Extracellular DNA plays a key role in the structural stability of sulfide-based denitrifying biofilms.

Sulfide-based biofilm processes are increasingly used for wastewater denitrification, yet little is known about the extracellular polymeric substance (EPS) composition of sulfide-oxidizing biofilms. This can have an important impact on biofilm mechanical strength and stability. In this research, the properties and roles of EPS components in biofilm stability were investigated. Weak biofilm stability characterized by high roughness and numerous "needle" structures was visualized by optical coherence tomography (OCT) and microscopy. A high abundance of extracellular DNA (eDNA) and a low protein to polysaccharide ratio were found in the biofilm. The roles of eDNA, protein and polysaccharide in biofilm cohesion and adhesion were identified through enzyme treatment and atomic force microscopy (AFM). The enzymatic hydrolysis of eDNA increased the elastic modulus of biofilms by 57 times and reduced the adhesion energy by 96%. The hydrolysis of proteins led to an increase of elastic modulus by 27 times and a loss of adhesion energy by 95.5%. The enzymatic hydrolysis of polysaccharides caused minimal changes in elastic modulus and adhesion energy. These results suggest that eDNA was the key EPS component for biofilm cohesion and adhesion, possibly because it provided special binding sites and can form strong cross-linking with magnesium or other multivalent cations. This study provided new insights into the role of eDNA in biofilm stability and shed light on the development of sulfide-based denitrifying biofilms.