Microbial Ecology of Granular Sludge

Doctoral thesis, 2019

Granular sludge is an efficient and compact biofilm process for wastewater treatment. Despite the well-established methods for granule cultivation, the ecological processes underpinning the microbial community assembly during granulation are poorly understood. Unveiling fundamental aspects of the microbial ecology of granular sludge will contribute to the improvement of the granulation methods and to the technological upgrade. In this thesis, reviews of the available literature were undertaken to assess critical points of current knowledge about the combination of aerobic granular sludge and membrane filtration, and to gain further knowledge on the ecology of the granular sludge and the granular structure. In parallel, three sequencing batch reactors were employed in different experiments and molecular biology techniques, such as high-throughput DNA sequencing, fluorescence in-situ hybridization and confocal laser scanning microscopy, were used. The reproducibility of the reactors was tested, showing the reactors to be generally reproducible for the abundant community members and for the reactor functions when constant conditions were applied. However, when subjected to periodic disturbances, the replicate reactors did not display a high degree in reproducibility in microbial community. Granulation responded to deterministic factors driven by the reactor conditions. During the start-up of the reactors, microorganisms were washed-out randomly and the granulation started as a response to the shear forces applied in the reactor. Simultaneously, there was a deterministic selection of microorganisms involved in aggregate development and for those that were well adapted to grow at the specific reactor conditions. It was also observed that stochastic processes, i.e. drift, had considerable effect on the less abundant community members. Moreover, stochasticity seemed to be important when the community was subjected to periodical disturbances. Also, bacterial predators appeared as part of the core community and they were found to predate on bacteria that were exerting important reactor functions. Ammonia-oxidizing bacteria were observed in the inner locations of the granules, which did not follow the commonly accepted multilayer model of stratification of different functional groups. The granules were able to withstand high pressures showing a high stability and strength when submitted to different water fluxes. In a separate study, it was shown that the choice of bioinformatics pipelines and dissimilarity indices affects the conclusions drawn from experimental data and the use of Hill-based indices was proposed for robust data analysis.