AbstractUsing microsensors O2concentrations were measured in photosynthetically active microbial mats of up to 3 mM, corresponding to a partial pressure of 3 bar. This could damage mats by internal gas formation, and be inhibitory by formation of reactive oxygen species (ROS) and reduced effectivity of RuBisCo. The reliability of the electrochemical microsensors was checked by creating elevated O2concentrations in a water volume placed inside a pressure tank. A microsensor mounted with the tip in the gassed water bath showed a response linearly proportional to 5.5 mM corresponding to 4 bar pure O2pressure. After release of the pressure the O2concentration reduced quickly to 2.5 mM, then stabilized and subsequently reduced slowly over 14 hours to approximately 2 mM. We concluded that the very high O2concentrations measured in phototrophic microbial mats are real and O2oversaturation in mats is a stable phenomenon. As consequence of high O2concentrations, net production of H2O2occurred. The accumulation was, however, limited to the respiratory zone under the photosynthetic layer. Despite the high gas pressure inside mats, no disruption of the mat structure was apparent by bubble formation inside the mats,and bubbles were only observed at mat surfaces. Additions of H2O2to high concentrations in the water column were efficiently removed in the photosynthetically active zone. As the removal rate was linearly proportional to the H2O2influx, this removal occurred possibly not enzymatically but by abiotic processes. Phototrophic microorganisms can produce O2at high rates under strongly elevated O2levels, despite the decreased efficiency due to the unfavorable kinetics of RuBisCo and energy costs for protection. Under non-limiting light conditions, this apparent dilemma is, however, not disadvantageous.ImportanceBiofilms are often used in photobioreactors for production of biomass, food or specialty chemistry. Photosynthesis rates can be limited by high O2levels or high O2/CO2ratios which are especially enhanced in biofilms and mats, due to mass transfer limitations. High O2may lead to reactive O2species (ROS) and reduce the efficiency of RuBisCo. Moreover, gas formation may destabilize their structure. Here we show that extremely high levels of O2are possible in mats and biofilms without ebullition, and while maintaining very high photosynthetic activity.