Hydrogen (H-2), a possible future clean energy carrier, requires process-based improvement routes for cutting down the production cost. The impact of nitrogen (N-2) sparging on H-2 production during co-culture system of Enterobacter aerogenes and Clostridium butyricum from crude glycerol (CG) was studied to reduce the overall process cost. H-2 production using 1% CG under nitrogen sparged medium before autoclaving resulted in 1.2 mmol-H-2/mol of glycerol in comparison to 1.5 mmol-H-2/mol of glycerol without sparging. In the presence of air ranging from 5 mL to 75 mL in the headspace volume, H2 production increased to a maximum of 26.14 mmol/L with 1.4 g/L of ethanol production. The concentration of 1,3-propanediol with N-2 sparging was around 3.0 g/L and decreased to 0.5 g/L due to presence of 75 mL of air in the headspace. This observation can be attributed to a shift from reductive to oxidative metabolism of glycerol. A process-based improvement strategy to optimize H-2 formation resulted in metabolic pathway shift from reductive to oxidative with increase in H-2 production. Synergistic influence of co-culture system in absence of expensive reducing agent and without nitrogen sparging step can offer a better process-based economic strategy for H-2 production, minimize the metabolite production and increase field-scale application of biodiesel plant. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.