Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20–40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation – ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW – composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

Aerobic granular sludge (AGS) is a promising alternative wastewater treatment to the conventional activated sludge system allowing space and energy saving. Basic understanding of AGS has mainly been obtained using simple wastewater containing acetate and propionate as carbon source. Yet, the aspect and performances of AGS grown in such model systems are different from those obtained in reactor treating real wastewater. The impact of fermentable and hydrolyzable compounds on already formed AGS was assessed separately by changing the composition of the influent from simple wastewater containing volatile fatty acids to complex monomeric wastewater containing amino acids and glucose, and then to complex polymeric wastewater containing also starch and peptone. The reversibility of the observed changes was assessed by changing the composition of the wastewater from complex monomeric back to simple. The introduction of fermentable compounds in the influent left the settling properties and nutrient removal performance unchanged, but had a significant impact on the bacterial community. The proportion of Gammaproteobacteria diminished to the benefit of Actinobacteria and the Saccharibateria phylum. On the other hand, the introduction of polymeric compounds altered the settling properties and denitrification efficiency, but induced smaller changes in the bacterial community. The changes induced by the wastewater transition were only partly reversed. Seven distinct stables states of the bacterial community were detected during the 921 days of experiment, four of them observed with the complex monomeric wastewater. The transitions between these states were not only caused by wastewater changes but also by operation failures and other incidences. However, the nutrient removal performance and settling properties of the AGS were globally maintained due to the functional redundancy of its bacterial community.

Complete genomes can be recovered from metagenomes by assembling and binning DNA sequences into metagenome assembled genomes (MAGs). Yet, the presence of microdiversity can hamper the assembly and binning processes, possibly yielding chimeric, highly fragmented and incomplete genomes. Here, the metagenomes of four samples of aerobic granular sludge bioreactors containing Candidatus (Ca.) Accumulibacter, a phosphateaccumulating organism of interest for wastewater treatment, were sequenced with both PacBio and Illumina. Different strategies of genome assembly and binning were investigated, including published protocols and a binning procedure adapted to the binning of long contigs (MuLoBiSC). Multiple criteria were considered to select the best strategy for Ca. Accumulibacter, whose multiple strains in every sample represent a challenging microdiversity. In this case, the best strategy relies on long-read only assembly and a custom binning procedure including MuLoBiSC in metaWRAP. Several high-quality Ca. Accumulibacter MAGs, including a novel species, were obtained independently from different samples. Comparative genomic analysis showed that MAGs retrieved in different samples harbour genomic rearrangements in addition to accumulation of point mutations. The microdiversity of Ca. Accumulibacter, likely driven by mobile genetic elements, causes major difficulties in recovering MAGs, but it is also a hallmark of the panmictic lifestyle of these bacteria.

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Shooting ranges represent about 10% of polluted sites in Switzerland. This pollution, mainly consisting of lead (Pb) and antimony (Sb), can spread into the local groundwater, wildlife, and plants. In this study, shooting range stop butt soils (elevated soil mount behind the target) of 3 sites (Versoix, Cartigny and Nyon, Western Switzerland) were investigated for metal contamination and culturable lead-resistant bacteria in contaminated soils. The ICP-MS analysis of surface stop butt soils (0-5 cm) indicated high metal concentrations especially for Pb and Sb; e.g. the Pb concentration ranging from [61,135 to 395,651], [23,821 to 201,268] and [120 to 27,517] mg kg-1 in the soil from the sites of Versoix, Cartigny and Nyon, respectively. Molecular analysis of 16S rRNA gene demonstrates the presence of bacteria from diverse classes: Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli that exhibited high Pb MIC value from 1,036 to 2,694 mg kg-1. The isolated strains could be the subject of further studies to evaluate their role in bioremediation of lead contaminated soil