Nitrification in wastewater treatment at its biological oxygen limit

Abstract

Using low dissolved oxygen (DO) concentrations in activated sludge could improve the energy and environmental sustainability of wastewater treatment plants (WWTPs). At present there is limited information about the influence of low DO on nitrifying bacteria: the mode of acclimatization of ammonia-oxidizing bacteria (AOB); the mechanistic basis for the biological adaptation of AOB under those conditions; and the impact of low DO on the emissions of the greenhouse gas (GHG). We operated four reactors in continuous mode at low (0.2 mg/L) and high (6.5 mg/L) DO. Improvements in ammonia (NH3) and nitrite (NO2 - ) oxidation were observed in reactors at low DO after two months, though neither form of nitrogen was removed completely. Quantitative PCR of the NH3 monooxygenase (amoA) and NO2 - oxidoreductase (nxrB) genes showed similar cell concentrations of AOB and nitriteoxidizing bacteria (NOB) cells in all reactors, though AOB biomass declined in the initial phase at low DO. The AOB found at low DO reactors were phylogenetically associated with the Nitrosomonas europaea/Nitrosococcus mobilis lineage and appeared to be selected from the seed, representing a very small percentage of the original community. The effect of initial cell concentration on AOB adaptation was modelled using two species, linking adaptation time and the initial concentration of the species selected. High growth yields in the AOB adapted to low DO conditions suggested a mixotrophic metabolism. The production of nitrous oxide (N2O) was observed in batch experiments using activated sludge samples from both DO conditions. Higher production was observed in low DO experiments achieving 402 nmol/L.h against 11 nmol/L.h of N2O in high DO. However, this production represented a very small percentage (1.5% and 0.03% in low and high DO respectively) of the total NH3 removed, suggesting the occurrence of simultaneous nitrification and denitrification. The impact of N2O production was estimated by two methods and suggested that plants must save 0.16 kWh/m3 of treated wastewater or 0.02 kWh/m3 of treated wastewater per each mg of NH3 oxidized, to make climate sense. Fluorescence in situ hybridisation (FISH) and fluorescent-activated cell sorting (FACS) were combined to analyse AOB genomes in reactors operated at low and high DO. AOB cells fixed in ethanol revealed positive signal with high fluorescent signal intensity when using Cy5 and SYTO 9 and when cells were pre-treated with ultrasonication. AOB were enriched, forming up to 14% of the total sorted population, but it was still not sufficient to guarantee a good assembly and thus reconstruct genomes. Though the feasibility of FISHFACS to separate targeted uncultivated populations was demonstrated, optimization of the protocol is still needed.