There is great potential to use free nitrous acid (FNA), the protonated form of nitrite (HNO2), as an antimicrobial agent due to its bacteriostatic and bactericidal effects on a range of microorganisms. However, the antimicrobial mechanism of FNA is largely unknown. The overall objective of this thesis is to elucidate the responses of two model bacteria, namely Psuedomonas aeruginosa PAO1 and Desulfovibrio vulgaris Hildenborough, in wastewater treatment in terms of microbial susceptibility, tolerance and resistance to FNA exposure.
The effects of FNA on the opportunistic pathogen P. aeruginosa PAO1, a well-studied denitrifier capable of nitrate/nitrite reduction through anaerobic respiration, were determined. It was revealed that the antimicrobial effect of FNA is concentration-determined and population-specific. By applying different levels of FNA, it was seen that 0.1 to 0.2 mg N/L FNA exerted a temporary inhibitory effect on P. aeruginosa PAO1 growth, while complete respiratory growth inhibition was not detected until an FNA concentration of 1.0 mg N/L was applied. The FNA concentration of 5.0 mg N/L caused complete cell killing and likely cell lysis. Differential killing by FNA in the P. aeruginosa PAO1 subpopulations was detected, suggesting intra-strain heterogeneity. A delayed recovery from FNA treatment suggested that FNA caused cell damage which required repair prior to P. aeruginosa PAO1 showing cell growth.
To further understand the inhibitory mechanisms of FNA on the model denitrifier P. aeruginosa PAO1 in wastewater treatment, genome-wide transcriptome analyses, coupled with a suite of physiological detections were conducted. The responses of P. aeruginosa PAO1 were detected in the absence and presence of an inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. Respiration was likely inhibited as denitrification activity was severely depleted in terms of decreased transcript levels of most denitrification genes. As a consequence, the tricarboxylic acid (TCA) cycle was inhibited due to the lowered cellular redox state in FNA exposed cultures. Meanwhile P. aeruginosa PAO1 rerouted its carbon metabolic pathway from the TCA cycle to pyruvate fermentation with acetate as the end product to survive the FNA stress. Moreover, protein synthesis was significantly decreased while ribosomes were preserved. These findings improved our understanding of P. aeruginosa PAO1 in response to FNA.
Hydrogen sulfide produced by sulfate reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide induced concrete corrosion. FNA was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, knowledge of the antimicrobial mechanisms of FNA are largely unknown. Here we report the multiple-targeted antimicrobial effects of FNA on the SRB Desulfovibrio vulgaris Hildenborough by determining growth, physiological and gene expression responses to FNA exposure. The activities of growth, respiration and ATP generation were inhibited when exposed to FNA. These changes were reflected in corresponding transcript levels detected during exposure. Removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterised hybrid cluster protein since the genes coding for these proteins were highly expressed. During FNA exposure lowered ribosome activity and protein production were detected. Additionally, conditions within the cells were more oxidising and there was evidence of oxidative stress.
A sequential window acquisition of all theoretical mass spectra (SWATH-MS) quantitative proteomics investigation was performed to gain a comprehensive and systematic understanding of the antimicrobial mechanisms of FNA on D. vulgaris Hildenborough. Protein expression dynamics were determined when D. vulgaris Hildenborough was exposed to FNA concentrations of 0, 1.0, 4.0, and 8.0 μg/L for periods of 2, 8 and 12 h. Based on the interpretation of the measured protein changes the responses of D. vulgaris Hildenborough to different FNA levels over incubation time were revealed. During exposure to 1.0 μg N/L FNA, only the proteins involved in nitrite reduction (nitrite reductase and the poorly characterized hybrid cluster protein) showed obvious increased expression levels. In the presence of 4.0 and 8.0 μg N/L FNA, an increase of proteins levels for nitrite reduction was also evident. The abundance of proteins involved in the sulfate reduction pathway (from sulfite to hydrogen sulfide) and lactate oxidation pathway (from pyruvate to acetate) were firstly lowered by FNA at 8 h incubation, and then recovered at 12 h incubation. During FNA exposure, lowered ribosomal protein levels were detected while the total protein levels for viable cells remained constant. Additionally, there was evidence that proteins corresponding to the genes DVU0772 and DVU3212 play a critical role in defending oxidative stress caused by FNA.
The outcomes of this thesis advance our understanding of P. aeruginosa PAO1 and D. vulgaris Hildenborough in response to FNA, and contribute towards the potential application of this environmentally sustainable antimicrobial agent for improving wastewater treatment technologies, such as sewer corrosion control and odor elimination in wastewater treatment.