Structural Changes in Cell-Wall and Cell-Membrane Organic Materials Following Exposure to Free Nitrous Acid

Previous studies demonstrate that free nitrous acid (FNA, i.e., HNO₂) is biocidal for a range of microorganisms. The biocidal mechanisms of FNA are largely unknown. In this work, it is hypothesized that FNA will break bonds in molecules found in the cell envelope, thus causing cell lysis. Selected molecules representing components found in the cell envelope were treated with FNA at 6.09 mg N/L (NO₂^– = 250 mg N/L, pH 5.0) for 24 h (conditions typically used in applications) to evaluate the hypothesized chemical interactions. Molecular changes were observed using analytical techniques including proton (¹H) nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization mass spectrometry (ESI-MS). It was found that FNA broke down a range of cell envelope molecules. The spectral data demonstrated that the FNA reactions proceeded via two general pathways. One consisted of electrophilic substitution, whereby the nitrosonium ion (NO⁺) was the reactive electrophile. The other was via oxidative reactions involving nitrogen radicals (e.g., •NO₂ and •NO) formed from the decomposition of FNA. We further revealed that it was HNO₂ that caused the breakdown, rather than the exclusive action of the acid (H⁺) or nitrite (NO₂^–) counterparts. The fragmentation of these representative cell envelope molecules provides insight into the biocidal effects of FNA on microorganisms.