There are a number of applications in which free nitrous acid (FNA), the protonated form of nitrite (HNO2), is used as an antimicrobial agent due to its bacteriostatic and bactericidal effects on a range of microbes. However, details of the antimicrobial mechanisms of FNA are largely unknown. Such details are important for optimising the application of FNA and for understanding possible resistance to the agent.

The overall objective of this research is to elucidate the responses of various microbes such as Psuedomonas aeruginosa PAO1 (P. aeruginosa), Desulfovibrio vulgaris Hildenborough (D. vulgaris) as well as determining the responses of organisms directly in mixed culture activated sludge used in wastewater treatment. The project aims to determine the microbial molecular and physiological mechanisms that may contribute to tolerance and resistance to FNA exposure. The outcome of these studies will improve our understanding of microbial responses to FNA, and contribute towards improved control of sewer corrosion and odour management in wastewater treatment.

Another potential application of FNA is for control of nitrifying microorganisms in wastewater treatment systems. The activated sludge process is used in nitrogen removal from wastewater. The nitrifying bacterial community in the activated sludge of wastewater treatment processes contain the Ammonia oxidising bacteria (AOB) and the Nitrite oxidising bacteria (NOB). Nitrite is produced by AOB through the oxidation of ammonia. In a nitrifying bacterial community FNA exerts a stronger biocidal effect on the NOB than AOB. The reason for this difference in tolerance is unknown especially considering NOB has the molecular pathways/mechanisms by which it can tolerate higher nitrite concentrations. The removal of NOB from the activated sludge system is beneficial for wastewater treatment since it saves on running costs. We study the effects of FNA on nitrifying community i.e. both AOB and NOB on exposure to FNA using metagenomics and SWATH metaproteomics techniques.  


  • Australian Centre for Ecogenomics
  • University of East Angila
  • Université Du Luxembourg

Project Outcomes


  1. The effects of FNA were studied on the opportunistic pathogen P. aeruginosa, a well-studied denitrifier, model biofilm microbe, and environmentally and medically important microorganism. By applying different levels of FNA we revealed that the antimicrobial effect of FNA is concentration-determined and population-specific. It was seen that 0.1 to 0.2 mg N/L FNA exerted a temporary inhibitory effect on P. aeruginosa growth, while complete respiratory growth inhibition occurred at FNA concentration of 1.0 mg N/. FNA at 5.0 mg N/L caused complete cell killing and likely cell lysis. Differential killing of FNA in the P. aeruginosa PAO1 subpopulations was detected, suggesting intra-strain heterogeneity with regard to tolerance to FNA.

  2. To determine the antimicrobial mechanisms of FNA we are studying the response of P. aeruginosa using transcriptomics coupled together with a suite of physiological measurements.  These responses were detected in the absence and presence of an inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. Transcripts exhibited significant changes in abundance in the presence of FNA, 167 genes were up-regulated and 424 genes down-regulated. Our study reveals that respiration was severely depleted, carbon metabolism was reverted from the TCA cycle to pyruvate fermentation with acetate as the end product, and protein synthesis was significantly decreased. These findings improve our understanding of P. aeruginosa in response to the FNA stress, and provide insight for application of FNA as an antimicrobial agent.

  3. Hydrogen sulfide produced by the sulfate reducing bacteria (SRB) in sewers causes odour problems and asset deterioration due to the sulfide induced concrete corrosion. Recent research at the ACWEB demonstrates that FNA is an effective antimicrobial agent to alleviate hydrogen sulfide production in sewers. Consequently, it is of great importance to determine details of the antimicrobial effects of FNA on SRB for continued improvement of the application of this antimicrobial agent. We are examining the bacteriostatic/bactericidal effects of FNA on the model SRB D. vulgaris. Responses to FNA are being revealed by analyses of transcriptomic and proteomic analyses, along with a series of growth and physiological assays. The results imply that FNA exerts multiple antimicrobial effects, these included:

    1. Inhibition of growth and causing cell death.
    2. Severely lowered respiration and ATP production.
    3. Increased activity of nitrite removal by nitrite reductase and a potentially novel mechanism.
    4. Lowered ribosome activity.
    5. Oxidative stress.
    6. Damage to proteins.



  • Gao S., Fan L., Yuan Z., Bond P.L. (2014). The concentration-determined and population-specific antimicrobial effects of free nitrous acid on Pseudomonas aeruginosa PAO1. Applied Microbiology and Biotechnology, 99:2305-2312.
  • Gao S., Fan L., Peng L., Guo J., Agulló-Barceló M., Yuan Z., Bond P.L. (2016). Determining multiple responses of Pseudomonas aeruginosa PAO1 to an antimicrobial agent, free nitrous acid. Environmental Science & Technology. DOI: 10.1021/acs.est.6b00288.
  • Wang, D., Wang, Q., Laloo, A., Xu, Y., Bond, P.L., Yuan, Z., (2016) “Achieving Stable Nitritation for Mainstream Deammonification by Combining Free Nitrous Acid-Based Sludge Treatment and Oxygen Limitation.” Sci Rep.

Poster presentations

  • Gao S., Fan L., Peng L., Guo J., Yuan Z., Bond P.L. (2015). Global transcriptome response to the antimicrobial agent free nitrous acid (FNA) by Pseudomonas aeruginosa PAO1 via RNA sequencing. Poster presentation at 6th Congress of European Microbiologists (FEMS 2015), Maastricht, The Netherlands, 7-11th June, 2015.
  • Gao S., Fan L., Peng L., Guo J., Yuan Z., Bond P.L. (2015). Determining growth inhibition mechanisms of the antimicrobial agent free nitrous acid (FNA) on Pseudomonas aeruginosa PAO1. Poster presentation at 115th general meeting of American Society for Microbiology (ASM 2015), New Orleans, Louisiana, United States, May 30th – 2nd June, 2015.
  • Gao S., Fan L., Yuan Z., Bond P.L. (2015). The concentration-determined and population-specific antimicrobial effects of free nitrous acid on Pseudomonas aeruginosa PAO1. Best Journal paper (Advanced Water Management Centre) at Faculty of Engineering, Architecture and Information Technology (EAIT) postgraduate conference 2015, Brisbane, Australia. 10th June, 2015.
  • Laloo A E., Wang Q., Vanwonterghem I., Steen J., Hugenholtz P., Yuan Z., Bond P.L (2015) “ The muddle of metagenome binning from an enriched nitrifying community. “ Computational Biology and Bioinformatics Student Symposium (COMBINE), October 2015, University of Sydney.
  • Laloo A E., Wang Q., Vanwonterghem I., Steen J., Hugenholtz P., Yuan Z., Bond P.L (2015) “ The muddle of metagenome binning from an enriched nitrifying community. “ Australian Bioinformatics and Computational Biology Society (ABACBS) Conference, October 2015. Garvan Insitute, Sydney. 

Project members

Professor Zhiguo Yuan

Centre Dir & ARC Laureate Fellow
Australian Centre for Water and Environmental Biotechnology


  • Associate Prof. Paul Wilmes, Université Du Luxembourg
  • Professor Philip Hugenholtz, Australian Centre for Ecogenomics, University of Queensland
  • Professor David Richardson, University of East Anglia

Other members

  • Shuhong Gao
  • Andrew Laloo
  • Dr Qilin Wang