Production of hydrogen from crude glycerol via an integrated process of L-malate biosynthesis by Escherichia coli and photofermentation by Rhodobacter capsulatus

Biorefinery is an innovative concept for resource utilization. To further increase its efficiency, the integration of different biomass conversion technologies (integrated biorefinery) is essential. Biodiesel, a traditional biofuel, generates glycerol as an inevitable byproduct; making it an attractive carbon source for biorefineries due to its abundance, low price, and high degree of reduction. In this work, we propose an integrated two-step process of dark fermentation with Escherichia coli to produce L-malate from glycerol and photofermentation by Rhodobacter capsulatus to convert this L-malate into hydrogen, a clean and sustainable energy carrier. To this end, we optimized an E. coli L-malate-producing strain by overexpressing GlpK in the M4-Δiclr/pBAD-pck strain and scale up the process from shake flask to bioreactor using waste crude glycerol from a biodiesel biorefinery instead of pure glycerol. Under the optimized conditions, E. coli produced up to 11 g/L L-malate in 24 h. The culture medium from this dark fermentation was used to formulate an L-malate enriched medium for Rhodobacter that led to the production of 58.0 ± 6 mM H₂ in 90 h.