Weissbrodt, David2009-02-162009-02-162009-02-162006https://infoscience.epfl.ch/handle/20.500.14299/35312• Goal The goal of the project was to produce biomolecular fingerprints in order to describe the structure of microbial community which develops during aerobic sludge granulation, initiated in sequencing batch wastewater treatment processes. Four SBR were run under different conditions of cultivation. Pulse and anaerobic feeding modes were applied, as well as mesophilic (20-30 ° C) and thermophilic (50 °C) conditions. Analytical methods of microscopy and molecular biology have been adapted to this purpose : optical microscopy, DNA extraction, polymerase chain reaction (PCR), gel electrophoresis, terminal restriction fragment length polymorphism (T¬RFLP), cloning and sequencing of fragments of Eubacteria 16S rRNA encoding genes. These analytical tools were applied to the characterization of the microbial architecture of the different states of the biomass present within the biosystems, such as dense and well settling granules, fluffy and non settling granules, suspended flocs and wall biofilms. • Results The inoculum of Morges municipal WWTP was containing a majority of heterotrophic bacteria, inducing a non optimal quality of aerobic granules and a low performance in the simultaneous removal of carbon, ammonium and phosphates. The optical microscopic analysis have shown that well settling granules possess a compact inner architecture, while fluffy granules, which are washed out of the SBRs, are mainly composed of filamentous bacteria. Yeast-like fungi were also detected by PCR/Gel electrophoresis in fluffy granules as well as in compact granules. Archaea bacteria were not present in the cultivated aerobic granules. The T-RFLP fingerprints of well settling granules and non settling consortiums include the same major terminal restriction fragment (TRF, in bases pairs) of Eubacteria 16S rDNA : after HaeIII digestion, TRF (74, 198, 211, 217 and 255 bp) were detected in pulse feeding mesophilic mode, and TRF (196, 198, 210, 214, 217, 245 and 321 bp) were detected in anaerobic feeding mesophilic mode. T-RFLP fingerprints were useful for the study of the temporal evolution of aerobic granules. Heterotrophic eubacterial species linked to TRF 198 bp are predominant in compact granules, while heterotrophic filamentous eubacterial species linked to TRF 210-211 bp are predominant in filamentous granules. The analysis of the presence of nitrifying bacteria is currently in progress, as well as cloning and sequencing of fragments of Eubacteria 16S rRNA encoding genes. • Conclusion The quality of the inoculum seems to be an operational key parameter. It should contain heterotrophic, nitrifying and phosphate-accumulating bacteria to allow their growth in multi-layered granules, competent for simultaneous C-, N- and P-removal. Analysis of scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) coupled to confocal microscopy have to be pursued to localise the microbial layers and functions in the deepness of aerobic granules architecture. The development, the application and the use of biomolecular identity cards (fingerprinting) constitute an essential tool for following and anticipating the evolution of one aerobic granulation process, and to describe the relationship between the different microbial layers.Wastewater treatmentsequencing batch reactor (SBR)aerobic sludge granulationmechanism of granulationpulse feedanaerobic feedmesophiliathermophiliamicrobial diversitystructure of microbial communitymolecular biologymicroscopySEMFISHDNA extraction16S rDNA PCR amplificationT-RFLP fingerprintcloning and sequencingstudent work::master thesis