Mechanism of action of Free Nitrous Acid (FNA) on nitrifiers​​​​​​​

Abstract

The biocidal agent Free Nitrous Acid (FNA) exerts a broad antimicrobial effect on bacteria, although susceptibility to FNA varies considerably among the microorganisms that have been studied. This has led to its usage in a broad range of applications for wastewater treatment processes (WWTP), such as the control of microbially induced sewer corrosion to enhanced biodegradability of activated sludge as well as achieving reduced N2O production in the activated sludge process. Once inside the cell FNA dissociates to form various reactive nitrogen and oxygen species (RNS and ROS) that have been speculated to enhance the toxicity of FNA although the chemistry of how this happens has not been well characterised. These free radicals can cause direct oxidative damage to cellular proteins, membrane and wall components, and nucleic acids. Furthermore, FNA has been hypothesised to act as a protonophore by collapsing the proton membrane potential and thereby inhibiting ATP production.Among nitrifiers found in activated sludge of wastewater treatment processes (WWTP), nitrite oxidising bacteria (NOB) are more susceptible to FNA compared to ammonia oxidising bacteria (AOB). However, the molecular mechanisms governing this atypical tolerance of AOB to FNA are yet to be understood. The sensitivity of NOB to FNA is unexpected, as the NOB population generally has more genes that code for nitrite detoxification enzymes compared to AOB.I hypothesise that AOB uses a potent innate stress response mechanism different to known nitrite detoxification pathways for persistence to FNA. The lack of persistence mechanisms within NOB to FNA treatment is hypothesised to be due to a lack of an innate, robust stress response mechanism. Additionally, it is hypothesised that FNA targets multiple sites within a microorganism possibly through reactive intermediates.This thesis studied the varying effects of the antimicrobial FNA on activated sludge mixed cultures containing AOB and NOB using an integrated metagenomics and label free quantitative metaproteomic approach. My research shows evidence that FNA exhibits a strong oxidative stress response on bacteria. The Nitrosomonas genus of AOB on exposure to high levels of FNA maintains internal homeostasis by upregulating a number of known oxidative stress enzymes such as pteridine reductase, S-adenosylmethionine synthase and cytochrome c551 peroxidases. The denitrifying enzyme nitrite reductase was also upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Additionally enzymes involved in energy generation were upregulated on FNA exposure suggesting a higher energy requirement within Nitrosomonas to detoxify FNA. The heightened expression of DNA and protein repair enzymes provided evidence for FNA damage on DNA and proteins. Interestingly phage prevention proteins, which are associated with the activation of temperate phage were also upregulated suggesting an active suppression of lysis within Nitrosomonas. Reduction in iron bioavailability within Nitrosomonas was observed after exposure to FNA. Nitrosomonas coped with the lack of internal cellular iron by upregulating iron transport proteins such as the ABC iron transporter and siderophore transport proteins.On the other hand the Nitrobacter genus of NOB was observed to have very poor stress response mechanisms to FNA toxicity, compared to Nitrosomonas. Limited proteins with oxidative stress functions were detected. Of the 9 proteins detected with known antioxidant functions, all of them were downregulated on exposure to increasing FNA concentration. Additionally, DNA and protein repair enzymes were not upregulated on exposure to FNA. Pathways involved in ATP generation were downregulated on FNA exposure. Proteins that are involved in ion/small molecule transport across the cell membrane were upregulated on exposure to FNA, suggesting strongly that FNA caused osmotic stress within Nitrobacter. In addition, enzymes involved in biofilm formation within Nitrobacter were downregulated on exposure to increasing FNA concentration suggesting a poor stress response by Nitrobacter.Overall findings from this thesis improved our understanding of mechanism of FNA action on bacteria in general and nitrifiers in particular. This thesis clearly underpins the mechanisms of persistence within the AOB Nitrosomonas that allow it to grow at high concentrations of FNA. Additionally, the sensitivity of NOB Nitrobacter to FNA was attributed to a poor stress response. This knowledge contributes positively towards the applications of FNA in sustainably improving the nitrogen removal process in WWTP, particularly nitrogen removal using the activated sludge process.