| In recent years,with the exploitation and smelting of mines,excessive use of pesticides and fertilizer,heavy metal contamination in farmland in China has become more and more serious.Rape is one of the main oil crops in China and is easy to accumulate heavy metals when it is grown in heavy metal-polluted farmland.Excessive accumulation of Cd and Pb in rapeseed not only causes reduced the yield,but also poses great threat to human health.Immobilization technology of heavy metal by bacteria is a realistic remediation strategy for the slightly metal-contaminated farmland soils.Plant growth-promoting bacteria can not only immobilize heavy metals,reduce the uptake of heavy metals by plants and improve the quality of the soil,but also promote plant growth.Immobilization of heavy metals by bacteria will be important in the remediation and safe production of crops in heavy metal-contaminated soil.In this paper,345 Cd-and Pb-resistant bacteria were isolated from the soils located at a lead and cadmium mining area in Qixia,Nanjing.Among the heavy metal-resistant bacteria,18 strains had the ability to produce IAA,siderophore,ACC deaminase and arginine decarboxylase.The effect of strains CL-1,X30,and H6 on the removal of Cd and Pb in the solution was evaluated.In the presence of Cd(5 mg L-1)and Pb(20 mg L-1)and strains CL-1,X30,and H6,the removal rates of Cd and Pb were 92.6-94.6%,89.2-85.3%%,and 81.2-84.3%,respectively.The effects of strains CL-1 and X30 on rape biomass and Cd and Pb accumulation were studied in solution.Strains CL-1 and X30 could promote rape growth(28.6-67.2%)and reduce Cd and Pb accumulation(15.9-59.6%)compared to the controls.16S rDNA sequence analysis showed that strains CL-1 and X30 belonged to Serratia liquefaciens and Bacillus thuringiensis,respectively.The biological characteristics and the effect of strains CL-1 and X30 on heavy metal adsorption were studied.Strain CL-1 is short rod shaped,resistant to Cd2+(700 mg L-1)and Pb2+(2400 mg L-1),and produced polysaccharides and arginine decarboxylase.Strain X30 is long rod shape,resistant to Cd2+(400 mg L-1)and Pb2+(1800 mg L-1),and produced biofilm,polysaccharide and arginine decarboxylase.Electron microscopy and infrared spectroscopy analyses showed that the cell surface of strains CL-1 and X30 could adsorb Cd2+and Pb2+.The functional groups such as OH-,N-H,C=O and P-O on the cell walls of strains CL-1 and X30 were involved in the adsorption of Cd2+and Pb2+.In the presence of Cd2+(50 mg L-1)and Pb2+(50 mg L-1),the intracellular Cd(55.5%)and Pb(19.2%)contents of strain CL-1 were significantly lower than those of strain X30.The contents of Cd(42.2%)and Pb(24.3%)on the cell walls of strain CL-1 were significantly higher than those of strain X30,indicating that strain CL-1 immobilized Cd and Pb mainly through the cell wall adsorption.Strain X30 immobilized Cd and Pb mainly through the cell wall adsorption and intracellular accumulation.Strain CL-1 can produce putrescine,while stran X30 can produce spermine and spermidine.Strains CL-1 and X30 could increase solution pH(from 6.98 to 8.18-8.41)and reduce the concentrations of water-soluble Cd(48.3-86.9%)and Pb(43.2-81.7%).The effects of strains CL-1 and X30 on the growth and Cd accumulation in rape(Qinyou-10)in the Cd(0,1,and 3 mg kg-1)-added soils were studied in pot experiments.Under different Cd-added soils,strains CL-1 and X30 significantly increased the biomass(18.7-36.4%)of rape and reduced rapeseed Cd content(25.3-72.5%)of rape compared to the controls.Furthermore,strain CL-1 had significantly higher ability to increase the biomass(17.3-24.3%)and reduce the Cd content(20.4-34.9%)of rape compared to strain X30.The DTPA-extractable Cd content was significantly reduced(17.3-54.6%)in the rhizosphere soils in the presence of strains CL-1 and X30 compared to the control.The content of putrescine(98.6-105%)and the proportion(23.3-49.5%)of arginine decarboxylase-producing bacteria,and the pH in the rhizosphere soil were significantly higher in the presence of strain CL-1 compared to the controls.The content of spermine(82.6-103%),spermidine(91.1-99.1%)and the proportion(21.3-39.6%)of arginine decarboxylase-producing bacteria and the pH in the rhizosphere soil were significantly higher in the presence of strain X30 compared to the controls.The effect of strains CL-1 and X30 on the bacterial community compositions in the rhizosphere soils of rape was evaluated by Miseq next-generation sequencing of 16S rRNA genes.The results showed that strains CL-1 and X30 significantly increased the abundance of the bacterial community of the rhizosphere soil of rape under low Cd-added soil,but did not change the bacterial community compositions of the rhizosphere soil under high Cd-added soil.A field experiment was designed to study the effects of strain CL-1,X30 and the combination of these strains and biochar on the growth and heavy metal uptake of rape plants(Qinyou-10 and Fengyou-737).The results showed that strain X30 or CL-1 did not increase the yield of rapeseed,while the compound strains(X30+ CL-1)significantly increased the yield of rapeseed(28.4-31.2%)and these strains combined with biochar also significantly increased the yield of rapeseed(18.9-44.2%)compared to the control.Compared to the control,the single strain X30 or CL-1,the compound strains(X30+CL-1)and the strains combined with biochar could significantly reduce the accumulation of Cd(18.6-73.6%)and Pb(23.6-48.2%)in the rapeseed of Qinyou-10.In addition,the compound strains and the strains combined with biochar significantly reduced the accumulation of Cd(16.6-43.2%)and Pb(17.6-34.2%)in the rapeseed of Fengyou-737.In all treatments,the combined application of mixed strains and biochar was the best to increse the biomass and reduce the rapeseed Cd and Pb uptake of the rape plants.Compared to the control,the compound strains(X30+CL-1)and the combined application of mixed strains and biochar could decrease DTPA-extractable Cd(48.6-73.6%)and Pb(31.9-48.2%)contents of the rhizosphere soil and increased the proportion(25.8-42.4%)of the small aggregates,the content of organic matter(21.4-33.3%)and pH,thus reducing the accumulation of Cd and Pb in the rapeseed.Compared to the control,the compound strains(X30+CL-1)and the strains combined with biochar could significantly increase the pH of the rhizosphere soil of rape by increasing the content of polyamines(53.3-145%)and NH4+(108-304%),as well as the ratio of polyamines-producing bacteria(20.2-49.6%)and urease-producing bacteria(26.8-69.2%).By using the homologous recombination,we knocked out the aguA gene involved in putrescine production and the potE gene related to the transfer of putrescine in strain CL-1 to study the effect of the mutant strains on the uptake of Cd and Pb of rape(QY-10).The results showed that,after 48 h culture,the content of putrescine was decreased by 70.3%and 94.2%in the presence of strains CL-lAaguA and CL-lApotE,respectively.However,strain CL-1?potE also produced spermidine(2.2 mg L-1)and subspermidine(2.1 mg L-1)in the solution.The pH in the solution was lower in the presence of strains CL-1 ?aguA(7.75)and CL-1?potE(7.52)than in the presence of strain CL-1(8.53)and the complemented strains(8.49),indicating that strain CL-1 could increase pH in the solution through the putrescine production.Compared with the wild and complemented strains,the ability of strains CL-1?aguA and CL-1?pot to immobilize Cd and Pb was significantly decreased by 35.6-64.1%,indicating that the putrescine secreted by strain CL-1 increased its ability to immobilize Cd and Pb.Compared with the wild and complemented bacteria,the ability of strains CL-lAaguA and CL-lApotE to reduce Cd and Pb accumulation in the rape was significantly decreased by 56.2-66.6%and 54.3-155%,indicated that the putrescine secreted by strain CL-1 significantly increased its ability to reduce Cd and Pb uptake of the rape. |
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