Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics
in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we
investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria
(Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching
peak values of up to 30 mol H2 liter1 at about 1-mm depth below the mat surface, slowly decreasing to about 11 mol H2
liter1 just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 mol H2 liter1, was found in
the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation
indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier
findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples
from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of
other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3.