Although simultaneous P-removal and nitrate reduction has been observed in laboratory studies as well as full-scale plants, there are contradictory reports on the ability of PAO I to efficiently use nitrate as electron acceptor. Such discrepancy could be due to other microbial groups performing partial denitrification from nitrate to nitrite. The denitrification capacities of two different cultures, a highly enriched PAO I and a PAO I-GAO cultures were assessed through batch activity tests conducted before and after acclimatization to nitrate. Negligible anoxic phosphate uptake coupled with a reduction of nitrate was observed in the highly enriched PAO I culture. On the opposite, the PAO I-GAO culture showed a higher anoxic phosphate uptake activity. Both cultures exhibited good anoxic phosphate uptake activity with nitrite (8.7 ± 0.3 and 9.6 ± 1.8 mgPO4-P/gVSS.h in the PAO I and PAO I-GAO cultures, respectively). These findings suggest that other microbial populations, such as GAOs, were responsible to reduce nitrate to nitrite in this EBPR system, and that PAO I used the nitrite generated for anoxic phosphate uptake. Moreover, the simultaneous denitrification and phosphate removal process using nitrite as electron acceptor may be a more sustainable process as can: i) reduce the carbon consumption, ii) reduce oxygen demand of WWTP, and iii) due to a lower growth yield contribute to a lower sludge production.

Populations of "Candidatus Accumulibacter", a known polyphosphate-accumulating organism, within clade IC have been proposed to perform anoxic P-uptake activity in enhanced biological phosphorus removal (EBPR) systems using nitrate as electron acceptor. However, no consensus has been reached on the ability of "Ca. Accumulibacter" members of clade IC to reduce nitrate to nitrite. Discrepancies might relate to the diverse operational conditions which could trigger the expression of the Nap and/or Nar enzyme and/or to the accuracy in clade classification. This study aimed to assess whether and how certain operational conditions could lead to the enrichment and enhance the denitrification capacity of "Ca. Accumulibacter" within clade IC. To study the potential induction of the denitrifying enzyme, an EBPR culture was enriched under anaerobic-anoxic-oxic (A2O) conditions that, based on fluorescence in situ hybridization and ppk gene sequencing, was composed of around 97% (on a biovolume basis) of affiliates of "Ca. Accumulibacter" clade IC. The influence of the medium composition, sludge retention time (SRT), polyphosphate content of the biomass (poly-P), nitrate dosing approach, and minimal aerobic SRT on potential nitrate reduction were studied. Despite the different studied conditions applied, only a negligible anoxic P-uptake rate was observed, equivalent to maximum 13% of the aerobic P-uptake rate. An increase in the anoxic SRT at the expenses of the aerobic SRT resulted in deterioration of P-removal with limited aerobic P-uptake and insufficient acetate uptake in the anaerobic phase. A near-complete genome (completeness = 100%, contamination = 0.187%) was extracted from the metagenome of the EBPR biomass for the here-proposed "Ca. Accumulibacter delftensis" clade IC. According to full-genome-based phylogenetic analysis, this lineage was distant from the canonical "Ca. Accumulibacter phosphatis", with closest neighbor "Ca. Accumulibacter sp. UW-LDO-IC" within clade IC. This was cross-validated with taxonomic classification of the ppk1 gene sequences. The genome-centric metagenomic analysis highlighted the presence of genes for assimilatory nitrate reductase (nas) and periplasmic nitrate reductase (nap) but no gene for respiratory nitrate reductases (nar). This suggests that "Ca. Accumulibacter delftensis" clade IC was not capable to generate the required energy (ATP) from nitrate under strict anaerobic-anoxic conditions to support an anoxic EBPR metabolism. Definitely, this study stresses the incongruence in denitrification abilities of "Ca. Accumulibacter" clades and reflects the true intra-clade diversity, which requires a thorough investigation within this lineage.

Recent research suggests future alterations in rainfall patterns due to climate variability, affecting public safety and health in urban areas. Urban growth, one of the main drivers of change in the current century, will also affect these conditions. Traditional drainage approaches using grey infrastructure offer low adaptation to an uncertain future. New methodologies of stormwater management focus on decentralized approaches in a long-term planning framework, including the use of Green Infrastructure (GI). This work presents a novel methodology to select, evaluate, and place different green-grey practices (or measures) for retrofitting urban drainage systems. The methodology uses a hydrodynamic model and multi-objective optimization to design solutions at a watershed level. The method proposed in this study was applied in a highly urbanized watershed to evaluate the effect of these measures on Combined Sewer Overflows (CSO) quantity. This approach produced promising results and may become a useful tool for planning and decision making of drainage systems.