Urease Producing Bacteria Combined With Sheep Manure Organic Fertilizer Inhibit The Absorption Of Cd And Pb In Lettuce

With the rapid development of economy,environmental pollution is becoming more and more serious and the pollution of heavy metals(Cd and Pb)in vegetable fields is becoming more and more serious.As one of the essential foods in people's daily life,the quality and safety of vegetables had attracted more and more attention.Therefore,how to reduce the absorption of heavy metals in vegetable fields is an urgent problem to be solved.Microbial passivation remediation technology had attracted more and more attention because of its advantages of environment-friendly,low cost and low soil damage.Heavy metal immobilizing and plant growth promoting bacteria can not only reduce the absorption of heavy metals in vegetables,but also improve the nutritional quality of vegetables and soil quality.In our study,urease producing bacteria with Cd and Pb fixation ability were screened out from heavy metal contaminated vegetable rhizosphere soil by urease producing characteristics of microorganisms.The urease-producing bacteria and sheep manure organic compound fertilizer were prepared,and the fermentation and storage conditions were optimized.Finally,the mechanisms of compound bacterial fertilizer inhibiting the absorption of Cd and Pb in lettuce were studied by pot experiment.In this paper,16 villages in Nanyang suburb were selected to investigate the pollution status of vegetable soil and vegetables.At the same time,the land accumulation index method and Hakanson potential ecological risk index method were used to evaluate the heavy metal pollution of vegetable soil,and the single pollution index and Nemerow pollution index were used to evaluate the heavy metal pollution of vegetables.The results showed that,among the 16 sampling points,only the content of Cd in the soil sample of C5sampling point(0.32 mg kg^-1)exceeded the national screening value of soil pollution risk of agricultural land(0.3 mg kg^-1).The content of Pb was lower than 70 mg kg^-1,which is the national screening value of agricultural land soil pollution risk.The I_geo of Pb in vegetable soil in Nanyang suburb belonged to Grade I mild pollution,and about 37.5%of Cd belonged to Grade I mild pollution.The potential ecological risk index of Pb in soil belonged to low grade as a whole,while the potential ecological risk index of Cd at sampling points C5,C12 and C13 belonged to heavy grade.The contents of Cd and Pd in different vegetable sampleswere were leafy vegetables>solanaceous fruits>rhizomes,the comprehensive pollution degree were leafy vegetables>solanaceous fruits>rhizomes.Risk coefficient(HQ)and hazard index(HI)were less than 1,which will not caused harm to human body and affect human health.A total of 8 urease-producing functional strains were screened by urease screening medium in vegetable rhizosphere soil contaminated by heavy metals.Among them,strain CH5 had high urease-producing capacity and the removal rate of heavy metal Cd was more than 95%.The highest concentrations inhibiting its growth of Cd and Pb were 200 mg kg^-1and 2000 mg kg^-1.The iron carrier capacity of strain CH5 was++++,the IAA production capacity was 35.85 ms cm^-1min^-1,and the strain CH5 was resistant to ampicillin,kanacillin,chloramphenicol and streptomycin.The strain CH5 was identified belong to Enterbacter bugandensis.Strain CH5 was selected as the test strain,and the fermentation medium was screened.Response surface methodology was used to optimize the different components of the medium to determine the optimal ratio of each component of the fermentation medium.The results showed that the YN medium was the initial fermentation medium for the tested strain CH5,and its carbon and nitrogen sources were molasses and ammonium sulfate.The ionic components of magnesium sulfate and dipotassium hydrogen phosphate were retained.The optimal content of the medium was designed by response surface methodology:molasses 0.0130(g L^-1),ammonium sulfate 0.245(g L^-1),magnesium sulfate0.162(g L^-1),sodium chloride 1.84(g L^-1),yeast extract 0.084(g L^-1)and dipotassium hydrogen phosphate 0.1473(g L^-1).The optimal fermentation conditions for strain CH5were p H 6.0,temperature 28?,inoculation amount of 1%,liquid loading amount of 20m L.The bacterial age was 15 h and the rotation speed was 180 rpm min^-1.The strain CH5was prepared with different organic fertilizers(cow manure,sheep manure and pig manure),and the preservation technology was studied.The highest number of bacteria in CH5+sheep manure bacteriums was 1.486×10⁸ CFU g^-1,and the number of bacteria was significantly reduced after 270 d storage.The p H value of sheep manure organic fertilizer was 7.41 weakly alkaline.Therefore,the choices of sheep manure organic fertilizer was the most suitable carrier for strain CH5 was considered comprehensively.The potted experiment results of lettuce showed that under the stress of Cd 0.5+Pb 5(mg kg^-1),compared with the control,CH5+sheep manure decreased the centents of Cd(34.7%?67.3%)and Pb(71.5%?75.3%)of lettuce.It also increased the dry weight(33.3%?71.4%)of lettuce roots and leaves.The centents of the protein,total soluble sugar,and Vitamin C of lettuce leaves were increased by 40.1%?44.9%and 189.5%.The contents of extractable Cd and Pb in soil acid decreased by 14.3%and 35.8%,the contents of reducible Cd and Pb decreased by 35.3%and 39.4%,the contents of oxidizable Cd and Pb increased by 18.6%and 45.3%,and the contents of residual Cd and Pb increased by 23.5%and 49.3%.The p H value(from 6.23 to 6.78)and the content of organic matter(120.0%)were increased in the presence of CH5+sheep manure.In addition,the activity of soil urease was reduced by 41.3%in the treatment of CH5+sheep.The ammonium nitrogen was increased by 28.0%,nitrate nitrogen was decreased by 4.0%,and the ratio of ammonium nitrogen to nitrate nitrogen was increased by 36.9%under the treatment of CH5+sheep manure.Under the stress of Cd 5+Pb 20(mg kg^-1),compared with the control,CH5+sheep manure decreased the centents of Cd(48.5%?85.6%)and Pb(60.1%?69.8%).It also increased the dry weight(42.9%?80.0%)of lettuce.The centents of the protein,total soluble sugar,and vitamin C were increased by 39.7%,40.8%and 66.7%.The contents of extractable Cd and Pb in soil acid decreased by 19.6%and 37.8%,the contents of reducible Cd and Pb decreased by 12.3%and 37.8%,the contents of oxidizable Cd and Pb increased by 18%and 41.2%,and the contents of residual Cd and Pb increased by17.9%and 34.7%.The p H value(from 6.25 to 6.88)and the content of organic matter(43.3%)in the lettuce rhizosphere soil were increased in the presence of CH5+sheep manure.In addition,the conten of ammonium nitrogen was increased by 73.6%,the conten of nitrate nitrogen was decreased by 4.8%,and the ratio of ammonium nitrogen to nitrate nitrogen was increased by 88.9% under the treatment of CH5+ sheep manure.