Decoupling methanogenesis to boost hydrogen production via microalgae–wastewater co-digestion in a continuous stirred reactor

• Co-culturing microalgae and activated sludge boosts H 2 content in biogas. • Separate influent streams in CSTR operation shifts metabolic activity from CH 4 to H 2 . • Metabolic shift produced ∼ 9% of H 2 with remaining content of O 2 , and N 2 in gas output. • CH 4 was fully inhibited and CO 2 dropped from 72% to 1% in biogas content. • Higher COD and TOC contents in digestate confirmed reduced CO 2 conversion. Co-culturing microalgae with wastewater is a promising approach for H 2 production via anaerobic digestion in batch reactors; however, continuous-flow operation requires careful optimization of key parameters to enhance hydrogenogenic activity and suppress methanogenesis. This study investigates the operation of a continuous flow stirred tank reactor (CSTR) under quasi-steady-state for different pH ranges for converting wastewater activated sludge into a continuous stream of H 2 via co-digestion with microalgae ( Chlorella vulgaris ). The experimental setup was divided into two operational phases. During Phase I, influent streams were pre-mixed outside the reactor with controlled pH in different ranges inside the reactor. In Phase II, influent streams were kept separate prior to reactor feeding without a pH control inside the reactor. Various operational parameters such as bioreactor configuration for influents, pH, chemical oxygen demand (COD), total organic carbon (TOC), optical density (OD), and chlorophyll concentrations revealed significant metabolic shift towards H 2 production during CSTR operation. It was observed that during the uncontrolled pH stage of Phase II, metabolic activity shifted completely towards H 2 production up to ∼ 9% (∼72 mL/L/day), minimal CO 2 from ∼ 72% (∼232 mL/L/day) to ∼ 1% (∼3.14 mL/L/day), and complete inhibition of CH 4 in final gas composition. Similarly, during Phase II, higher COD concentration of ∼ 22 g/L and ∼ 3 – ∼3.5 g/L of TOC suggest carbon deposition in the system and reduced CO 2 conversion during H 2 production. These findings suggest that influent handling significantly affects microbial community dynamics and potential for operational modifications to optimize H 2 production from organic substrates in anaerobic systems.