Figure 2: Photosynthetic hydrogen production and oxygen evolution.

Quantification of the H₂ase mRNA synthesized by (a–d) C. vulgaris YSL01 and (e–h) C. vulgaris YSL16 during photoautotrophic cultivation under atmospheric and anaerobic conditions. The kinetics of hydrogen production by two different microalgal isolates was monitored for up to 34 days under (a,e) atmospheric and CO₂-limited (closed system) as well as (c,g) anaerobic, 10% CO₂, and photoautotrophic conditions using crimp-sealed culture bottles. The highest hydrogen content achieved by C. vulgaris YSL01 and YSL16 under atmospheric condition was 0.17 and 0.23%, whereas under the anaerobic condition at 10% CO₂, maximum hydrogen contents of 24.6 and 18.2% were achieved, respectively. (b,f) During the period of aerobic hydrogen production, both normalized HYDA expression level and in vitro H₂ase activity were quantitatively determined. HYDA was expressed at various concentrations of oxygen, and its relative mRNA concentration displayed a linear correlation with H₂ase activity. In vitro H₂ase activity was significantly decreased as the oxygen levels increased (≥21%; P<0.05). (d,h) Under anaerobic conditions, H₂ase activity and mRNA expression peaked after 4–6 days of cultivation and then dropped significantly owing to the gradual accumulation of oxygen. The value at each time point represents the average of three independent experiments±s.e. The size of the error bars is smaller than the symbol size.