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

## TL;DR

Researchers developed a two-step process to produce hydrogen from crude glycerol using bacteria, improving biorefinery efficiency and sustainability.

## Contribution

A novel integrated biorefinery process using E. coli and Rhodobacter capsulatus for hydrogen production from crude glycerol.

## Key findings

- E. coli produced up to 11 g/L of L-malate in 24 hours using crude glycerol.
- Rhodobacter capsulatus converted L-malate into 58.0 ± 6 mM hydrogen in 90 hours.
- The process was optimized and scaled up from shake flasks to bioreactors.

## Abstract

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.

The online version contains supplementary material available at 10.1186/s12934-025-02866-y.