Abstract A diatom biofilm was grown in a chamber developed for culture of biofilms in chemical gradients. The diatoms grew on a polycarbonate membrane filter which separated a sterile reservoir, with added phosphate, from a reservoir without phosphate. Within 3 weeks of inoculation, a thick biofilm developed on the surface of the filter. The biofilms were homogeneous and therefore suitable for calculations of O
2 diffusion fluxes from concentration profiles of O
2. Profiles of O
2, pH, and gross photosynthesis at different light intensities and liquid medium concentrations of dissolved inorganic carbon and O
2 were measured with microelectrodes. Respiratory activity in a layer of the biofilm was determined as the difference between gross photosynthesis and outflux of O
2 from that layer. The photosynthetic activity in a well-developed biofilm grown at 360 μEinst m
−2 s
−1 and 2.4 mM HCO
3− was limited by the supply of inorganic carbon. Exposure to light above 360 μEinst m
−2 s
−1 stimulated gross photosynthesis as well as respiratory processes without affecting net outflux of O
2. Higher concentrations of inorganic carbon, on the other hand, enhanced gross photosynthesis without concurrent increase in respiratory rate, resulting in an increased outflux of O
2. High concentrations of O
2 in the liquid medium decreased the net outflux of O
2 with little effect on the gross photosynthesis. The effects of inorganic carbon and O
2 on the metabolic activities of the biofilm were consistent with the presence of photorespiratory activity.
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