Photogranules are spherical aggregates formed of complex phototrophic ecosystems with potential for “aeration-free” wastewater treatment. Photogranules from a sequencing batch reactor were investigated by fluorescence microscopy, 16S/18S rRNA gene amplicon sequencing, microsensors, and stable- and radioisotope incubations to determine the granules’ composition, nutrient distribution, and light, carbon, and nitrogen budgets. The photogranules were biologically and chemically stratified, with filamentous cyanobacteria arranged in discrete layers and forming a scaffold to which other organisms were attached. Oxygen, nitrate, and light gradients were also detectable. Photosynthetic activity and nitrification were both predominantly restricted to the outer 500 µm, but while photosynthesis was relatively insensitive to the oxygen and nutrient (ammonium, phosphate, acetate) concentrations tested, nitrification was highly sensitive. Oxygen was cycled internally, with oxygen produced through photosynthesis rapidly consumed by aerobic respiration and nitrification. Oxygen production and consumption were well balanced. Similarly, nitrogen was cycled through paired nitrification and denitrification, and carbon was exchanged through photosynthesis and respiration. Our findings highlight that photogranules are complete, complex ecosystems with multiple linked nutrient cycles and will aid engineering decisions in photogranular wastewater treatment.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen-limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen-replete (N+) and limiting (N−) conditions was assessed in sequencing batch reactors. Photogranules under N− condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and chemical oxygen demand removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N− photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N 2 -fixation. These were higher at N− (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N 2 -fixation.