Isolation and characterization of microbial communities from a constructed wetlands system: A case study in Tunisia

Constructed wetlands (CWs) have received increasing attention in the last decade due to their high potential for wastewater treatment. The nitrogen removal performance is always the main focus when evaluating the treatment ability of CWs. In this work, we aimed to isolate and identify the heterotrophic bacteria using the 16S rRNA gene sequencing method and to characterize the microbial communities removing nitrogen from wastewater and rhizosphere of constructed wetlands and quantified the heterotrophic nitrification on a microtiter plate by applying a spectrofluorometric assay. A number of 35 isolates within the obtained bacterial collection (380 isolates), were selected for molecular classification based on their genetic patterns after amplification of ITS fragments. Results obtained from the PCR amplification of the 16S-23S rDNA gene followed by the partial sequencing of the 16S rDNA genes confirmed the affiliation of the isolates to γ-Proteobacteria, Firmictes, α-Proteobacteria, Actinobacteria and δ-Proteobacteria. Batch tests were carried out to investigate the capacity for heterotrophic nitrification in the pure culture. The batch test results indicate that nitrifying bacteria can utilize the organic carbon as the source of assimilation when it grows on glucose and ammonium chloride medium accompanying the formation of NO2 (nitrified products > 80%). Quantification of bacterial biofilms grown on microtiter plate showed that six strains were strongly adhesive to polystyrene microtiter plate with an OD595 ranging between 3.031 and 4.0368 and only one strain was weak biofilm forming. These bacteria might be used to activate nitrifying bioreactor for a tertiary domestic wastewaters treatment.