| In recent years,with the continuous development of the economy,emissions of sanitary sewage and industrial wastewater had improved continuously,which caused the serious nitrogen pollution in the surface water.In cold region,the temperature of the surface water will remain between 0-2℃ for quite a long period in winter,which causes great difficulties for the traditional biological nitrogen removal process based on autotrophic bacterium to treat the nitrogen pollution in cold water.Compared with autotrophic bacterium,the heterotrophic bacterium can adapt various environments and has stronger tolerance to the low temperature.This study aimed to isolate a novel psychrotrophic heterotrophic bacterium with significant nitrogen removal capacity.We made a comprehensive investigation on the nitrogen ability of the strain.A deep research on the mechanism of the nitrogen removal capacity at low temperature was made through molecular biology techniques.Study on this novel bacterium will enrich the treatment methods for cold water with micro pollution,which has important significance for drinking water security in cold region.In this paper,a psychrotrophic nitrogen removal bacterium,named M-11,was isolated from the filter material.The filter material was from a pilot plant reactor which treat the nitrogen pollution of the cold water.The strain was identified through morphological characteristics,BIOLOG analysis and 16 S r DNA analysis.The effect of different environment factor for the nitrogen removal performance and cell growth of the strain was also investigated.Results showed M-11 belonged to the specie of Janthinobacterium.It could remove 98% ammonium in 35 h of the water with the initial concentration of 5 mg/L,meanwhile,1mg/L N2 O was generated during this process.Neutral and alkalescent environments were beneficial to the cell growth and nitrogen removal performance of the strain.Suitable kinds of carbon sources（glycerol and glucose）and high C/N ratio would obviously increase the nitrogen removal capacity of the strain.M-11 was a facultative anaerobe and the anaerobic environment would inhibit the cell growth and nitrogen removal capacity of the strain.The nitrification was almost stagnant under anaerobic condition and the assimilation had become the main way for ammonium removal.Different from the aerobic denitrification bacterium in previous reports,nitrite was not accumulated during the denitrification of nitrate under aerobic condition.Conversely,as the increase of the conversion rate of nitrate,an obvious accumulation of nitrite was observed during the denitrification of nitrate under anaerobic condition.Meanwhile,the production of N2 O increased obviously under anaerobic condition.Secondly,the whole genome of the strain Janthinobacterium sp.M-11 was sequenced.Results showed the length of the genome was 6,394,979 bp with the GC content of 62.36%.Results of gene annotation of COG,KEGG and GO showed the number and kind of the functional gene were both higher than other cold-resistant heterotrophic bacteria reported previously.Genome of M-11 contained 32 gene islands,which contained the genes of Tra C、Tra E and Tra L（encoding conjugation transfer protein）.Results of Genome collinearity analysis between M-11 and other five strains of Janthinobacterium in NCBI database showed that the genome of M-11 was more similar to the genome of Janthinobacterium svalbardensis.3 cold shock genes and 6 heat shock genes were found in the genome which would improve the adaptive capacity of M-11 for cold environment.Meanwhile,the genome of M-11 contained 18 antifreeze protein genes such like aspartic acid,glycine and methionine,which would reduce the catalytic energy of catalytic process.In that case,the activity of M-11 at low temperature further improved.Results of transcriptome at different temperatures showed that 122 genes were up-regulated and 58 genes were down-regulated with the temperature increasing from 2℃ to 25℃.Heat shock protein genes were up-regulated to prevent the coagulation of protein as the temperature increasing.Genes of chemotaxis expressed obviously under low temperature,which meant M-11 would transfer to the more suitable living environment to accelerate the cell growth and metabolism rate of the strain.Significant differences were found in expression of transport protein genes between 2℃ and 25℃,which indicated M-11 would change the transportation systems at low temperature to guarantee the material exchange capacity of the strain.When the temperature decreased from 25℃ to 2℃,proportion of unsaturated fatty acid and long-chain fatty acid in the cell membrane both increased.The change of fatty acid would reduce the melting point of the whole cell membrane which maintained the membrane fluidity and selective permeability of M-11 at low temperature.Base on the analysis of nitrogen metabolic pathway and the PCR result of amo A gene,the inhibition of anaerobic environment for ammonia monooxygenase was the primary cause for the stagnation of nitrification.Genes of periplasmic nitrate reductase（nap A）and membrane-bound nitrate reductase（nar G）were both amplified from M-11.Combined with the analysis of denitrification metabolic pathway,under anaerobic condition,the nar G gene would express preferentially and accelerate the denitrification rate of nitrate,which caused the accumulation of nitrite.Under aerobic condition,the denitrification enzymes of M-11 were inhibited by the dissolved oxygen.This inhibition would decrease the denitrification efficiency,which was the primary cause for the low production of N2O under aerobic condition.Finally,results of continuous flow-reactor showed M-11 would significantly increase the ammonium removal performance for the cold water.After running continuously for 80 days,the ammonium concentration of the reactor inoculated with M-11 decreased from 1mg/L to 0.6 mg/L,and the ammonium removal efficiency of reactor with no inoculation was only 8%.Results of microbial community structure showed Janthinobacterium became the predominant bacterium in the reactor.Meanwhile,the inoculation of M-11 increased the species richness and biomass,which enhanced the stability of microflora. |
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