Research On The Aerobic Denitrification And Cold Adaptation Mechanism Of A Psychrotrophic Bacillus Simplex

Along with the intensification of human activities and rapid development of industrialization,excessive nitrogen compounds are continually being discharge into various aqueous systems,posing a severe threat to both ecological equilibrium and humans’health.Therefore,how to effectively and sustainably remove nitrogen pollutants from the water bodies is an important challenge for environmental governance.Biological means is widely used for nitrogen removal by taking of its advantages of high efficiency,low energy consumption and environment friendly.The novel nitrogen removal technology,especially the discovery of microorganism with the ability of denitrification under aerobic conditions provided a new possible way for biological denitrification treatment and made it become a research hotspot in the field of wastewater treatment.However,in winter or for areas experiencing low temperature that have demand of biological nitrogen treatment,the growth and denitrification metabolism of the mesophilic denitrifying bacteria would be inhibited under such condition.Therefore,isolating cold-adapted denitrifying microorganisms and analyzing the aerobic denitrification and cold adaptation mechanism of the strain could provide a theoretical basis and technology support for better realizing efficient nitrogen removal in cold regions.In the study,a cold-adapted Bacillus strain with aerobic denitrification ability was isolated,then the cold tolerance mechanism of this functional strain during aerobic denitrification was explained in combination with experimental results obtained from cold adaptation and analysis of transcriptomic data.The function of the predicted candidate denitrification genes was verified and the nitrogen removal efficiency of the modified biochar immobilized this Bacillus was evaluated.The main results are as follows:（1）A cold-adapted bacterium strain,Bacillus simplex H-b,was isolated and identified with the potential to conduct heterotrophic nitrification and aerobic denitrification in the temperature range from 5 to 37°C.At 5°C,the removal efficiencies of NO₃^--N,NO₂^--N,and NH₄⁺-N were 40.07%,61.56%,and 73.83%,respectively,in 168 h.The strain also showed good removal capacity on the mixture nitrogen sources and the actual nitrogen contaminated sewage at low temperature.Moreover,by the determination of N₂ content,the estimation of nitrogen balance,the amplification of functional genes（nap and nor）and the characterization of key enzyme activities（HAO,NR,and NIR）,the specific denitrification pathway of strain H-b was confirmed as follows:NO₃^-～NO₂^-～NO～N₂O～N₂.At low temperature,strain H-b could achieve the better nitrogen removal performance under the condition with sodium succinate as carbon source,Na Cl concentration of 0-10 g/L,C/N ratio of 15-20,p H of 7-9,initial NO₃^--N concentration of 10-60 mg/L,shaking speed of 150-200 rpm and concentration of Cu²⁺,Zn²⁺,and Ni²⁺≤2 mg/L.（2）The whole genome of strain H-b consists of a circular chromosome with a total length of 5499613 bp.A total of 5706 coding genes were predicted and 33 genes that may be involve in the cold tolerance of strain H-b.Then transcriptomics and nitrogen removal characterization experiments were conducted at different temperatures（5°C,20°C,and 30°C）.The result indicated strain H-b mainly regulated the expression levels of genes encoding membrane transport,cofactor and vitamin synthesis,and transcriptional regulators to survive under cold stress.In addition,ATP（adenosine triphosphate）formation,utilization of nitrous substances,EPS（extracellular polymeric substances）formation,fatty acid composition,nucleotide precursor synthesis,translation,oxidative and temperature stress response mechanism were also adjusted.Overall,several regulatory strategies were adopted by strain H-b,which worked synergistically to resist the damage which caused by low temperatures.（3）Through sequence alignment and the analysis of protein conserved domains,25candidate denitrification genes were predicted in the genome sequence of strain H-b.Protoplast transformation method for this strain was established and then 25 gene knock strains were successfully constructed by the homologous recombination way.It was found that there was an obviously delay in the growth of H-bΔnap A2 at low temperature,meanwhile,the tolerance of complementation strains to low temperature has been restored somewhat.The above results suggested that nap A2 is an important gene for strain H-b to grow and perform denitrification at low temperature.The mutant strain with the knockout of cba A1 was unable to grow and remove nitrogen in the NO₃^--N and NO₂^--N media,while the utilization of inorganic nitrogen source and N₂ production by the denitrification pathway can be restored to some extent in the complementation strain,indicating cba A1 has an important function in the inorganic nitrogen metabolism of strain H-b.In addition,when the gene nor D,nor Q2,cba A2,cba A3,cba B1,nos F1,nos F3,nos F6,nos F02,nos Y1,and nos Y2 were knocked out,the N₂ production of these mutants through denitrification were reduced by 6.06-32.89%.（4）Four kinds of metal modified biochar were prepared by using walnut shell.Only zinc and iron modified biochar have the NO₂^--N adsorption performance and 29.13%and 92.44%of NO₂^--N could be removed from the solution respectively in 48 h.Based on the analysis of the adsorption characteristics and kinetics of the two kinds of modified biochar,iron modified biochar with higher NO₂^--N adsorption capacity used as the carrier to prepare the immobilized B.simplex H-b.The immobilized bacterial cells performed better NO₂^--N removal efficiency than that of the free cells and the modified biochar under the temperature of 5-37°C and the initial NO₂^--N concentration of 5-100 mg/L.Moreover,the complex which high biomass immobilized on the modified biochar could achieve higher NO₂^--N removal performance,especially in the temperature≤20°C or the initial NO₂^--N concentration≥40 mg/L.In addition,the immobilized bacterial cells could be reused in successive removal experiments,and the removal efficiency of NO₂^--N could reach 97.62%for the tenth cycle.