Enhanced biological phosphorus removal (EBPR) is one of the most sustainable processes to remove phosphorus (P) from wastewater. In EBPR, phosphorus is accumulated intracellularly by polyphosphate accumulating organisms (PAOs) in the presence of oxygen. The accumulated polyphosphate in PAOs can then be removed when the sludge containing the PAOs is from the tank.

SCELSE research overturned the accepted paradigm that the EBPR process cannot thrive in tropic regions due to higher ambient temperatures, since temperature >25°C typically favours glycogen-accumulating organisms (GAOs), which outcompete PAOs. EBPR also typically requires anaerobic/aerobic or anaerobic/anoxic cycling conditions to proceed. However, the presence of EBPR activity has been observed in a Singapore wastewater treatment plant (WWTP) with an ambient temperature range of 30–32°C adopted the Modified Ludzack-Ettinger (MLE) system, which contains only anoxic/aerobic zones without a defined anaerobic zone.

To understand this seeming contradiction, SCELSE investigated EBPR activities in a laboratory-batch study using sludge obtained from the activated sludge (AS) tank in a local WWTP. As EBPR activities occurred in the tank without defined anaerobic zones, the organisms responsible for these activities were investigated. The presence of Ca. Accumulibacter as the only PAO group in the sludge was confirmed, with Type II Accumulibacter consistently detected at higher abundance than Accumulibacter Type 1. Among the multiple Accumulibacter strains observed, Clade IIC was also detected as the most abundant PAO in the sludge at both sampling dates. Type II Accumulibacter taxa are considered non-denitrifying PAOs (non-DPAOs) since they cannot reduce nitrate while Type I was identified as DPAO since these taxa are able to couple P uptake with nitrate reduction.

Non-DPAOs were more abundant than DPAOs in the samples. Hence, it was postulated that non-DPAOs were the organisms driving the EBPR observed in the tank without the anaerobic zones. Indeed, when either acetate or propionate was used in batch laboratory experiments, EBPR activities occurred under anaerobic/aerobic conditions using acetate or under anoxic/aerobic conditions using either acetate or propionate. The presence of EBPR in the anoxic/aerobic reactor fed with propionate suggested the participation of non-DPAOs since DPAOs could only use acetate and not propionate. The ability of non-DPAOs to use both acetate and propionate provides a competitive advantage over DPAOs.

Comparable EBPR activities between anaerobic/aerobic and anoxic/aerobic conditions when acetate was used as a carbon source further supported the hypothesis that non-DPAOs could perform EBPR under anoxic/aerobic conditions. Hence, non-DPAOs in the sludge recognised the anoxic condition as pseudo-anaerobic.

EBPR was demonstrated to occur in conditions conventionally believed to be unfavourable to the process (i.e., higher ambient temperature and lack of a defined anaerobic zone). Hence, this discovery has created the possibility for wider application of this method in tropical countries like Singapore.

Fluorescence imaging of activated sludge from local WWTP. Bacteria hybridisedwith EUBMix probe (green), Accumulibacter-PAOs hybridised with PAOmix (Red), and Accumulibacter-PAOs Type II hybridised with ACC-II-444 (blue). Cells hybridised to both PAO probes appear magenta.