Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3-37%), nucleic acids (9-50%), and carbohydrates (3-21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.