Use of free nitrous acid from partial nitrification reactor for the sanitization of digester effluents and Class A biosolids production

Free ammonia-free nitrous acid based partial nitrification in sequencing batch membrane aerated biofilm reactor.

Kinetic and stoichiometric characterisation of enriched Nitrosomonas and Nitrobacter cultures by decoupling the growth and energy generation processes

We investigated the effects of free ammonia (FA) and free nitrous acid (FNA) concentrations on the predominant ammonia-oxidizing bacteria (AOB) and the emission of nitrous oxide (N2O) in a lab-scale sequencing batch reactor for partial nitrification. The reactor was operated with stepwise increases in the NH4+ loading rate, which resulted in a maximum FA concentration of 29.3mg-N/L at pH 8.3. Afterwards, FNA was increased by a gradual decrease of pH, reaching its maximum concentration of 4.1mg-N/L at pH 6.3. Fluorescence in situ hybridization indicated that AOB remained predominant during the operation, achieving specific nitrification rates of 1.04 and 0.99g-N/g-VSS/day at the highest accumulations of FA and FNA, respectively. These rates were in conjunction with partial nitrification efficiencies of >84%. The N2O emission factor of oxidized NH4+ was 0.90% at pH7.0, which was higher than those at pH 8.3 (0.11%) and 6.3 (0.12%), the pHs with the maximum FA and FNA concentrations, respectively. High-throughput sequencing of 16S ribosomal RNA genes showed that increases in FNA drastically changed the predominant AOB species, although increased FA produced no significant changes. This study demonstrates that the FNA concentration and pH are the main drivers that determine the predominant AOB species and N2O-emission in a partial nitrifying bioreactor.

Insights on biological phosphorus removal with partial nitrification in single sludge system via sidestream free ammonia and free nitrous acid dosing

Effect of free ammonium and free nitrous acid on the activity, aggregate morphology and extracellular polymeric substance distribution of ammonium oxidizing bacteria in partial nitrification

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 mg/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 mg 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 mg 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.

Accelerating enrichment of ARGs and MGEs with increasing ammonium removal during partial nitrification treating high-strength ammonia wastewater.

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.

Intelligent FA/FNA alternating strategy for nitrite-oxidizing bacteria inhibition: Data-driven prediction and process control.

Hydrogen sulfide produced by sulfate-reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide-induced concrete corrosion. Free nitrous acid (FNA) was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, details 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 the 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 the transcript levels detected during exposure. The removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterized hybrid cluster protein, and 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 oxidizing, and there was evidence of oxidative stress. Based on an interpretation of the measured responses, we present a model depicting the antimicrobial effects of FNA on D. vulgaris These findings provide new insight for understanding the responses of D. vulgaris to FNA and will provide a foundation for optimal application of this antimicrobial agent for improved control of sewer corrosion and odor management.IMPORTANCE Hydrogen sulfide produced by SRB in sewers causes odor problems and results in serious deterioration of sewer assets that requires very costly and demanding rehabilitation. Currently, there is successful application of the antimicrobial agent free nitrous acid (FNA), the protonated form of nitrite, for the control of sulfide levels in sewers (G. Jiang et al., Water Res 47:4331-4339, 2013, http://dx.doi.org/10.1016/j.watres.2013.05.024). However, the details of the antimicrobial mechanisms of FNA are largely unknown. In this study, we identified the key responses (decreased anaerobic respiration, reducing FNA, combating oxidative stress, and shutting down protein synthesis) of Desulfovibrio vulgaris Hildenborough, a model sewer corrosion bacterium, to FNA exposure by examining the growth, physiological, and gene expression changes. These findings provide new insight and underpinning knowledge for understanding the responses of D. vulgaris to FNA exposure, thereby benefiting the practical application of FNA for improved control of sewer corrosion and odor.

Low alkalinity, free ammonia, and free nitrous acid cooperatively stabilize partial nitrification under excessive aeration condition

Achieving efficient nitrogen removal from real sewage via nitrite pathway in a continuous nitrogen removal process by combining free nitrous acid sludge treatment and DO control

Producing free nitrous acid – A green and renewable biocidal agent – From anaerobic digester liquor

Qualitative and quantitative analysis of extracellular polymeric substances in partial nitrification and full nitrification reactors

Evaluating the effects of nitrogen loading rate and substrate inhibitions on partial nitrification with FISH analysis