Phylogenetic Changes And Functional Characteristics Of The Nitrifying Bacteria Under Copper Inhibition In Activated Sludge

Activated sludge process is currently one of the most widely used biological wastewater treatment processes in the world.Ascribing to the complexity of microbial community structures in activated sludge,the relationships between the nutrient removal performance and the shifts in bacterial community of the activated sludge system are largely unexplored.In this study,the distribution charateristics of copper(Cu)in activated sludge,and the effects of Cu on the nitrification performances in sequencing batch reator(SBR)and anoxic/oxic(A/O)reactor were investigated.Illumina high throughput sequencing and quantitative polymerase chain reaction(q-PCR)technologies were applied to analyze the dynamic changes in the community structures of overall microbes and nitrifiers and functional gene transcriptions in activated sludge.Quantitative analysis was conducted to investigate the correlations between the transcriptional levels of the functional genes and physiological metabolisms of nitrifiers.An A/O process with an external sludge return system was developed to enhance the nitrogen removal and sludge settleability,and the performance and microbes in the new system was investigated.The batch experiment showed that the intracellular Cu inhibited the respiration activities of the nitrifying bacteria and the inhibition fitted a saturation-type biological toxicity model.The intermittent Cu gradient loadings in the SBR revealed that the transcription of amoA and nxrB genes was sensitive to Cu exposure and positively correlated with the specific oxygen uptake rates of ammonia oxidizing bacteria(AOB)(sOURNH4)and nitrite oxidizing bacteria(NOB)(sOURNO2),respectively.According to the principal regression analysis model,the inhibitory impacts of Cu on nitrifying bacteria could be mainly attributed to intracellular Cu.The 13.0 mgCu/g·MLVSS Cu loadings impoverished microbial diversity and altered the microbial community.Zoogloea,Thauera and Dechloromonas(genera within the Rhodocyclaceae family of the β-proteobacteria class)were the dominant genera in the presence of Cu.The abundances of Unclassified bacteria at the phylum level increased substantially with increasing Cu loadings.The new local nitrifying bacterial 16 S rRNA database for sequence BLAST searches improved the resolution of nitrifiers.Nitrosomonas and Nitrospira were the predominant nitrifiers in the SBR.In the A/O experiment,it was found that the variations in transcriptional levels of amoA,nosZ,napA and nirS genes during the inhibition and recovery processes were similar: they first increased and then decreased.While,the transcriptional levels of nxrB and nirK genes kept decreasing.The changes of the overall microbial structures were different from those in the SBR test.AOB and NOB populations had different Cu tolerance concentrations.When the total concentration of Cu was 16.50 mg/L,the abundance of AOB decreased,but the abundance of NOB significantly increased.Furthermore,the recovery ability of AOB was stronger than that of NOB after being seriously inhibited by Cu.The A/O process with an external sludge return was found to have advantages in improving nitrification efficiency and inhibiting sludge bulking under inhibitory condition and low temperature condition.