Long-term continuous hydrogen production from cheese whey: Operational transitions, reactor restart without external inoculation, and microbial community profiles at selected operational states.

Cheese whey is an attractive substrate for dark fermentative hydrogen production, but stable continuous operation remains limited by operational instability and microbial competition. This study investigated long-term hydrogen production from cheese whey in a continuous stirred-tank reactor, under sequentially modified conditions involving hydraulic retention time (HRT), organic loading rate (OLR), sludge morphology, and inoculation strategy. With thermally pretreated anaerobic sludge as external inoculum, the combined HRT/OLR condition at 9 h provided the most favorable balance between hydrogen yield and production rate, with a hydrogen molar yield (HMY) of 1.03 mol H2/mol lactose added and a specific hydrogen production rate (SHPR) of 0.85 L H2/g volatile suspended solids (VSS)/d. Stepwise OLR reduction before restart lowered ethanol accumulation but did not consistently improve hydrogen production, while sludge granulation was mainly associated with biomass retention. The highest performance was achieved after complete reactor restart at an OLR of 26.7 g chemical oxygen demand/L/d without external inoculum, relying solely on the endogenous microbiota of the whey used in this study, with HMY of 2.69-2.99 mol H2/mol lactose added, SHPR of 3.45-5.67 L H2/g VSS/d, and hydrogen content of 47-50 %. Similarly high hydrogen-production performance was observed after a second complete restart. Because several factors changed simultaneously, their individual contributions cannot be distinguished. These results show that high continuous hydrogen production can be established without external inoculation under the tested restart conditions.