Reactive nitrogen species from free nitrous acid (FNA) cause cell lysis

Free nitrous acid (FNA, i.e. HNO₂) has been demonstrated to have broad biocidal effects on a range of microorganisms, which has direct implications for wastewater management. However, the biocidal mechanisms still remain largely unknown. This study aims to test the hypothesis that FNA will induce cell lysis via cell membrane perforations, and consequently cause cell death via proteolysis, through the use of two model organisms namely Escherichia coli K12 and Pseudomonas putida KT2440. A combination of analytical techniques that included viability assays, atomic force microscopy (AFM), protein abundance assays and proteomic analysis using Quadruple-Orbitrap™ Mass spectrometry was used to evaluate the extent of cell death and possible cell lysis mechanisms. FNA treatment at 6.09 mg/L for 24 h (conditions typically applied in applications) induced 36 ± 4.2% and 91 ± 3.5% cell death/lysis of E. coli and P. putida, respectively. AFM showed that the lysis of cells was observed via perforations in the cell membrane; cells also appeared to shrink and become flat following FNA treatment. By introducing a reactive nitrogen species (RNS) scavenger to act as a treatment control, we further revealed that it was the nitrosative decomposition species of FNA, such as ^.NO that caused the cell lysis through the destruction of protein macromolecules found in the cell membrane (proteolysis). Subsequently, the RNS went on to cause the destruction of protein macromolecules within the cells. The death of these model organisms E. coli and P. putida following exposure to FNA treatment provides insights into the use of FNA as an antimicrobial agent in wastewater treatment.