Kinetic characterization of nitrifying pure cultures in chemostate

The process of nitrification in wastewater treatment is widely accepted as a two-step process. In the first step the ammonia is oxidized to nitrite, a process considered to be carried out mainly by the Nitrosomonas sp., while in the second step the Nitrobacter sp. oxidizes the nitrite to nitrate. Both species are autotrophic (chemolithotrophic) and they use CO2 as the only carbon source for growth and maintenance, as well as, inorganic reduced nitrogen compounds to satisfy their basic needs for energy. In the present work, experiments were carried out in a chemostate reactor, using a specific synthetic medium, in order to study the kinetic characteristics of nitrifying pure cultures. In the first set of experiments, the nitrosifying bacteria Ns. europaea were used, while in the second set we employed the nitrifying bacteria Nb. winogradkyi. Subsequently, a specially prepared mixed culture, consisting of the two above mentioned species was studied, in order to evaluate the possible interactions between them. In order to determine the influence of the pH on the growth rate of pure cultures and to determine the optimum pH value, a series of chemostate experiments was conducted with gradual changes of the pH. The optimum pH was determined at 7.5-7.6. The nitrosifying bacteria oxidize ammonia to nitrous acid, whereas the nitrifying bacteria oxidize nitrous acid to nitrate. Their growth rate and kinetic behavior depend on the concentration of the energy source and also on the concentration of the dissolved oxygen and CO2. Therefore, the kinetics can be described by means of the Monod equation. The half-saturation coefficient for the energy source was determined by non-linear regression of the steady state data, which provided the corresponding values of Κm,NH3 = 0.62 mg ΝH3 l-1 and Κm,HNO2 = 21.8 μg HΝO2 l-1 for each pure culture, on the actual substrates for the specific species. The influence of the dissolved oxygen concentration on the microbial activity was studied under controlled conditions in the chemostate, i.e. pH=7.6, T=30°C and HRT=14 h. The results for several steady state conditions and for different dissolved oxygen concentrations provided the value Km,O2 = 0.408 mg O2 l-1 for Ns. europaea. Under similar conditions for the culture of Nb. winogradskyi, altering only the retention time in the chemostate i.e. pH=7.6, T=30°C and HRT=28 h, the results provided the value Km,O2 = 1.657 mg O2 l-1. The influence of the CO2 concentration and its limiting role on the bacterial growth was also investigated, under steady state conditions, as it is important for the synthesis of new cells by the autotrophic bacteria. The values of Km,CO2 for Ns. europaea and Nb. winogradskyi were calculated to 3.8μmol l-1 and 0.37μmol l-1 respectively, which indicates that the substrate affinity for the Ns. europaea is higher by one order of magnitude than the one for the Nb. winogradskyi.