The co-culture of microalgae and bacteria is the most effective and appropriate pathway for synergetic H 2 production using photofermentative and biophotolytic metabolic pathways. The integration of wastewater activated sludge as a waste resource in co-culture acts as a bacterial partner. This study assessed the co-culture of Chlorella vulgaris and real wastewater activated sludge with different carbon substrates for the improvement of H 2 production and simultaneous organics removal. Three different inoculum ratios of 1.5:1, 1:1, and 1:1.5 v/v (microalgae/activated sludge) were used with glucose, sorbitol, and mannitol at 10 g/L as exogenous carbon substrates. A high-value activated sludge of 15.1 g chemical oxygen demand (COD)/L was used as the bacterial source. The highest H 2 production of 1246 mL/L was observed in 1:1.5 v/v inoculum ratio using glucose, followed by 831 and 340 mL/L in co-cultures with sorbitol and mannitol addition, respectively. The inoculum ratio of 1:1.5 v/v also achieved ∼33% COD and ∼47% TS removal efficiencies in O 2 -deficient tris-acetate-phosphate (TAP) medium. This study demonstrated, based on experimental data, that the model glucose supplementation was hierarchically the most appropriate carbon substrate for a higher H 2 yield and substantial organics removal in co-culture. A lower H 2 yield is expected when decomposing actual food waste containing different sugars and complex organics.