Optimization of hydrogen production using a coculture of Chlamydomonas reinhardtii and activated sludge bacteria

The production of biophotolytic hydrogen (H₂) relies on the effective management of oxygen (O₂) levels. Coculturing bacteria with microalgae helps mitigate the excess O₂ produced by algal cells. After depleting O₂, the bacteria activate the enzyme hydrogenase in microalgae, leading to H₂ production. In this study, Chlamydomonas reinhardtii was cocultured with indigenous bacteria from activated sludge at varying algae-to-bacteria ratios (1:1, 1:1.5, 1:2, 1:2.5, and 1:3 v/v), with an illumination intensity of 2.8 mmol/m²/s (31 × 10³ lux). The 1:1.5 v/v ratio yielded the highest H₂ volume (1162 mL/L) and the highest O₂ concentration (153.2 mL/L) over a 6-day period. Production of all gaseous components ceased for all ratios as the pH dropped below 4 due to acetate accumulation, and the concentration of acetate reached approximately 1 g/L by the end of each experiment. Gas composition analysis after the first day of coculture revealed that H₂, CO₂, N₂, and O₂ constituted 25%–46%, 20%–40%, 5%–30%, and 1%–10% of the total gas volume, respectively. Glucose (10 g/L) was introduced as an external carbon source for all cultures. After 6 days, the coculture maintained a high total organic carbon (TOC) level of 3.1 g/L, whereas the initial TOC ranged between 3.9 and 4.3 g/L. The findings illustrated a significant correlation between H₂ production, acetate accumulation levels, and O₂ consumption. The algae–activated sludge coculture method substantially enhanced H₂ production compared with previously published methods employing only one or two types of bacterial cultures, underscoring its potential for more efficient biophotolytic H₂ production.