CONICET | Buscador de Institutos y Recursos Humanos

NITRIFICATION FAILURE IN AN INDUSTRIAL FULL-SCALE ACTIVATED SLUDGE LINKED TO LOW DIVERSITY AND LOW GROWTH YIELD OF AMMONIA OXIDATION BACTERIA (AOB) Eva L. M. Figuerola and Leonardo Erijman Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET-UBA) Vuelta de Obligado 2490 (1428) Buenos Aires Telephone: + 54 11 4783-2871 ext 35 e-mail: erijman@dna.uba.ar Topic: Full-scale applications of population dynamics principles; combined engineering and microbiological approaches Type of presentation: poster Instability of biological nitrification is usually brought about by changes in environmental conditions, such as temperature, pH and the presence of toxic compounds that affect the diversity and activity of nitrifier bacteria. 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 an petroleum refinery, which receives mainly hydrocarbons (average 6.1 ± 3.9 mg l-1) and a high load of ammonia (81 ± 33 mg l-1). The WWTP treated approximately 3.4 million liters of wastewater per day, operated with a hydraulic retention time of 40 h and had mean cell residence time of 48.5 days. Loss of nitrification did not appear to correlate with process pH or temperature. The basin pH was maintained around 7.1 ± 0.3 by the addition of NaOH. Process temperature was 34 ± 3 °C. A statistically significant correlation was found between the reduction in ammonia oxidation and the levels of phenol concentration in the treated wastewater. We performed a molecular diversity analysis of ammonia oxidizing bacteria (AOB) present in the sludge of the full-scale facility. 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 450 bp of the amoA genes 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 the second population was related to the Nitrosomonas nitrosa linage. The total number of ammonium oxidizing bacteria, determined by real time PCR, ranged from 4.7 x 1010 cells ml-1 to 2.6 x 1011 cells ml-1, corresponding to 0.6 to 2.1% of total bacteria, in four samples taken along a period spanning two years of plant operation. This result was confirmed using primers targeting a conserved 16S rRNA gene fragment of Betaproteobacteria ammonia-oxidizing bacteria. Novel PCR primer sets were designed to target signature DNA sequences in the amoA gene of the two detected taxa, and used to quantify both groups using real time PCR. The number of cells determined as belonging to the N europaea-related taxa was in close agreement with total AOB, whereas populations belonging to N. nitrosa-related taxa were one order of magnitude less abundant. These values were used to estimate the observed biomass yield coefficient for the dominant AOB (Yobs, AOB) as 0.015 kg VSS/kg N. We could not detect the presence of ammonia-oxidizing archea (AOA), or anaerobic ammonia oxidation (anammox) in the system. Therefore, in the absence of other nitrification processes (e.g. heterotrophic nitrification), 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.