Isolation Of Biofilm-Producing Bacillus Strains And They Reduced Cadmium Content Of Leafy Vegetables And The Mechanisms Involved

The degree of heavy metal pollution in cultivated land from industrial and agricultural activities is deepening in China,and the pollution of cadmium(Cd)is particularly serious.The vegetable land is a kind of agricultural soil with high intensity and great influence on humans.The excessive accumulation of Cd in vegetables grown in Cd-contaminated vegetable fields not only causes the vegetables to reduce production,but also poses a great threat to human health.The passivation and remediation technology of heavy metal contaminated soil can repair with production.It has strong operability,and has received much attention in recent years.Some rhizosphere bacteria can colonize well on the root of plants,and their extracellular polymers and cells can effectively adsorb heavy metals.We screened the biofilm-producing Bacillus spp.to study their effect and mechanisms involved in promoting vegetable growth and reducing Cd uptake of vegetables,providing a theoretical basis and technical approach for the remediation of heavy metals-contaminated vegetable fields and the safe production of vegetables.89 Cd and Pb resistant bacterias were screened from the rhizosphere soil of Chinese cabbage contaminated by heavy metals.Among them,7 strains could produce biofilm.Bacillus velezensis B9 and B25,B.subtilis B12,and B.siamensis B36 have the strongest ability of biofilm formation,Cd fixation in solution,and producing IAA,siderophore and urease.Under hydroponic conditions,compared with the non-inoculated control,the strains B9,B12 and B25 could significantly increase the biomass of above-ground tissues of Chinese cabbage(41%-95%),reduce the Cd content of above-ground tissues(43%-73%),and colonize the roots of the Chinese cabbage and form biofilms.Cd contaminated farmland soil in Liuhe,Nanjing were used as test soils.Strains B9,B12 and B25 were used as test strains.The culture experiment was carried out to study the effect and mechanism of inoculation treatment on growth and reducing Cd content of Chinese cabbage(Brassica campestris L)and Leaf lettuce(Lactuca sativa).Compared with the non-inoculated control,the inoculation treatment significantly increased the above-ground tissues biomass of Chinese cabbage by 63%-93%,and strains B12 and B25 significantly increased the above-ground tissues biomass of Leaf lettuce by 65%and 92%,respectively.In present of strains B9,B12 and B25,the Cd contents in the above-ground tissues of Chinese cabbage were significantly reduced by 23%-48%,and the Cd content of the above-ground tissues of the Leaf lettuce were significantly reduced by 25%-39%.The tested strains could colonize the roots of two vegetables and form biofilm-like structures.Moreover,inoculation of strains B9,B12 and B25 could significantly increase the proportion of biofilm-forming bacteria,soil polysaccharide content,sucrase and urease activity in rhizosphere soil,and also significantly increase the pH of rhizosphere soil,ultimately decrease the available Cd content in soil.The mutant strain(GFP gene marker)with enhanced(?ywcC)and attenuated(?slrR)ability of biofilm formation from strain B12 was constructed by homologous reorganization.Compared with the wild strain,the Cd immobilization ability of strain B12?slrR was significantly decreased and the motility was enhanced.The Cd immobilization ability of strain B12?ywcC was significantly improved,and the motility was significantly weakened.The Cd-contaminated farmland soils in Liuhe,Nanjing were used as tested soils.Culture experiments were carried out with strains B12,B12?slrR and B12?ywcC for further studying the important role of Bacillus spp.colonize the roots of vegetables and form biofilms for decreasing Cd content of Chinese cabbage and Leaf lettuce in soil.Compared with the dead bacterial-inoculated controls,strains B12 and B12?ywcC significantly increased the biomass of above-ground tissues of Chinese cabbage and Leaf lettuce by 79%-130%and 40%-98%,respectively.Stains B12 and B12?ywcC significantly reduced the Cd content of above-ground tissues of Chinese cabbage and Leaf lettuce by 35%-50%and 29%-39%,respectively,and many cells of the strains were observed to form different long-chain and cell cluster structures on roots of Chinese cabbage and Leaf lettuce,forming a distinct biofilm structure.The effect of the strain B12?slrR on reducing Cd content of above-ground tissues and roots of vegetables was weaker than that of other strains,and only sporadic root hair colonization.The inoculation treatment(B12 and B12?ywcC)could significantly increase the soil polysaccharide content,sucrase and urease activity and pH in rhizosphere soil,ultimately decrease the available Cd content in rhizosphere soil and Cd content of vegetables.Strains B12 and B12?ywcC reduced the Cd content of the above-ground tissues of Chinese cabbage to 0.043 mg·kg-1 and 0.032 mg·kg-1,respectively,reaching the limit of Cd 0.05 mg·kg-1 as defined by the WHO Food Safety Standard(CF/5 INF/1).