Study On The Nitrogen Removal Characteristic And Aerobic Denitrification Mechanism Of Bacillus Subtilis JD-014

The releasing of severe excessive amounts of nitrogenous substances into aqueous systems is one of the important reasons for the continuous degradation of the water environment.As a result,the ecological balance as well as the public health is being disrupted and threatened.Thus,how to effectively control the nitrogen pollution in water has become a significant challenge in the field of environmental management.Biological technology,which has been widely applied,has been proved to the most economical,efficient and environmentally friendly method for nitrogen removal treatment.Specially,isolated microbes with the capability of aerobic denitrification have shown great advantages and application potential for optimizing current bioremediation process.For these functional strains,a systematic analysis of the aerobic denitrification metabolic pathway and regulatory mechanism could further improve their nitrogen removal efficiency and provide important reference for the practical application of these kind of bacterium.In this study,on the basis of the isolated aerobic denitrifying Bacillus strain,techniques including genomic and transcriptomic were utilized comprenhensively to screen key genes involved in the nitrogen metabolism pathway.The gene function and also the regulatory mechanism of nitrogen removal were further verified and revelead through the molecular manipulation method.The main results and described as following:1.Four novel denitrifying Bacillus strains were isolated from different environmental samples.Based on 16S r DNA sequence alignment and phylogenetic analysis,they were initially identified as Bacillus pumilus JD-005,Bacillus subtilis JD-014,Bacillus paralicheniformis JD-016 and Bacillus subtilis JD-017.The four strains could efficiently degrade the different forms of nitrogen sources,such as nitrate,nitrite and ammonium.At the same time,they also exhibited good aerobic denitrification characteristics in multiple nitrogen coexistence.Bacillus subtilis JD-014,which showed the most efficient nitrogen removal performance,was chosen as the target strain to confirm the aerobic denitrification metabolic pathway by the evaluation of the production of gaseous nitrogen,the estimation of nitrogen balance and the analysis of the functional genes.2.The genome of strain JD-014 was successfully sequenced using the Pac Bio RS II,and the genome data had been uploaded to NCBI under the accession number CP045478-CP045479.The complete genome of JD-014 consists of one 4,215,417 bp circular chromosome and one 84,215 bp circular plasmid,with GC content of 43.51%and 35.11%,respectively.A total of 4324 protein coding genes(CDSs)were predicted on the genome,in which 4223 harbors on the chromosome and 101 on the plasmid.Through annotation and comparative analysis,there were 43 genes involved in the nitrogen metabolism pathway,among which 9 selective putative genes were related to denitrification process.By comparing JD-014 with the closely related strain B.subtilis 168,the homology of the denitrification functional genes between these two is 100%,but the nitrogen removal performance of them is quite different.The difference both in cell morphology during the denitrification process and the composition of the genome between JD-014 and strain 168 might contribute to their variance in denitrification capability.3.The candidate functional genes involved in the aerobic denitrification pathway of JD-014 were knocked out by the homologous recombination method that using the thermosensitive plasmid p NZT and FLP/FRT site-specific recombination system,and the complementation was also conducted to validate the function of them.The results showed that the NO₃^--N removal efficiency decreased by 12.72-31.64%after knocking out the 4 candidates encoding the nitrate reductase,For the other two putative genes encoding nitric oxide reductase and two genes encoding nitrous oxide reductase,there was a decrease of 14.32-18.42%of N₂production observed for three of these four deletion mutant strains,while one candidate encoding the nitric oxide reductase was found to have no significant effect on the production of N₂.In addition,a new key gene nirsir encoding nitrite reductase was screened out in the nitrogen removal metabolism of JD-014.When the gene nirsir was knocked out,the mutant cannot degrade nitrite anymore.It should be noticed that the nitrite removal characteristic could be restored in the retro-complementation mutant,and it could also produce N₂ through aerobic denitrification.The removal efficiency of nitrite and the production of N₂ could be improved after further overexpression.The results indicated that nirsir was an important functional gene in the nitrogen metabolism of JD-014.4.A transcriptome analysis of JD-014 during the aerobic denitrification process was carried out and it was found that a total of 567 genes was differentially expressed during the nitrogen removal process induced by low concentration of nitrate,of which 349 genes were up-regulated and 218 genes were down-regulated.While in the process of removing nitrate with high concentration,more differentially expressed genes were found,with 599 up-regulated genes and 447 down-regulated genes.The analysis of DEGs related to the denitrification process suggested that the up-regulated DEGs involved in the ETC system and carbon metabolism could provide more electron and energy for the aerobic denitrification,while through the up-regulation expressed DEGs related to cell motility,JD-014 could have better environmental adaptability in the degradation process of nitrogen pollutants.Meanwhile,the aerobic dentification pathway would also be affected by the differential expression of genes that related with the membrane transport and transcription regulation.5.When incubating JD-014 with sodium succinate or sodium citrate as carbon source,C/N ratio of 10 to 25,temperature of 37?,shaking speed of 150 to 200 r/min,and salinity of less than 1%,the optimal aerobic denitrification performance of JD-014 could be achieved.Moreover,JD-014 also showed great tolerance to nitrite.Even when the initial NO₂^--N concentration was 200 mg/L,the nitrogen removal efficiency could still reach about 50%.In addition,a continuous reactor was set-up to investigate the performance of JD-014 in the treatment of artificial simulated wastewater.It was found that the maximum nitrogen removal efficiency of nitrate and nitrite was 98.91%and 81.99%,suggesting that there was great application potential of JD-014 in the large-scale nitrogen removal process.