Mechanistic Study On Adaptive Response And Recovery Potential Of Nitrosomonas Europaea Under Metallic Nanoparticle Stress

Due to their unique physical and chemical properties,nanoparticles?NPs?have been widely used in the fields of daily-life,industry and scientific research.The increasing application of NPs will make them inevitably released into the municipal sewage water network.As a result,diverse microorganisms used for pollutant removal in biological wastewater treatment plants?WWTPs?are potentially threatened by the NPs due to their bio-toxicities.In this study,herein,biotoxicity effects of three typical metal nanoparticles?n-TiO₂?n-CeO₂ and n-ZnO?on a typical ammonia oxidizers-Nitrosomonas europaea were comprehensively investigated under different test conditions.The recovery capacities of the NP-impaired cells were also assessed.Genome-wide microarray techniques were further used for the exploration of the transcriptional regulations of the NP-impaired N.europaea cells,as well as the associated adaptive regulation and recovery mechanisms.Low concentrations?1 or 10 mg/L?of n-TiO₂?n-CeO₂ and n-ZnO did not exert obvious toxic effects on cellular metabolic activities after their long-term exposure,except that 10 mg/L n-ZnO resulted in a slight decrease in cell density and membrane integrity.High concentration?50 mg/L?of the three NPs all significantly inhibited the cell density,membrane integrity,ammonia oxidation performances and ammonia monooxygenase?AMO?activities.The cellular physiological activities were significantly inhibited by n-TiO₂ only after 7-d continuous exposure,and then gradually recovered after 40-d incubation,indicating that the n-TiO₂impaired cells had strong resistance and recovery potentials;when under stress of n-CeO₂ stress,the cellular activities reached a steady state around 12-d incubation and did not continue to decline,suggesting that N.europaea cells were tolerant to n-CeO₂ stress;while after n-ZnO's7-d exposure,the cells completely lost their metabolic activities,which indicated that n-ZnO was relatively much more toxic than n-CeO₂ and n-TiO₂ under the same test conditions.High concentrations of dissolved oxygen?DO?would help enhance cellular resistance/adaption capacities and recovery potentials under NP stress in this study.The different DO concentrations?0.5-2.0 mg/L?generally did not affect the long-term effects of 50mg/L n-ZnO and n-CeO₂ on N.europaea,and all the cellular physiological and metabolic activities were significantly inhibited.Nevertheless,the cells were more susceptible to n-ZnO,n-CeO₂ and n-TiO₂ stress under low DO conditions,and n-TiO₂ impaired cells required more time to adapt to and recover from the stress.Twelve-h Batch recovery test results indicated that when the ammonia removal rate was inhibited by 10%in the chemostat,the physiological and metabolic activities of 50 mg/L n-ZnO and n-CeO₂ impaired cells significantly recovered during the incubation time regardless of the DO levels.However,the high DO cultured cells displayed relatively higher recovery rate than that of low DO cultured cells.Therefore,high DO level was beneficial to cellular resistance or recovery from NPs stress.In addition,with the prolonged NP exposure time and increasing inhibitions on bacterial metabolic activities,n-ZnO finally displayed irreversible damage on cells under both low and high DO concentrations.Genome-wide microarray profiling results reflected that all the three NPs could interrupt cellular diverse biological metabolic pathways,which included:a)destroying cell membrane structure and interfering with cell membrane-related metabolic functions;b)destroying the structure of functional proteins and inhibiting enzymatic activities;c)affecting electron transfer chain and energy production/conversion;d)binding with genetic material and damage DNA/RNA structure.However,the expression levels and sensitivities of specific functional genes in different metabolic pathways varied from different kinds of NPs,which might be related to NPs'self-characteristics and bacterial damage extent.Moreover,the NP impaired cells could stimulate the transcriptional activities of various anti-toxic systems to resist NPs stress,such as toxin-antitoxin,reactive oxygen species quenching and toxicant efflux systems.Furthermore,various metabolic pathways regulations,such as osmotic equilibrium adjustment,aminoacyl-tRNA biosynthesis and DNA repair were actively involved with cellular adaption/recovery in response to NP stress.It was also found that the metal ion scavenging gene merPT were significantly up-regulated only under n-ZnO stress,supporting that the dissolved Zn²⁺released from n-ZnO was one of the key toxicity sources.The cytochrome maturation related genes ccmACEF,chemotaxis genes cheARW,and protein modification genes groEL/ES were dramatically up-regulated only under n-CeO₂ stress,suggesting that they might be specific stress response genes to n-CeO₂ stress.Moreover,the cell signal sensing genes NE0666/0667 and radical kinase encoding gene NE0221 were notably up-regulated only after n-TiO₂ exposure,indicating that these genes might be specific adaptive regulator under n-TiO₂ stress.Based on the transcriptional profiling,potential universal and specific biomarkers for NP contamination early warning and monitoring were preliminary screened.The universal biomarkers mainly include:respiratory chain regulator SCO1,G3P acyltransferase gene plsX,ammonium transporter gene Rh50,ribosomal protein gene rpmF,GTP-binding protein gene lepA and elongation factor cysN;The specific biomarkers mainly include:metallo-beta-lactamase superfamily gene NE2571?n-ZnO stress?,signal-sensing regulatory gene NE0667and glucose-methanol-choline reductase gene NE1237?n-TiO₂ stress?,and DNA structure modification gene NE2544,protein chaperonin gene groEL and putative ferredoxin protein gene NE1874?n-CeO₂ stress?.In summary,the toxicity effects of three typical NPs on N.europaea were extensively explored under different conditions?e.g.different NP or DO concentrations and NP exposure time?.The stress tolerance and recovery potentials of the NP impaired cells were assessed,as well as cellular adaptive response and recovery mechanisms using genome-wide expression profiling techniques.Besides,potential universal and specific biomarkers for indicating NP stress were preliminary screened.The findings of this study provide theoretical and technical supports for NP toxicity risk assessment,NP contamination control,and establishment of safety early warning monitoring system in the BNR system in response to potential NP stress.