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> Abstract Nitrification in freshwater, a key process in the nitrogen cycle, is now well known to take place predominantly on suspended particles and in sediment. Nitrobacter is the most commonly isolated nitrite oxidizing bacteria from water environments. Three methods for counting nitrite oxidizing communities (especially Nitrobacter) in sediment were investigated: MPN-Griess, fluorescent antibodies (immunofluorescence), and a more recent molecular method coupling specific DNA amplification by PCR and statistical MPN quantification. After preliminary adjustments of the MPN-PCR technique, the detection level and the yield of each method were determined by inoculating a sediment with a pure Nitrobacter culture. The best recovery yield was obtained with the immunofluorescence technique (21.3%) and the lowest detection level was reached with the MPN-Griess method (10(3) Nitrobacter/g dry weight sediment). The MPN-PCR method resulted in the lowest recovery yields and needs further adaptation to become a reliable and precise tool for investigations of nitrifying bacteria in sediment.

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Sediments were collected in three small water courses upstream and downstream a water treatment plant discharge, in one highly fertilized eutrophic pond, and from one oligotrophic brook. They were analysed for ammonium oxidation activity, for nitrite oxidation activity and for their respective nitrifying community size. The relationships between variables was investigated by a principal components analysis of the physicochemical and biological data.Upstream sites of treatment plant discharges looked similar to an oligotrophic river, but the downstream sites have certain similarities with a highly eutrophic pond. Globally, whatever the hydrosystem, the organic carbon and organic nitrogen content of the sediments were highly correlated with nitrifying activity and nitrifiers number (significance level α = 0.001), but there was no significant correlation between the nitrifiers number and the related activity. The hypothesis of a role of the grain size on the nitrification communities was tested. A Mann-Withney U test (α = 0.05) indicated that, whatever the sediment, the two fractions of sand (500-200 µm and 200-50 µm) were non significantly different for nitrification. Conversely, the silt-clay fraction was significantly different from sands for all the nitrification's parameters. This silt-clay fraction exhibited the highest functional nitrifying community per mass unit indicating that the silt fraction is a main contributor to the nitrification process in river sediment. Finally, for each river, there were significant differences (at α = 0.05) between the nitrifiying communities in downstream and upstream sediments. In the 3 studied rivers, upstream sites were significantly higher in terms of activity and in terms of number of nitrifying bacteria. The effect of wastewater effluents discharge seems to be site specific.

Nitrobacter, a ubiquitous nitrite oxidizer in natural and anthropized environments, is commonly studied as the model genus performing the second stage of nitrification. In rivers, wastewater treatment plant discharges may affect the nitrite-oxidizing activity and the responsible genera that are largely associated with sediment. We used a laboratory batch culture approach with Nitrobacter wynogradskyi ssp. agilis strain AG and Nitrobacter hamburgensis strain X(14) to characterize the possible stress effect of wastewater effluent on these populations and to study the possible competition between an effluent strain (X(14)) and a sediment strain (AG) over a 42-day incubation time. Immunofluorescence enumerations of each strain showed that they both survived and settled in the sediment, indicating that there was no significant stress effect due to chemical changes caused by the effluent. The development of the strains' density and activity was directly correlated with the available nitrite concentration. Nevertheless, the potential specific activity was not constant along the so-called mixotrophic (non-limiting nitrite concentration) and heterotrophic (nitrite depletion) conditions. This illustrates the inducibility of the nitrite oxidoreductase and indicates the metabolic versatility of the strains. In our experimental conditions, the preferentially autotrophic AG strain appeared more competitive than the preferentially mixo- or heterotrophic X(14) strain, including in heterotrophic environment.