Effects Of Stenotrophomonas Rhizophila JC1 And Its Extracellular Polysaccharide On Growth Of Medicago Sativa Under Pb²⁺ Stress

With the increasing attention to health and environmental protection issues,the remediation of soil polluted by different heavy metal is a hot research topic currently,and researchers are focusing on finding suitable and efficient methods to remediate soil heavy metal contamination.Pb is one of the main elements of heavy metal pollution in soil,and its toxicity is very strong,which can directly damage human brain cells,causing intellectual disability and elderly dementia.It was found that physical remediation and chemical remediation can easily bring secondary pollution and destroy the original ecological balance and soil structure.while the combined plant-microbial remediation can not only complement each other,but also improve the remediation effect of heavy metal soil pollution.This study used the bacteria-Stenotrophomonas rhizophila JC1 isolated from heavy metal contaminated soil and its metabolite extracellular polysaccharide（EPS）were used as research materials to study their effects on seed germination and seedling growth of alfalfa（Medicago sativa L）before and after Pb²⁺stress.The research results are as follows:1.After treating the 100 mg/L Pb²⁺solution with JC1 strain for 24 hours,the removal efficiency of Pb²⁺from the solution was as high as 79.8%.Based on the results of scanning electron microscopy,it was found that the surface of the bacterial changed from smooth to rough before and after treatment,and a large number of particles gathered on the surface of the strain,indicating that the strain has a good adsorption effect on Pb²⁺.But desorption occurs gradually with the increase of adsorption duration.At the same time,it was found that the metabolic product EPS of the strain had a high adsorption efficiency of 85.73%for 150 mg/L Pb²⁺solution at 4 hours,indicating that the strain and its EPS have great potential for heavy metal adsorption and removal.2.The study on the growth promoting ability of strain JC1 showed that it has the ability to produce indoleacetic acid（IAA）,siderophore,ACC（1-aminocyclopropane-1-carboxyl）deaminase,nitrogen fixation,cellulase,xylanase,protease formation of biofilm,and has strong natural resistance to ampicillin.Based on anti SMASH online analysis software,the genome of JC1 was predicted to contain two clusters of genes associated with siderophore synthesis,in which the genes iuc B and iuc D were responsible for regulating the biosynthesis of siderophore.By comparing the complete genome sequence of strain JC1 with antibiotic resistance genes in the CARD database,it was deduced that there were 39 antibiotic resistance genes in the genome,mainly through four mechanisms:efflux pumps that confers antibiotic resistance,antibiotic target modifications,antibiotic inactivation,and antibiotic resistance gene mutations,causing strain JC1 to develop ampicillin resistance.Among them,there were 24 genes encoding antibiotic resistance efflux pumps,accounting for 61.54%of the total resistance genes.3.The single factor optimization method,combined with Plackett Burman and Box Behnken experimental optimization methods,was used to optimize the fermentation medium components and fermentation conditions for EPS production by strain JC1.The strain was inoculated with 4%（V/V）inoculum in a medium composed of 7%（m/V）fructose,6%（m/V）yeast extract,0.8 g/L Na Cl,0.6 g/L Mg SO₄·7H₂O（p H 4）.When cultured at 30℃,200 r/min for 24 hours,the EPS yield reached 3.1292 mg/m L,and the sugar production rate is 61.60%,which is 5.09 times the sugar production rate before optimization.The monosaccharide composition and molecular weight distribution of EPS were determined by High Performance Liquid Chromatography（HPLC）analysis,which confirmed that EPS was composed of mannose,glucosamine,glucose and galactose,with the molar percentage of 12.20:1:22.29:1.68.Based on anti SMASH online analysis,it was predict that the genome of strain JC1contained two gene cluster related to EPS synthesis.By comparison with the data in the carbohydrate database CAZY,it was concluded that the strain JC1 contained a large amount of gene information related to the synthesis of sugar nucleotide precursors and glycosyltransferases,indicating that the strain had strong EPS synthesis ability.4.Pb²⁺can inhibit the seed germination and growth of alfalfa,and when the concentration was greater than 1000 mg/L,seed germination was completely inhibited.The germination rate,germination potential,seedling root length,and fresh weight of aboveground and underground tissues all showed a decreasing trend with the increase of Pb²⁺concentration.After Pb²⁺stress,seedlings will activate their own antioxidant system to resist the toxicity of Pb²⁺.When the concentration of Pb²⁺was less than 200 mg/L,superoxide dismutase（SOD）,peroxidase（POD）,and catalase（CAT）can remove excessive free radicals and H₂O₂ in the plant,and alleviating the stress of Pb²⁺on alfalfa.However,with the continues increase of Pb²⁺concentration,the content of soluble protein decreases,the antioxidant enzyme system was damaged,the POD and CAT activity decreases,and a large amount of H₂O₂accumulates in the plant,membrane damage intensifies,and the content of alondialdehyde（MDA）was increased,which affected the normal growth and metabolism of seedlings.Pb²⁺content in seedlings was determined by flame atomic spectrophotometer,it was found that the transfer and enrichment abilities of seedlings to Pb²⁺were the strongest at the concentration of 400 mg/L and 50 mg/L,respectively.5.In the absence of external nutrients,comprehensive analysis of changes in root length,fresh weight,soluble protein and antioxidant enzyme system of alfalfa seedlings showed that strain JC1 inhibited the normal growth of alfalfa seedlings.When the strain JC1 and Pb²⁺acted together on alfalfa seeds,the germination rate,germination potential,root length and fresh weight all showed a decreasing trend with the increase of the concentration of strain JC1,while the content of soluble protein,MDA and the activities of antioxidant enzyme showed an increasing trend.Compared with the changes in various indicators when Pb²⁺acted on the seeds,strain JC1 could alleviate the adverse effects of Pb²⁺on alfalfa growth.The chlorophyll content increased first and then decreased with the increase of bacterial solution concentration.When the OD₆₀₀ of the strain JC1 was greater than 0.8.Therefore,the high concentration of strain JC1could promote the photosynthesis of alfalfa.6.When EPS solution concentration≤5 mg/m L,the seed germination rate,germination potential,root length,fresh weight,soluble protein,and chlorophyll content of seedlings all increased,indicating that low concentrations of EPS solution（≤5 mg/m L）could promote seed germination,while high concentrations can inhibit its germination.When EPS and Pb²⁺acted together on seeds,compared to the changes in various indicators when Pb²⁺acted on seeds,the germination rate,germination potential,root length,fresh weight,soluble protein and chlorophyll content of the seeds increased,while the content of MDA,the activity of POD,SOD,and CAT decreased,which proved that EPS could alleviate the inhibition of Pb²⁺on seed germination,enhance seedling photosynthesis,and alleviate the toxicity of Pb²⁺to alfalfa seedlings.Furthermore,it was found that the transfer and enrichment ability of seedlings to Pb²⁺was consistent with the transport and enrichment results of seedlings to Pb²⁺under different concentrations of Pb²⁺stress.