Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge

The composition and the distribution of extracellular polymeric substances (EPS) and pellets of autotrophic nitrifying biofilm and activated sludge were investigated in this work. Fourier-transform Infrared Spectroscopy, fluorescent in situ hybridization and fluorescence staining were used to examine proteins, carbohydrates, humic substances and DNA being present in the biofilms and the sludge samples. To investigate extraction efficiency and its effect on characterization of tightly bounded EPS, four extraction methods (ethylenediamine tetraacetic acid (EDTA), NaOH, cationic exchange resin (CER), ultrasound) were compared. EDTA and ultrasound showed more effective extraction ability than NaOH and CER. NaOH and ultrasound extraction led to high activity of glucose-6-phosphate dehydrogenase from cell lysis, which was confirmed by fluorescence staining analysis. Ultrasound and NaOH extraction yielded 18% and 11% of dead cells in biofilm, respectively, whereas they obtained 11% and 9% of dead cells in activated sludge, respectively. Four layers of extractible products were separated from autotrophic nitrifiering flocs. The extraction results indicated that extraction yield in different layers varied in a wide range: 3–6% of soluble EPS, 5–10% of loosely bound EPS, 34–67% of tightly bound EPS and 30–60% of pellets.