Optimized biohydrogen production from sewage sludge: Advanced pretreatment strategies in dark fermentation and microbial electrolysis cells

• The implementation of pre-treatments enhanced protein and carbohydrate accumulation by 90–95%. • CCPH-Pre-treatment yielded 1,962 mg/L TVFA post-dark fermentation (11.4-fold× untreated sludge) Acetic/propionic acids comprised 80% of TVFAs as dominant electron-donor metabolites. • CCPH-Pre-treatment achieved 172.5 A/m³ current density with 133.071 ml/g VS bio-H₂ in MECs. • Firmicutes dominated the genus in all pre-treated substrates (60–88% vs. 10.5% untreated). • CCPH-Pre-treatment integrates into the DF and MEC for efficient bio-H₂ production, optimizing substrate solubilization and microbial activity in sewage sludge energy recovery. The production of biohydrogen from sewage sludge presents a promising avenue for sustainable energy conversion and pollution control. This study systematically evaluates biohydrogen (bio-H₂) production through dark fermentation (DF) and single-chamber microbial electrolysis cells (MECs), employing diverse substrate pretreatment methods, including acidic, ultrasonic, heat, alkaline, and a novel combined CalciumCarbonate-2Potassiumhydroxide (CCPH) pretreatment. The findings reveal that these pretreatments significantly enhance the accumulation of proteins, carbohydrates, and volatile fatty acids (VFAs), with the DF phase further augmenting the solubilization of these critical components. Notably, 90–95% of carbohydrate and protein accumulation occurs during the pretreatment phase, which concurrently suppresses methanogenesis in sewage sludge, thereby optimizing conditions for bio-H₂ production in subsequent MEC processes. Among the pretreatments, CCPH-P demonstrates exceptional performance, achieving a total volatile fatty acid (TVFAs) concentration of 1,962 ± 124 mg/L post-dark fermentation, representing an 11.4-fold increase compared to untreated sludge. In MEC experiments, CCPH-P sludge attains a maximum current density of 172.5 A/m³ and an average bio-H₂ yield of 133.071 ml/g VS, underscoring its efficiency in electrochemical hydrogen recovery. Acetic and propionic acids, derived from sludge fermentation, emerge as the predominant electron donors, constituting approximately 80% of the total VFAs content across all pretreated substrates. Metagenomic analysis further reveals that Firmicutes exhibit the highest relative abundance, ranging from 60% to 88% in pretreated substrates, compared to merely 10.5% in untreated sewage sludge. These results collectively indicate that CCPH-pre-treatment serves as a viable and efficient method for enhancing biohydrogen recovery from sewage sludge through DF and MEC systems, thereby advancing the potential for waste-to-energy applications.