9Using microsensors O 2 concentrations were measured in photosynthetically active microbial mats of up to 10 3 mM, corresponding to a partial pressure of 3 bar. This could damage mats by internal gas formation, and 11 be inhibitory by formation of reactive oxygen species (ROS) and reduced effectivity of RuBisCo. The 12 reliability of the electrochemical microsensors was checked by creating elevated O 2 concentrations in a 13 water volume placed inside a pressure tank. A microsensor mounted with the tip in the gassed water bath 14 showed a response linearly proportional to 5.5 mM corresponding to 4 bar pure O 2 pressure. After release 15 of the pressure the O 2 concentration reduced quickly to 2.5 mM, then stabilized and subsequently reduced 16 slowly over 14 hours to approximately 2 mM. We concluded that the very high O 2 concentrations 17 measured in phototrophic microbial mats are real and O 2 oversaturation in mats is a stable phenomenon. 18As consequence of high O 2 concentrations, net production of H 2 O 2 occurred. The accumulation was, 19 however, limited to the respiratory zone under the photosynthetic layer. Despite the high gas pressure 20 inside mats, no disruption of the mat structure was apparent by bubble formation inside the mats,and 21 bubbles were only observed at mat surfaces. Additions of H 2 O 2 to high concentrations in the water column 22 were efficiently removed in the photosynthetically active zone. As the removal rate was linearly 23 proportional to the H 2 O 2 influx, this removal occurred possibly not enzymatically but by abiotic processes. 24Phototrophic microorganisms can produce O 2 at high rates under strongly elevated O 2 levels, despite the 25 decreased efficiency due to the unfavorable kinetics of RuBisCo and energy costs for protection. Under 26 non-limiting light conditions, this apparent dilemma is, however, not disadvantageous. 27 28 Importance Biofilms are often used in photobioreactors for production of biomass, food or specialty 29 chemistry. Photosynthesis rates can be limited by high O 2 levels or high O 2 /CO 2 ratios which are 30 especially enhanced in biofilms and mats, due to mass transfer limitations. High O 2 may lead to reactive 31 O 2 species (ROS) and reduce the efficiency of RuBisCo. Moreover, gas formation may destabilize their 32 structure. Here we show that extremely high levels of O 2 are possible in mats and biofilms without 33 ebullition, and while maintaining very high photosynthetic activity.