional groups critical for microbe–material interactions. Here, a lignin-derived biochar (LSB) was prepared via low-temperature pyrolysis (250 °C) to preserve its redox functionality. The addition of LSB enhanced biohydrogen production by 16.2% and improved xylose utilization from 68.62% to 76.95%. Mechanistic investigations revealed that LSB stimulated the secretion of humic-like substances in extracellular polymeric substances (EPS) and functioned as an effective electron shuttle, thereby promoting extracellular electron transfer (EET). Correspondingly, intracellular NADH concentration and the NADH/NAD + ratio increased by 52.99% and 44.97%, respectively. Electron balance analysis further demonstrated a metabolic shift toward acetate and hydrogen production, with their electron equivalent fractions increasing to 9.7% and 9.5%, respectively. These results highlight a low-temperature lignin valorization strategy for regulating microbial electron flux, offering a sustainable approach to enhance biohydrogen production.