https://www.selleckchem.com/products/gw-441756.html Previous studies demonstrate that free nitrous acid (FNA i.e. HNO2) detaches sewer biofilms, breaks down flocs of waste activated sludge (WAS) and enhances biogas production from WAS. This suggests possible interactions of FNA with organic extracellular polymeric substances (EPS) that bind the cells into biofilms or sludge flocs. This study evaluates the chemical interactions and reaction mechanisms between FNA and molecules representative of key EPS in biofilm and sludge flocs. Molecules chosen to represent components found in the extracellular polymeric matrix were treated with FNA at 6.09 mgN/L (NO2- = 250 mgN/L, pH = 5.0 ± 0.2, T = 22 °C) for 24 hours (conditions typically used in applications) so as to consider the hypothesized chemical interactions and the consequent reaction pathways. A number of analytical techniques were employed to measure the molecular changes in the EPS molecules including; proton (1H) nuclear magnetic resonance spectroscopy (NMR), electrospray ionisation mass spectrometry (ESI-MS) and gel permeation chromatography (GPC). The results demonstrated that FNA broke down a range of large EPS molecules including carbohydrates, protein and lipids to smaller molecules. Two mechanistic pathways have been proposed including electrophilic substitution, whereby the nitrosium ion (NO+) was the reactive electrophile, and oxidative radical reactions, through which the nitrogen radicals (.NO2, .NO) and reactive nitrogen intermediates (RNIs) (e.g. N2O3 and N2O4) formed from the decomposition of FNA became part of the reaction products. Larger, more complex organic molecules such as humic acid, required higher concentrations of FNA (6.09 mgN/L or greater) to cause molecular breakdown, whereas smaller molecules, such as calcium alginate, was broken down at lower concentrations (3.04 mgN/L). The study contributes to the understanding of the fundamental mechanisms behind the application of FNA for biofilm c