CONICET | Buscador de Institutos y Recursos Humanos

Ammonia removal via nitrification is an important service provided by many wastewater treatment plants. It is a two-step process, usually rate-limited by the oxidation of ammonia to nitrite performed by ammonia-oxidizing bacteria (AOB). Frequent instability of biological nitrification is brought about by small changes in environmental conditions, such as temperature, pH and the presence of toxic compounds, that influence the growth and activity of AOB. The way in which the environment will affect the process will ultimately depend on the diversity and ecophysiology of nitrifiers existing in each particular ecosystem. Therefore, there is a strong need to understand the ecological basis for instability that would allow to predict and eventually avoid operational failures. We have investigated the process of ammonia oxidation in a full-scale activated sludge from an oil refinery, which receives a high load of free ammonia, hydrocarbons and phenol and suffers from repeated periods of nitrification failure. A significant correlation between NH4-N and phenol concentration in the treated wastewater was observed. NO3-N and phenol concentration exhibited the same correlation, albeit negative. We performed a thorough molecular diversity analysis of ammonia oxidation bacteria present during a period of full nitrification. DNA was isolated from a sludge sample from the aeration basin of the WWTP and used as template for PCR, with primers targeting highly conserved regions in the gene coding for the enzyme ammonia oxidase (amoA). PCR-amplified fragments of 453 bp were used to construct a clone library. Nucleotide sequences and the derived amino acid sequences were determined for a total of 110 clones. AmoA genes were divided into only two OTUs separated by a genetic distance of 0.06. Dominant OTU (77% of the clones) was related to Nitrosomonas europaea, whereas the second OTU affiliated with the Nitrosomonas nitrosa linage. Novel PCR primer sets were designed to target signature DNA sequences in the amoA gene of the two detected taxons and quantified using real time PCR. The proportion of cells belonging to taxon 1 was estimated as 0.7% of total cell quantified with primers targeting total bacterial 16S rRNA. The percentage of AOB was 0,9% based on 16S rDNA assay. A value of 2,4% was obtained for the quantification of bacteria belonging Nitrosomonas genus by fluorescence in situ hybridization (FISH) using the Nitrosomonas-specific probe Nso190. According to these results, AOB cell numbers are approximately three times lower than the predicted values determined by a theoretical process-based estimates of AOB biomass. If other nitrification processes (e.g., heterotrophic nitrification) are discarded, we propose that AOB adapted to perform under harsh conditions have very low yield, providing one possible explanation for why nitrification is unstable in the presence of toxic compounds such as phenol.