Identification Of Bacteria For Renovating Of Soil With Lead And Selection Breeding By Protoplast Fusion Technique

Lead pollution in soil has been a serious threat to human health,Remediation of lead by microorganisms is one of the methods to control soil lead pollution at present.We can select excellent strains with protoplast fusion technolngy.Therefore,this research will perform lead resistant ability of CGMCC13007 and repair soil lead pollution ability of CGMCC5515 for protoplast fusion.The aim of this work was to screen fusants with the ability to tolerate lead and the ability to repair lead by protoplast fusion.The related resulted are as follows:Firstly this paper identifies the two strains of resistant lead which have been screened: by culturing characteristics and morphological observation,physiological and biochemical characteristics and identification of genetic characterization,we can analyze the two strains of bacteria were Bacillus cereus and Bacillus subtilis.Then we determine the conditions of preparation and fusion of protoplasts: lysozyme concentration was 4 mg/mL,enzymolysis temperature was 36 ℃,enzymolysis time was 60 min,the concentration of PEG 6000 was 36%,the fusion conditions was the ice bath 5min,protoplast formation rate can reach 68% and 73% respectively,the regeneration rate reached 23.73% and 58.23%.The conditions of screening fusion: containing 1mg/L gentamicin,1 mg/L rifampicin and 1mg/L TTC the high regeneration medium for preliminary screening and the second screen with 1800mg/L lead hypertonic medium.Finally we obtained four regenerated fusants and were inoculated with 1mg/L gentamicin,1mg/L rifampicin,1 mg/L TTC and 1800 mg/L nutrient agar plate for lead,but all of them were not successful.Maybe they are heterokaryon.Finally,we confirm some conclusions that the optimum concentration of lead in soil seed germination of Chinese cabbage was 800 mg/kg,the germination rate was 50%;seed soaking with these two strains of bacteria in lead soil on the germination rate were 65% and 78.5%;graphite furnace atomic absorption spectrometry analyses that the of mixed these two strains of bacteria can make the soil containing 500 mg/kg reduce to 46.48 mg/kg.And the soil reaches to the secondary standard according to the soil national standard <GB15618-1995>.