Development Of Microbial Inoculants For Enhancing Plants Extraction Of Heavy Metals

With the development of society,human demand for metal smelting,pesticide use and industrial manufacturing has increased,resulting in a large amount of heavy metals being discharged into the soil,which seriously threatens the environmental ecosystem and human health,and improper disposal methods further aggravate pollution.Therefore,remediation of soil contaminated by heavy metals has become an urgent problem.The traditional phytoremediation is an economical,effective and environmentally friendly method for remediation of heavy metal pollution.However,it is often limited by plant biomass and heavy metal accumulation capacity,resulting in low remediation efficiency.Therefore,The combined remediation of plant-microorganisms for heavy metal contaminated soil has attracted much attention in recent years.In order to solve the problem of low accumulation of heavy metals by hyperaccumulators,this study developed microbial commercial inoculants with high-efficiency remediate function.Aiming at cadmium-contaminated soil,the microbial strains strengthening Solanum nigrum extract cadmium screened in the laboratory in the early stage are used to optimize the fermentation conditions,establish a large-scale fermentation production process of strains and preparation processing technology.In addition,the application technology of microbial inoculants in the farmland environment polluted by heavy metals in Huanggu Village,Zhuzhou,Hunan Province was studied to form a field application standard.The plant growth promoting rhizobacteria PGP5（Bacillus sp.）,PGP41（Arthrobacter sp.）,PGP6（Pantoea sp.）and the composite strain PGP6+5,which were screened in the laboratory,and selected as experimental materials to explore the optimal medium components,ratios and the best freezedrying protective agent.What is more,they were cultured with high cell density,combined with Cichorium intybus L.,Linum usitatissimum L.and Phytolacca Americana L.to remediate heavy metal polluted fields in Zhuzhou,Hunan.Explore the effects of combined remediation of strains and plants.The experimental results are as follows:In order to determine the optimal culture medium components and freeze-drying protective agents of single strains PGP5.PGP6.PGP41 and the composite strain PGP6+5,single-factor experiments and response surface methodology were used to screen the carbon source,nitrogen source,inorganic salts of the medium and protective agents.The results show that the experimental model designed by Design-Expert software fits well with the actual situation.In order to maximize the response value（OD600）,the optimal medium for PGP5 was glucose,6.707 g/L;soy peptone,27.573 g/L;KH₂PO₄+MgSO₄,2.312 g/L.The optimal medium for PGP6 was fructose,15.057 g/L;soy peptone,30 g/L;KH₂PO₄+MgSO₄,2.64 g/L.The optimal medium for PGP41 was fructose,17.057 g/L;soy peptone,14.868 g/L;KH₂PO₄+CaCO₃,1.938 g/L.The best medium for the composite strain PGP6+5 was fructose,19.386 g/L;soy peptone,22.541 g/L;KH₂PO₄+CaCO₃,2.957 g/L.Compared with the initial fermentation medium,the OD600 value of the four strains optimized by response surface methodology has been improved by at least 46.56%.After obtaining the optimal culture medium for the four strains,they were separately scaled up and cultured,and it was found that the fermentation tank had a shorter fermentation period and higher product concentrations than the shake flasks.For the screening results of protective agents,the best freeze-drying protective agent for PGP5 and PGP41 are glucose and trehalose,the best freeze-drying protective agent for PGP6 are sucrose,and the best freeze-drying protective agent for composite strain PGP6+5 are glucose and sucrose.The survival rates were above 78%.In the field contaminated by heavy metals in Zhuzhou,Hunan,Cichorium intybus L.,Linum usitatissimum L.and Phytolacca Americana L.were treated with single strains PGP5,PGP41 and the composite strain PGP6+5 to verify the effects of these strains on plant biomass and heavy metal accumulation.The results showed that Cichorium intybus L.,Linum usitatissimum L.and Phytolacca Americana L.treated with these strains significantly increased plant height,fresh weight,dry weight,and the Cd and Zn concentration of heavy metals in the shoots compared with the untreated group.What is more,the composite strain PGP6+5 has the best effect on promoting the biomass and heavy metal absorption of Cichorium intybus L.and Linum usitatissimum L.in the shoots.Compared with the untreated group,the fresh weight of Cichorium intybus L.and Linum usitatissimum L.in the shoots treated with the composite strain PGP6+5 increased by 11.93 times and 2.07 times,respectively,and the cadmium concentration of shoots increased by 125.42%and 109.42%,respectively.Based on this,Two sets of field application schemes for Cichorium intybus L.composite strain PGP6+5 and Linum usitatissimum L.-composite strain PGP6+5 can be established.