The Synergistic Effects Of Heavy Metal-immobilizing Bacterium Enterobacter Bugandensis TJ6 Combined With Calcium Polypeptides On Decreasing The Cadmium Uptake And Accumulation In Wheat And The Mechanism Underlying Passivation

Heavy metal-polluted soil remediation represents a large challenge for our society,the"producting while remediating"of moderately and mildly heavy metal-polluted cultivated land is an urgent research for food production and food security.Both domestic and international studies have shown that the in-situ passivation technology is the most primary approach currently to obatin safe agricultural products today.Due to the main drawbacks of many passivation technologies which may cause the new soil pollution,damage in soil structure and even inhibition of crop growth,biological in-situ passivation technology has become a new preferred method,but it is not easy to achieve stable passivation effect due to population advantages and environmental factors.Studies have found that it is better to combine microorganisms with other remediation technologies.Therefore,an efficient in-situ passivation microbial strain Enterobacter bugandensis TJ6 with ecological adaptability screened out fro m cadmium-contaminated soil in the main wheat producing area was used in combination with the protein polypeptide(calcium polypeptide,CPP)developed by our research group.The CPP not only has chemical passivation effect by its carboxyl and mercapto groups to precipitate heavy metal ions into metal salts but also all components can promote the growth of microorganisms and plants without residue and negative cumulative effect imposed over a long period of time.It is a new multifunctional passivator.To investigate the mechanism of TJ6 combined with CPP passivation of cadmium under solution conditions,the SEM,XRD,FTIR analysis were conducted.The changes of soil microecological population and the synergistic blocking mechanism under pot conditions and hydroponics conditions were studied through high-throughput and macro genome sequencing and the perspective of proteomics to the molecular mechanism on reducing cadmium uptake in wheat,respectively,so as to establish a new technical method for the passivation of cadmium polluted farmland.Specific studies are as follows:To investigate the diversity of culturable and heavy metal-immobilizing bacteria in wheat rhizosphere soil in high concentration of heavy metals and explore the microbial resources such as screening high effective passivation bacteria,three samples of wheat rhizosphere soil with different concentrations of heavy metals were collected from Xinxiang,Henan Province.The diversity of culturable bacterial and heavy metal-immobilizing bacteria in the rhizosphere soil of wheat was compared by the culturable separation technology and solution adsorption experiment.The results showed that the high concentration(25.3 mg kg^-1)of Cd(II)decreased the taxon and diversity of bacteria in wheat rhizosphere.?-Proteobacteria was the dominant genus in high concentration Cd(II)polluted soil,Acetobacter,Brevundimonas,Serratia,Arthrobacter and pseudoarchacter were the dominant genus.While Firmicutes was the dominant genus in low concentration Cd(II)polluted soil(0.6 mg kg^-1),and Bacillus was the dominant genus.Based on the ecological relationship between wheat rhizosphere microbial population and wheat,Considering the possibility of artificial culture and application in the future,a novel bacteria of Rhodobacter xinxiangensis TJ48^T and the other two strains of urease producing bacteria(Enterobacter bugandensis TJ6 and Bacillus megaterium HD8)screened by the ability of absorb cadmium/lead,tolerance to heavy metals,growth promotion and urease production from wheat rhizosphere heavy metal polluted soil.After test,the urease producing abilities of TJ6 and HD8 were 46.5 m S cm^-1 min^-1 OD^-1₆₀₀ and 33.2 m S cm^-1min^-1 OD^-1₆₀₀.In addition,these two urease producing bacteria had a physiological basis to promote the growth of crops by produce IAA,siderophore and ACC deaminase.The"wheat-strain"sand culture(Cd 3 mg L^-1)matching experiment results showed that compared with the control,inoculation with TJ6 significantly increased the dry weight of roots(26.1%)and shoots(47.8%)of the wheat and reduced the contents of Cd in roots(57.5%)and shoots(48.5%)of wheat.This indicates that TJ6 has good passivation effect on cadmium ions and promoting crop growth.It is screened from wheat rhizosphere and has biological safety and ecological adaptability.In order to study passivation mechanism of TJ6 and CPP immobilize Cd(II),the experiments of solution standing(Cd 3 mg L^-1)and soil culture(Cd 2.7 mg kg^-1)were carried out.Strain TJ6 has the ability to resist heavy metals such as Cd(500 mg L^-1),Pb(2300 mg L^-1),Cu(500 mg L^-1)and Zn(900 mg L^-1).The treatments of TJ6 and TJ6+CPP significantly reduced the content of Cd(73%-83.7%)and increased the p H(from 7.01 to8.02-8.25)and NH₄⁺(4.16-5.82 fold)of the solution.In addition,compared with TJ6,TJ6+CPP had a stronger ability to remove Cd in solution.SEM,FITR and XRD showed that TJ6 and TJ6+CPP reduced the content of Cd in the solution through cell wall adsorption,intracellular enrichment and induced precipitation of Cd CO ₃,and induced mineralization precipitation of Cd CO₃ was the main way to immobilize Cd for TJ6 and TJ6+CPP.Based on the wheat growing in soil e nvironment and passivation efficiency of TJ6+CPP combination in solution condition,soil static experiments with TJ6 and TJ6+CPP were incubated for 30 days.The results showed that TJ6+CPP reduced the exchangeable content of Cd(41.5%)and increased the carbonate bound and residual fraction contents of Cd(33.8%-26.7%)in soil.In addition,TJ6+CPP significantly increased the number of urease-producing bacteria and p H(from 6.42 to 7.34)in soil(conducive to the alkaline passivation of cadmium ions in so il through hydrolysis to hydrogen and then to oxidation).It also showed that TJ6 combined with CPP had a biochemical basis and synergistic effect for passivating cadmium ions.Based on the conventional planting background of wheat,the synergistic passivation of TJ6+CPP on cadmium uptake by wheat and the regulation of soil microbial population structure and mechanism were studied through the pot experiments(Cd 0 mg kg^-1,Cd 1 mg kg^-1 and Cd 3 mg kg^-1).The results showed that the treatments of TJ6,CPP and TJ6+CPP significantly(P<0.05)increased the biomass(18.2%-45.9%)and reduced the Cd(II)content(17.2%-65.3%)in grains,roots and shoots of wheat.TJ6+CPP had a better effect on increasing the biomass of whea t grain and reducing the Cd accumulation in wheat grain(0.17 mg kg^-1)lower than the national food safety standard of 0.2 mg kg^-1(GB 2762-2017)in the Cd concentration(1 mg kg^-1)soil,while the control is 0.49 mg kg^-1 and more than twice the legal limit of food safety standard.Through the study of the effect of CPP,TJ6 and TJ6+CPP on soil microecology regulation.All treatments significantly(P<0.05)reduced the DTPA content of Cd(17.6%-60.9%)in wheat rhizosphere soil by increasing p H value(from6.72 to 7.02-7.16)and organic matter content(12.3%-50.2%),as well as reducing the aggregate contents with larger particle size and increasing the aggregate contents with smaller particle size in rhizosphere soil.In addition,each treatment could significantly improve the urease activity(42.5%-79.6%),the proportion of urease-producing bacteria,the content of ure C gene,the content of NH₄⁺-N and NO₃^--N,and the ratio of NH₄⁺/NO₃^-in the rhizosphere soil.High throughput sequencing of 16S RNA showed that the treatment of CPP had no significant(P>0.05)effects on the microbial community diversity of wheat rhizosphere soil,while the treatments of TJ6 and TJ6+CPP significantly(P<0.05)increased the abundance of chlorofloxi,cyanobacteria and verrucomicrobia,and reduced the abundance of proteobacteria and actinobacteria.Macrogenomic sequencing showed that the treatments of TJ6 and TJ6+CPP improved the amino acid synthesis pathway,quorum sensing level,aspartic acid and glutamic acid metabolism and glycine metabolism of microorganisms and decreased the metabolism of amino acid,serine.The threonine decreased purine metabolism and oxidative phosphorylation by increasing the abundance of Gemmatimonas,Opitutus,Arenimonas and Arthrobacter in rhizosphere soil and decreasing the abundance of Sorangium,Nocardioides,Lysobacter and Actinoplanes.It is that the synergetic microecological regulation to reduce Cd accumulation in wheat.To further reveal the mechanism of TJ6+CPP on reducing cadmium uptake by wheat roots and the molecular mechanism of reducing cadmium transport from wheat roots to the aboveground parts through the unlabeled quantitative proteomics technology by hydroponics experiments excluding the influence of soil microecology.The results showed that under Cd stress(1 mg L^-1),compared with the control,the treatments of TJ6,CPP and TJ6+CPP significantly(P<0.05)increased the root biomass(7.5%-31.3%)and shoot biomass(15.5%-72.1%)of wheat,and reduced the roo t and shoot Cd(II)content(11.4%-50.9%)of wheat.The ability of the treatment of TJ6+CPP to promote growth and reduce Cd absorption of wheat was higher than that of the treatments of CPP and TJ6.In addition,TJ6 and TJ6+CPP significantly(P<0.05)increased the activities of SOD(42.7%-67.8%)and POD(75.6%-151%)in wheat leaves.GO analysis of differential protein showed that strain TJ6 improved the activity of peroxidase and oxidoreductase in wheat roots,improved the level of DN A replication a nd protein translation,protected the DN A of wheat roots from damage,and then improved the resistance and tolerance of wheat to heavy metals.On the other hand,strain TJ6 reduced the activity of Cd transporter by reducing the activity of MAP kinase and transaminase in wheat roots and the expression of S-adenosylmethionine,which catalyzed the formation of S-adenosylmethionine from methionine and ATP.The treatments of CPP increased the activities of peroxidase and oxidoreductase in wheat roots.Then,it b locked the absorption of Cd by changing the structure of wheat root membrane and the transporter in root cells.The treatment of TJ6+CPP could protect DNA and chromatin of wheat root from damage by improving DNA replication and protein translation of wheat root,and then improve the resistance and tolerance of wheat to heavy metals.KEGG analysis of differential protein showed that the treatments of CPP,TJ6 and TJ6+CPP increased jasmonic acid,abscisic acid,peroxidase,catalase,glutathione,malate dehydrogenase and isocitrate dehydrogenase through enhancing alpha-Linolenic acid metabolism,Glycolysis/Gluconeogenesis,Metabolism of xenobiotics by cytochrome P450 and Plant hormone signal transduction,and then inhibited Cd accumulation in wheat roots.