Nitrification failure in an industrial full-scale activated sludge linked to low growth yield of ammonia oxidation bacteria (AOB)

FIGUEROLA, E.L.M.; ERIJMAN, L.

Aalborg

Congreso; 5th IWA Specialist Conference on Activated Sludge Population Dynamics; 2009

International Water Association

Instability of biological nitrification is usually brought about by changes in environmental conditions, such as temperature, pH and the presence of toxic compounds. Therefore, a strong understanding of the ecological basis of autotrophic ammonia oxidation is critical to predict and hopefully avoid the risk of operational malfunction. We have investigated the process of ammonia oxidation in a full-scale activated sludge from a petroleum refinery, which receives mainly hydrocarbons (average 6.1 ± 3.9 mg/l and a high load of ammonia (81 ± 33 mg/l). The WWTP treated daily 3400 m3 of wastewater, operated with a hydraulic retention time of 40 h and had mean cell residence time of 48.5 days. Loss of nitrification did not correlate with process pH, temperature or dissolved oxygen concentration. Nitrification inhibition could be fitted to a pattern of non- competitive inhibition by phenol, where the concentration of phenol for which the rate of ammonium oxidation is one half of the maximum rate was 0.4 mg/L. We performed a molecular diversity analysis of ammonia oxidizing bacteria (AOB). DNA was isolated from sludge samples taken from the aeration basin and used as template for PCR, with primers targeting highly conserved regions in the gene coding for the enzyme ammonia monooxygenase (amoA). PCR-amplified fragments of approximately 440 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 belonged to two populations separated by a genetic distance of 0.06. The dominant population (77% of the clones) was related to Nitrosomonas europaea, whereas a minor population was related to the Nitrosomonas nitrosa linage. Total AOB number was determined at the time of stable nitrification using three different set of primers by real time PCR. Using primers that targeted a conserved 16S rRNA gene fragment of Betaproteobacteria AOB the concentration of positive cells was (9.8 +2.9)·10^10. Total AOB concentration was also estimated targeting the amoA gene, and the obtained number was (9.2 + 3.2)·10^10 cells/L. Finally, using novel PCR primer sets designed to target signature DNA sequences in the amoA gene of the major taxa detected in the clone library, the number of cells determined was (8.5 + 5.5)·10^10, in close agreement with total AOB. These values, in conjunction with operational data, were used to estimate the cell-specific ammonia oxidation rate as 0.66 fmol of NH4 to NO2 per hour, and the observed biomass yield coefficient for the dominant AOB (Yobs, AOB) as 0.0175 kg VSS/kg N.The presence of ammonia-oxidizing Archea or anammox in the system were not detected. We propose that AOB selected and adapted to perform under harsh conditions display a growth yield much lower than those normally used by engineers for plant design. This conclusion provides a possible explanation for the increase risk of failure in the presence of toxic compounds such as phenol.