Photogranules for wastewater treatment : From community assembly to targeted microbial functions

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat (waste)water and at the same time generate valuable biomass. The microbial community consists of phototrophs (e.g.., microalgae, cyanobacteria) and non-phototrophic microorganisms (e.g., nitrifiers, denitrifiers, PAOs) that form dark-green spheroid agglomerates. Photogranules have excellent settling properties and are easy to harvest. Introducing photosynthesis to the treatment process has the advantage of in-situ oxygenation. Oxygen produced by phototrophs is directly available for heterotrophs (and nitrifiers). In turn, carbon dioxide produced by respiration is sequestered via photosynthesis. This reduces (or eliminates) the need for energy intensive external aeration and simultaneously minimizes greenhouse gas emissions. Additionally, photogranules can use both light and organic carbon for metabolic energy and consequently can have higher biomass productivity compared to conventional treatment systems. This makes them especially suitable for nutrient recovery application.The aim or this thesis was to advance the field of phototrophic wastewater treatment by systematically exploring the microbial community assembly and functions of photogranules. To achieve this, we operated bioreactors that allowed photogranulation and subsequently explored the functional potential of photogranules for wastewater treatment by changing operation conditions. We integrated various approaches and techniques to investigate the microbial community composition and function, physical structure, and microscale functionality of photogranules. Ultimately, we provided a holistic overview on the microbial ecology and process engineering of photogranular technology to pave the way for more sustainable wastewater treatment.In chapter 2 we generated photogranules under hydrodynamic conditions in lab-scale bioreactors operated in sequencing batch mode. We used a diverse and species-rich inoculum from various sources and followed the microbial community assembly, from free-suspended cells to floccular aggregates and finally to photogranules. The main drivers for photogranulation were identified to be motile filamentous cyanobacteria and the excretion of extracellular polymeric substances (EPS). Operating the lab-scale bioreactors at low hydraulic retention time increased the selection pressure for well-settling biomass, which promoted photogranulation and decreased photogranule assembly time. Additionally, at low hydraulic retention times removal rates were increased due to higher nutrient loading.Wastewater can have various elemental compositions. In low N:P ratio wastewaters the microorganisms can face insufficient amounts of nitrogen for growth, which consequently hinders the biological treatment process. Therefore, in chapter 3 we investigated the effect of nitrogen limitation on the treatment performance and morphology of photogranules. We showed that part of the microbial community of photogranules can perform N2-fixation and can maintain treatment performance despite nitrogen limitation. N2-fixation was mainly attributed to the non-heterocyst forming motile filamentous cyanobacteria that was found to be important for the structural integrity of the photogranule. Long-term exposure to nitrogen-limiting conditions showed an effect on photogranule morphology. Photogranules exhibited a loose and open structure with occasional voids and crevices under these conditions. Therefore, photogranules had an increased surface area, which could have facilitated nitrogen uptake under low concentrations, and which could also have favoured N2-fixation.In chapter 4 we investigated the physical, chemical, and biological structure of photogranules and their metabolic functions on a microscale. This was achieved by applying microscopic and microsensor techniques and conducting incubations with isotopically labelled carbon or nitrogen substrates. We confirmed the structural role of gliding motile filamentous cyanobacteria by visualizing their complex interwoven network spanning through the entire photogranule. Additionally, we localized EPS excreted by cyanobacterial filaments and bacteria, which seemed to act like a glue holding the photogranule together and providing additional structural support. Due to light and substrate gradients most of the biological activity of microorganisms was confined to the outer 500 μm of the photogranule, while denitrification potentially extended further into the centre. Furthermore, we showed that processes for oxygen, carbon and nitrogen are internally coupled. This accounted especially for photosynthesis, nitrification, and heterotrophic activities.Photogranules showed high removal rates for carbon and nitrogen, but lower rates for phosphorus compared to other biological treatment systems. In chapter 5 we addressed this challenge by enhancing phosphorus removal with the introduction of polyphosphate accumulating organisms (PAOs). We accomplished this by mixing photogranules with aerobic granular sludge and operating the bioreactors under a feast-famine regime with an anaerobic (dark) and aerobic (light) phase. PAOs successfully integrated into photogranules and thereby increased phosphorus removal by 6x compared to photogranules without PAOs. The developed process was termed PG+. Further, we investigated the suitability of the PG+ process under a natural diurnal cycle introducing a 12h dark anaerobic phase during nighttime. Neither phototrophs nor PAOs were adversely affected by this anaerobic phase and therefore are suitable for operation under a natural diurnal cycle.Last, I summarized all our findings in chapter 6 and discussed them in detail. To support the discussion, I performed a meta-analysis on publicly available microbial community data of photogranules and used our lab-scale findings to perform a scenario analysis. The meta-analysis supported our findings on the importance of the microbial community compositions and functions, but also showed that photogranulation can be achieved by taxonomically different microbial communities. Further, it indicated possible key functions and traits that are necessary for photogranulation. Motile filamentous cyanobacteria were present in all investigated photogranules, which confirms their importance for photogranulation. Further, I revealed design principles for photogranular bioreactor configuration and modes of operation at large-scale. A sequencing batch reactor was regarded as not optimal for large scale application due to suboptimal use of sunlight during the day. Therefore, I proposed a continuous flow system, which allows the full use of all light available during the day. The scenario analysis illustrated the potential for large-scale application of photogranular technology and provided estimates for treatment performance and area needed. Under “low” light conditions the area required of a photogranule treatment plant was almost 4x higher compared to conventional treatment systems. Under “high” light conditions the footprint was reduced to less than twice. This would make photogranular technology especially suitable for low latitude countries where water sanitation is often still lacking and urgently needed