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Nitrite-oxidizing bacteria (NOB) are undesirable in the anaerobic ammonium oxidation (anammox)-driven nitrogen removal technologies in the modern wastewater treatment plants (WWTPs). Diverse strategies have been developed to suppress NOB based on their physiological properties that we have understood. But our knowledge of the diversity and mechanisms employed by NOB for survival in the modern WWTPs remains limited. Here, Three NOB species (NOB01–03) were recovered from the metagenomic datasets of a full-scale WWTP treating duck breeding wastewater. Among them, NOB01 and NOB02 were classified as newly identified lineage VII, tentatively named Candidatus (Ca.) Nitrospira NOB01 and Ca. Nitrospira NOB02. Analyses of genomes and in situ transcriptomes revealed that these two novel NOB were active and showed a high metabolic versatility. The transcriptional activity of Ca. Nitrospira could be detected in all tanks with quite different dissolved oxygen (DO) (0.01–5.01 mg/L), illustrating Ca. Nitrospira can survive in fluctuating DO conditions. The much lower Ca. Nitrospira abundance on the anammox bacteria-enriched sponge carrier likely originated from the intensification substrate (NO2−) competition from anammox and denitrifying bacteria. In particular, a highlight is that Ca. Nitrospira encoded and treanscribed cyanate hydratase (CynS), amine oxidase, urease (UreC), and copper-containing nitrite reductase (NirK) related to ammonium and NO production, driving NOB to interact with the co-existed AOB and anammox bacteria. Ca. Nitrospira strains NOB01 and NOB02 showed quite different niche preference in the same aerobic tank, which dominanted the NOB communities in activated sludge and biofilm, respectively. In addition to the common rTCA cycle for CO2 fixation, a reductive glycine pathway (RGP) was encoded and transcribed by NOB02 likely for CO2 fixation purpose. Additionally, a 3b group hydrogenase and respiratory nitrate reductase were uniquely encoded and transcribed by NOB02, which likely confer a survival advantage to this strain in the fluctuant activated sludge niche. The discovery of this new genus significantly broadens our understanding of the ecophysiology of NOB. Furthermore, the impressive metabolic versatility of the novel NOB revealed in this study advances our understanding of the survival strategy of NOB and provides valuable insight for suppressing NOB in the anammox-based WWTP.

ABSTRACT Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications. IMPORTANCE Hypochlorite is used in vast quantities for water disinfection, killing bacteria on surfaces, and washing and whitening. In pools, spas, and other waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance.

Trace concentrations of heavy metals cause health and environmental hazards. Specifically, trace concentrations of iron induce biofilm formation which is of great concern in water systems. To remove the iron, protein–polycation–clay composites were designed based on the hypothesis that the adsorbed apo-ferredoxin (apo-mFd: mFd protein without the 2Fe–2S cluster) chelates iron. Fe2+chelation by apo-mFd protein was established by optical spectroscopy and gel electrophoresis. To reduce its biodegradation, apo-mFd was adsorbed to montmorillonite and these composites were characterized by X-ray diffraction, zeta potential and adsorption isotherms. The apo-mFd–montmorillonite did not chelate iron with high efficiency; however, when a polycation–apo-mFd complex was adsorbed to the clay, the protein retained its chelating characteristics and specific interactions of iron with the biocomposite were established. Results present the innovative tailoring of bioactive protein–polycation–clay composites which may be applicable in many fields, e.g., protein immobilization, drug delivery and water treatment. [ABSTRACT FROM AUTHOR]