Study On Nitrate Reduction Pathway And Mechanism Of Secondary Salinization Soil By Bacillus Megaterium

The nitrate-type secondary salinization in greenhouse culture is increasingly acute.It not only harms the growth of crops,but also threaten the safety of agricultural products.Utilizing the assimilation of microorganisms to reduce the nitrate content in soil can make nitrogen immobilized by soil and is environmentally friendly.Hence this method has great potential for remediation of soil secondary salinization.In this study,the nitrate reduction pathway,the mechanism of nitrate reduction,the rhizosphere colonization pattern and the rhizosphere microecology remediation of a previously selected Bacillus megaterium NCT-2was carried out.The results can provide scientific basis for the development and practical application of microbial inoculants used in remediation of nitrate-type secondary salinization soil.The conclusions were summarized as follows:?1?B.megaterium NCT-2 can grow in the medium with nitrate as the sole nitrogen source.In the logarithmic phase under aerobiotic culture condition,NO₃^—N was completely degraded,and a small amount of NH₄⁺-N generated.It indicated that dissimilatory nitrate reduction to ammonium?DNRA?pathway may be existed under aerobiotic condition,however,nitrate assimilation pathway was still the principal pathway of nitrate reduction by NCT-2.Under anaerobic static culture condition,NO₂^—N content decreased while NH₄⁺-N content increased after eliminating consideration of nitrate assimilation.It indicated that NCT-2 can use nitrite to produce ammonium and further suggested that the presence of DNRA in NCT-2.Under anaerobic culture condition,no N₂O generated,which is the intermediate product of denitrification.It revealed that denitrification pathway is not existed in NCT-2.?2?The gene encoding assimilatory nitrate reductase from B.megaterium NCT-2 was cloned and over-expressed in Escherichia coli.The optimum co-expression condition was obtained with E.coli BL21?DE3?and 0.1 mM IPTG for 10 h when expression was carried out at 20°C and 150 rpm in Luria-Bertani?LB?medium.The molecular mass of nitrate reductase was 87.3 kDa and 80.5 kDa for electron transfer and catalytic subunit,respectively.The purified recombinant enzyme showed broad activity range of temperature and pH.The maximum activity was obtained at about 35°C and pH 6.2,which was similar to the condition in greenhouse soils.Maximum stimulation of the enzyme occurred with addition of Fe³⁺,while Cu²⁺caused the maximum inhibition.The optimum electron donor was the combined use of methyl viologen,Na₂S₂O₄ and EDTA.Kinetic parameters of K_m and V_max were determined to be 670?M and 58 U mg^-1,respectively.?3?The gene encoding assimilatory nitrite reductase from B.megaterium NCT-2 was cloned and over-expressed in Escherichia coli.The optimum co-expression condition was obtained with E.coli BL21?DE3?and 0.1 mM IPTG for 10 h when expression was carried out at about 20°C and 150 rpm in Luria-Bertani?LB?medium.The molecular mass of its large and small subunit was 88 kDa and 11.7 kDa,respectively.The purified recombinant enzyme showed broad activity range of temperature and pH.The maximum activity was obtained at 30°C and pH 6.5,which was similar to the condition in greenhouse soils.Maximum stimulation of the enzyme occurred with addition of Fe³⁺,while Cu²⁺caused the maximum inhibition.The optimum electron donor was the combined use of methyl viologen and Na₂S₂O₄.Kinetic parameters of K_m and V_max were determined to be 3.1 mM and 5.2 U mg^-1,respectively.?4?B.megaterium NCT-2 was marked with a green fluorescent protein?gfp?gene and was left to successfully colonize maize roots and the rhizosphere.Inoculation by gfp-tagged NCT-2 significantly promoted nitrate removal from the soil and improved plant growth.The laser scanning confocal results showed that NCT-2 was an endophyte that can colonize the meristematic and elongation zones of the root tip,and the middle segment of the root.Soil nitrate concentration had no significant effect on NCT-2 distribution.The gfp-tagged NCT-2 populations in the roots and rhizosphere soil first increased,but then decreased,and at the end of the experiment,colonization levels in the rhizosphere soil stabilized at⁵×10⁴ CFU g^-1 soil.However,the levels in the roots increased again to 1–3×10⁴ CFUg^-1 root in the different treatments.The NCT-2 population in the roots was significantly affected by nitrate content.A nitrate nitrogen of 72 mg kg^-1 was the optimum concentration for NCT-2 colonization of maize roots.?5?Sequencing of 16S rRNA gene amplicons from the maize rhizosphere soil revealed that the maize rhizosphere communities were dominated by Proteobacteria,Acidobacteria,Actinobacteria,Crenarchaeota,and Chloroflexi.The NCT-2 inoculants had no significant effect on the OTUs number and?diversity of rhizosphere bacterial community.Although the effect of nitrate addition on the OTUs number was insignificant,it significantly affected?diversity.Results of?diversity showed that inoculation with NCT-2 affected the bacterial community composition of maize rhizosphere microbiome and the composition changed along the nitrate gradient.The abundances of top 10 abundant phyla were significantly affected by NCT-2 inoculants.And NCT-2 inoculants also stimulated those genera involved in nitrogen transformation and antibiosis.Compared with the sterile NCT-2 treatments,the NCT-2 inoculants significantly decreased the electric conductivity and increased nitrate reductase in rhizosphere soil.Initial nitrate content,electric conductivity and nitrate reductase were closely related to Proteobacteria,Chloroflexi,Bacteroidetes,and Planctomycetes.These results indicated that NCT-2 inoculants significantly improved the physical-chemical properties and microbial diversity of rhizosphere soil.