Study On The Enrichment And Metabolic Pathways Of The Functional Bacteria With High Bromate Reducing Efficiency

Bromate is a disinfection by-product during the ozonation process of the water that contains bromide.It has been designated as a potential carcinogen by the International Agency for Research on Cancer(IARC).The removal of bromate can be conducted by physical,chemical and biological methods.The biological method is able to convert bromate into the non-toxic bromide ion by microorganisms with the organic matter or hydrogen as the electron donor.This method has the advantages of low energy consumption,no secondary pollution,and has a broad application prospect.However,in the study of microbial reduction of bromate,the diversity of bromate-reducing functional bacteria remains unclear,the bromate-reducing functional genes and metabolic mechanism are not yet uncovered,the correlation between the bromate reduction efficiency and functional gene expression and regulation are still need to be researched,these factors have restricted the development of biological removal of bromate technology and application.In this study,efficient bromate reducing bacteria were enriched and isolated from activated sludge of wastewater treatment plants(WWTPs)to explore the effects of different carbon sources on the diversity of bacteria and bromate reducing efficiency.The high-throughput sequencing based 16 S r RNA gene amplicon analysis and metagenomics were used to reveal the potential key genes and metabolic pathways for bromate reduction.In this study,activated sludge samples from Nanshan and Futian WWTPs in Shenzhen were collected.Inorganic carbon source(sodium bicarbonate)and organic carbon sources(acetate,glucose and lactate)were used as electron donors respectively to enrich bromate reducing bacteria in the anaerobic bioreactors with bromate as the electron acceptor.Among the inorganic carbon sources,the reduction efficiency of bromate from activated sludge samples from Futian WWTP was significantly higher than that from Nanshan WWTP.In the 8 h bromate reduction kinetics experiment,the reduction efficiency of the bacteria enriched from the activated sludge of Nanshan WWTP was 10.4%,and that of the bacteria enriched from the activated sludge of Futian WWTP was 69.9%.The results of metagenomic sequencing showed that there were significant differences in the structure of bacteria enriched in different kinds of activated sludge,in which the genera Tetrasphaera,Pseudomonas and Stenotrophomonas were the dominant species participating in bromate reduction.The analysis results of genomic metabolic pathways showed that the bacterial genomes belonging to the family Steroidobacteraceae,Chitinophagaceae and Sphingomonadaceae have carbon fixation key genes,which must be potential autotrophic bacteria and play a key role in the bromate reduction by using inorganic carbon sources.In the bromate reducing bacteria enriched with organic carbon sources as electron donors,the sequencing results of 16 S r RNA amplicons showed that the dominant bacteria enriched with different electron donors showed great differences.After 44 d enrichment,Enterobacteriaceae gradually dominated the microbial community in the glucose-fed bioreactor,while Campylobacteraceae and Rhodocyclaceae dominated in the acetate-and lactate-fed bioreactors,respectively.Two pure strains namely Glu3 and Lac1 belonging to Enterobacteriaceae,were isolated from the reactor using glucose and lactate as electron donors.Similarity comparison based on 16 S r RNA gene and whole genome sequences showed that Glu3 and Lac1 strains belonged to Klebsiella variicola and Raoultella electrica,respectively.Under 0.04 mmol/L of bromate exposure,the bromate reduction rate of strain Glu3 and Lac1 were 84.7% and 44.9% with glucose as the carbon source,while the reduction rates decreased to 14.1% and 27.7% with sodium lactate as the carbon source,respectively.The complete genomes of Glu3 and Lac1 strains were obtained by the combination of Illumina next-generation high throughput sequencing and Oxford Nanopore third-generation sequencing.The genome annotation suggested that both strains of Glu3 and Lac1 could utilize glucose through the glycolysis pathway and convert acetate to ethanol by fermentation.In addition,ethanol may also be utilized by both strains of Glu3 and Lac1,since the alcohol dehydrogenase(EC1.1.1.1)and the aldehyde dehydrogenase(EC1.2.1.-)encoding genes were identified in their genomes.However,the key enzyme of L-lactate dehydrogenase(EC1.1.1.27)was absent in the genome of strain Lac1 but present in the genome of strain Glu3.The results are consistent with the phenotype of bromate reduction efficiency of the two strains in different carbon sources.Glu3 has key functional genes for metabolizing glucose,acetate,lactate and ethanol in its genome,indicating that Glu3 has the potential to utilize a variety of carbon sources and has strong environmental adaptability.In the genomes of strains Glu3 and Lac1,genes belong to the dimethyl sulfoxide(DMSO)reductase family were predicted,including the alpha-subunit of nitrate reductase encoding gene nar G,the alpha-subunit of anaerobic dimethyl sulfoxide reductase encoding gene dms A,and the alpha-subunit of anaerobic selenate reductase encoding gene ynf E.Those genes are likely to be the key genes for bromate reduction.This study further revealed the biodiversity of bromate reducing bacteria,provided the bacterial strain and genomic information for further exploration and verification of the microbial metabolic pathway of bromate,and laid a theoretical foundation for the practical application and promotion of bromate reduction by biological method.