Photo-activated sludge (PAS) systems are an emerging wastewater treatment technology where microalgae provide oxygen to bacteria without the need for external aeration. There is limited knowledge on the optimal conditions for enhanced biological phosphorus removal (EBPR) in systems containing a mixture of polyphosphate accumulating organisms (PAOs) and microalgae. This research aimed to study the effects of substrate composition and light intensity on the performance of a laboratory-scale EBPR-PAS system. Initially, a model-based design was developed to study the effect of organic carbon (COD), inorganic carbon (HCO₃) and ammonium-nitrogen (NH₄-N) in nitrification deprived conditions on phosphorus (P) removal. Based on the mathematical model, two different synthetic wastewater compositions (COD:HCO₃:NH₄-N: 10:20:1 and 10:10:4) were examined at a light intensity of 350 µmol m⁻² sec⁻¹. Add to this, the performance of the system was also investigated at light intensities: 87.5, 175, and 262.5 µmol m⁻² sec⁻¹ for short terms. Results showed that wastewater having a high level of HCO₃ and low level of NH₄-N (ratio of 10:20:1) favored only microalgal growth, and had poor P removal due to a shortage of NH₄-N for PAOs growth. However, lowering the HCO₃ level and increasing the NH₄-N level (ratio of 10:10:4) balanced PAOs and microalgae symbiosis, and had a positive influence on P removal. Under this mode of operation, the system was able to operate without external aeration and achieved a net P removal of 10.33 ±1.45 mg L⁻¹ at an influent COD of 100 mg L⁻¹. No significant variation was observed in the reactor performance for different light intensities, indicating the EBPR-PAS system can be operated at low light intensities with a positive influence on P removal.

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.

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189 mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18 mg PO₄-P/g VSS/h. Up to 8 mg H₂S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic₆₀). At higher concentrations of sulphide, potassium (>16 mg H₂S-S/L) and phosphate (>36 mg H₂S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8 mg H₂S-S/L, as PAO I could be potentially inhibited.

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.