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Free nitrous acid (FNA) has been demonstrated to be effective in enhancing the degradability of waste activated sludge (WAS). Considering that extracellular polymeric substances (EPS) are a major component in sludge flocs, the chemical breakdown of EPS components by FNA has been hypothesized to account for the improvement of sludge biodegradability in addition to enhanced cell lysis. EPS extracted from WAS was treated with FNA at 2.0 mg HNO 2 -N per L (260 mg NO 2 À -N per L and pH 5.5).The molecular weight distribution of EPS showed the breakdown of macromolecules into smaller molecules. The chemical structure analysis of EPS using Fourier transform infrared spectroscopy ascribed the breakdown to FNA-induced deamination of proteins, amino sugars and nucleic acids, implying that the main targets of FNA in EPS are protein-like substances. Particle size distribution analysis on the original WAS with the same FNA treatment revealed that FNA treatment of sludge significantly reduces the flocs sizes, which supported that FNA breaks down EPS in activated sludge flocs.

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This study presents a novel pre-treatment strategy using combined free nitrous acid (FNA i.e. HNO2) and hydrogen peroxide (H2O2) to enhance methane production from WAS, with the mechanisms investigated bio-molecularly. WAS from a full-scale plant was treated with FNA alone (1.54 mg N/L), H2O2 alone (10–80 mg/g TS), and their combinations followed by biochemical methane potential tests. Combined FNA and H2O2 pre-treatment substantially enhanced methane potential of WAS by 59–83%, compared to 13–23% and 56% with H2O2 pre-treatment alone and FNA pre-treatment alone respectively. Model-based analysis indicated the increased methane potential was mainly associated with up to 163% increase in rapidly biodegradable fraction with combined pre-treatment. The molecular weight distribution and chemical structure analyses revealed the breakdown of soluble macromolecules with the combined pre-treatment caused by the deamination and oxidation of the typical functional groups in proteins, polysaccharides and phosphodiesters. These changes likely improved the biodegradability of WAS.