Enhancing Dark Fermentative Hydrogen Production from Corn Stover Using an Ethylene Glycol–NaOH Mixture Pretreatment

This study evaluated an ethylene glycol–sodium hydroxide (EG–NaOH, 8:1 molar ratio) mixture pretreatment for enhancing dark fermentative biohydrogen production from corn stover. Two separate single-factor screening series examined pretreatment temperature (30–110 °C at 3 h) and process time (1–9 h at 70 °C). In the temperature series, pretreatment at 70 °C for 3 h produced a reducing sugar concentration of 29.58 g L−1 after enzymatic hydrolysis and gave the highest reported measured hydrogen yield of 109.82 mL g−1 VS (549.09 mL), approximately 9.12 times that of the untreated control. In the independent pretreatment-time series, hydrogen yields ranged from 75.46 to 84.95 mL g−1 VS across 1–9 h. The 1 h treatment is therefore considered a candidate that prioritizes shorter process time rather than a validated optimum. For the 70 °C treatment in the temperature series, the modified Gompertz fit gave a fitted Pmax of 110.16 mL g−1 VS, a fitted Rmax of 3.30 mL g−1 VS h−1, and a fitted lag parameter λ of 4.63 h; these parameters are interpreted descriptively. Metabolite and oxidation–reduction potential (ORP) profiles changed during fermentation; however, the observed hydrogen production did not support a universal ORP threshold as a necessary or sufficient condition for hydrogen formation. Endpoint 16S rRNA gene sequencing showed a greater relative representation of several Clostridium groups in the treated profile, while PICRUSt2 predicted a higher relative representation of the hydrogenase-related COG4624 category (0.170% versus 0.0482% in the untreated profile). These results are descriptive and do not establish direct ecological filtering, differential hydrogenase expression, or increased enzyme activity. Overall, the EG–NaOH mixture pretreatment increased reducing sugar release and hydrogen production from corn stover, while process optimization, energy performance, and the proposed causal mechanisms require further validation.