Developmentof an immunosensor for enumeration of Nitrobacter

Sammanfattning: Eutrophication is a result of increasing discharges ofnutrients and is the main cause of the deterioration of theaquatic ecosystems. Major sources of nitrogen in coastal watersare wastewater treatment plants. A large number of wastewatertreatment plants have been upgraded to biologically removenitrogen using nitrification and denitrification processes.The aim of this thesis was to detect and enumeratenitrifying bacteria in wastewater treatment systems. Purecultures ofNitrobacterwere isolated from activated sludge and usedto produce monoclonal antibodies. Using these antibodies acompetitive ELISA (enzyme-linked immunosorbent assay) wasdeveloped, and successfully used to enumerateNitrobacterin pure cultures as well as in activatedsludge. This method was applied in a study whereNitrobacterwas quantified in the influent to Brommawastewater treatment plant.The principles of the ELISA were used to develop anamperometric enzyme-linked immunoassay. In this biosensor anelectrode made of glassy carbon is used, on which theimmunological reaction is carried out. An enzyme, conjugated tothe secondary antibody, catalyzes the formation of anelectroactive product, which can be amperometrically detected.Quantitative detection ofNitrobacterusing the immunosensor was compared to theELISA showing compatible results. The overall assay time wasshortened and a lower detection limit could be obtained byusing the biosensor.The cross-reactivity of a number of bacterial strains andisolates were determined. All strains of the nitrite-oxidizinggeneraNitrobacterandNitrococcusreacted strongly with the monoclonalantibody. The nitrite-oxidizingNitrospira moscoviensisas well as all theammonia-oxidizing and heterotrophic bacteria tested did notshow any affinity towards the antibody. The sensor was furtherused to enumerateNitrobacterin sludge samples from different wastewatertreatment plants in Sweden. By comparing the number ofNitrobacterobtained using the biosensor with theactivity of the samples, inhibition of the nitrification can bedetected. To identify the nitrification inhibitors in thewastewater, a procedure was presented for fractionation of thesample and location of the inhibitory compounds. In awastewater obtained from a plant for drying wood derived fuel,unsaturated fatty acids and monoterpenes were found toconstitute most of the inhibitory effect.Key words: biosensor, ELISA, enumeration, immunoassay,monoclonal antibodies, nitrification inhibition, nitrifyingbacteria,Nitrobacter, quantification, wastewater treatment.