Low-dissolved-oxygen nitrification in tropical sewage: an investigation on potential, performance and functional microbial community.

Intensive aeration for nitrification is a major energy consumer in sewage treatment plants (STPs). Low-dissolved-oxygen (low-DO) nitrification has the potential to lower the aeration demand. However, the applicability of low-DO nitrification in the tropical climate is not well-understood. In this study, the potential of low-DO nitrification in tropical setting was first examined using batch kinetic experiments. Subsequently, the performance of low-DO nitrification was investigated in a laboratory-scale sequential batch reactor (SBR) for 42 days using real tropical sewage. The batch kinetic experiments showed that the seed sludge has a relatively high oxygen affinity. Thus, the rate of nitrification was not significantly reduced at low DO concentrations (0.5 mg/L). During the operation of the low-DO nitrification SBR, 90% of NH4-N was removed. The active low-DO nitrification was mainly attributed to the limited biodegradable organics in the sewage. Fluorescence in-situ hybridisation and 16S rRNA amplicon sequencing revealed the nitrifiers were related to Nitrospira genus and Nitrosomonadaceae family. Phylogenetic analysis suggests 47% of the operational taxonomic units in Nitrospira genus are closely related to a comammox bacteria. This study has demonstrated active low-DO nitrification in tropical setting, which is a more sustainable process that could significantly reduce the energy footprint of STPs.

Long-term performance evaluation of EBPR process in tropical climate: start-up, process stability, and the effect of operational pH and influent C:P ratio.

To date, little information is known about the operation of the enhanced biological phosphorus removal (EBPR) process in tropical climates. Along with the global concerns on nutrient pollution and the increasing array of local regulatory requirements, the applicability and compliance accountability of the EBPR process for sewage treatment in tropical climates is being evaluated. A sequencing batch reactor (SBR) inoculated with seed sludge from a conventional activated sludge (CAS) process was successfully acclimatized to EBPR conditions at 28 °C after 13 days' operation. Enrichment of Candidatus Accumulibacter phosphatis in the SBR was confirmed through fluorescence in situ hybridization (FISH). The effects of operational pH and influent C:P ratio on EBPR were then investigated. At pH 7 or pH 8, phosphorus removal rates of the EBPR processes were relatively higher when operated at C:P ratio of 3 than C:P ratio of 10, with 0.019-0.020 and 0.011-0.012 g-P/g-MLVSS•day respectively. One-year operation of the 28 °C EBPR process at C:P ratio of 3 and pH 8 demonstrated stable phosphorus removal rate of 0.020 ± 0.003 g-P/g-MLVSS•day, corresponding to effluent with phosphorus concentration <0.5 mg/L. This study provides the first evidence on good EBPR activity at relatively high temperature, indicating its applicability in a tropical climate.

Examining substrate uptake patterns of Rhodocyclus-related PAO in full-scale EBPR plants by using the MAR-FISH technique.

While recognised as the important population responsible for enhanced biological phosphorus removal (EBPR), detailed knowledge on the physiology of Rhodocyclus-related polyphosphate accumulating organisms (PAO) has yet to be grasped. The objective of this study was to examine the in situ substrate uptake patterns of Rhodocyclus-related PAO present in full-scale EBPR plants by the combined technique of microautoradiography-fluorescent in situ hybridization (MAR-FISH). The presence of these PAO in the four investigated plants was confirmed by FISH and they constituted 17%, 9%, 8%, and 7% of the sludge community. By using MAR-FISH technique, Rhodocyclus-related PAO in all the plants demonstrated similar anaerobic substrate uptake patterns. They were capable of assimilating acetate, aspartate and glutamate under anaerobic condition but they showed negative uptake with palmitate. A significant fraction of the MAR-positive cells assimilated acetate, aspartate or glutamate was found to be Rhodocyclus-related PAO. Dual staining with DAPI and FISH showed that these PAO also accumulated polyphosphate aerobically with aspartate and glutamate as carbon source. The ability of assimilating amino acids besides acetate strongly indicates the versatile physiology of Rhodocyclus-related PAO, which could benefit them to achieve predominance in EBPR activated sludge.

The large PAO cells in full-scale EBPR biomass samples are not yeast spores but possibly novel members of the beta-Proteobacteria.

Large, homogenous clusters of coccobacilli were found to be abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. The identity and the in situ physiology of these dominant microorganisms were investigated in this study. These large clustered cells were revealed to be neither Gram positive nor Gram negative bacteria and contain polyP granules. Cells with similar features were also observed in some enhanced biological phosphate removal (EBPR) systems and reported as yeast spores and Rhodocyclus-related polyphosphate accumulating organisms (PAOs). In this study, fluorescent in situ hybridization (FISH) probing showed these cells were prokaryotic and members of the beta-Proteobacteria. However, these large clustered cells did not respond to the PAO mix FISH probes. The in situ physiology of these large cells was studied with FISH in combination with microautoradiography (MAR) in order to understand their substrate assimilation abilities under different conditions as well as their phosphate uptake ability. These cells were able to take up acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions. Nile Blue A staining in combination with MAR showed that cells incubated under anaerobic conditions contained polyhydroxyalkanoates (PHA) granules. In addition, MAR showed aerobic 33Pi assimilation with all these substrates, consistent with them supporting an EBPR capacity in these large cells. As well as raising doubts about a role for yeasts in EBPR, this study suggests that much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems, and emphasizes the benefits of using techniques like FISH/MAR and PHA staining/MAR to resolve the in situ physiology of the populations of interest there.