Isolation Of A Vanadium Reducing Bacterium Enterobacter Kobei And The Mechanism Of V(Ⅴ) Bioreduction

Vanadium is a relatively abundant transition metal element in the earth’s crust.It is dispersed in other minerals,rocks and sediments.It is widely used in steel production,aerospace,energy storage and materials.With the increase of vanadium demand in industrial production,vanadium pollution in atmosphere,water and soil is becoming more and more serious.Vanadium exists in nature mainly as V（Ⅴ）,V（IV）and V（III）valence states,among which V（Ⅴ）is the main form of vanadium in the environment,and its toxicity is much greater than that of low-priced vanadium and its bioavailability is high.The migration and transformation of vanadium is easily affected by environmental factors such as redox conditions and p H.In recent years,the reduction of V（Ⅴ）by microbial treatment to reduce its toxicity and migration has attracted wide attention.In this thesis,a vanadium reducing bacterium was Isolated from vanadium contaminated soil,and its species and genus were identified by molecular biology,physiology and biochemistry.The influence of different V（Ⅴ）concentration,inoculum amount and p H value on V（Ⅴ）bioreduction was investigated.The electron transfer process and reduction mechanism of the strain on V（Ⅴ）were investigated,and the toxicity changes of the strain after V（Ⅴ）reduction were studied.The research results provide reference for the mechanism research of microbial reduction of V（Ⅴ）and the remediation of vanadium pollution.The main research contents and results are as follows.Strains with V（Ⅴ）reducing capacity were screened and domesticated from vanadium-contaminated soil,and were isolated,purified and identified to explore the reducing capacity and reduction characteristics of the strains to V（Ⅴ）.The results showed that Enterobacter kobei was selected and it had good reducing ability in a relatively broad V（Ⅴ）concentration range.When the initial concentration of V（Ⅴ）was 160 mg/L,the V（Ⅴ）reduction rate reached96.3%after 7 d.Low concentration of V（Ⅴ）stimulated the growth of E.kobei.With the increase of V（Ⅴ）concentration,the growth of the strain was inhibited,and the redox potential decreased and p H value increased in the process of V（Ⅴ）microbial reduction.The reduction of V（Ⅴ）was greatly affected by the initial p H value,and the optimal p H value was8.0.The reduction of V（Ⅴ）was also related to the initial inoculation amount,and increased with the increase of the initial inoculation amount.Prepare different active cells and cell components to study their reduction of V（Ⅴ）,and analyze the changes of nicotinamide adenine dinucleotide（NADH）and nitrite reductase（NIR）during microbial reduction of V（Ⅴ）and the distribution of products after reduction.The results showed that the V（Ⅴ）reduction rate increased from 71.2%to 75.0%after the membrane permeability was reduced.Comparing the reduction of V（Ⅴ）by different subcellular components,it was found that both extracellular secretions and cytoplasmic components could reduce V（Ⅴ）in the presence or absence of glucose as electron donor,with reduction rates of 41.7%and 80.2%,respectively.However,no V（Ⅴ）reduction occurred in cell membrane components,indicating that there were substances in cytoplasmic and extracellular secretions that could directly reduce V（Ⅴ）.In the process of V（Ⅴ）reduction,the activity of nitrite reductase（NIR）and the content of nicotinamide adenine dinucleotide（NADH）were increased in different degrees,and were greatly affected by the concentration of V（Ⅴ）.In the process of V（Ⅴ）reduction,the contents of extracellular polymer of strain increased,which was conducive to the adsorption and transformation of strain V（Ⅴ）and its own detoxification.V（Ⅴ）is reduced to precipitate,which is distributed both inside and outside the cell.The products of V（Ⅴ）reduction were characterized by scanning electron microscope-energy scattering X-ray spectroscopy（SEM-EDS）,X-ray photoelectron spectroscopy（XPS）,Fourier infrared（FTIR）and transmission electron microscope-energy scattering X-ray spectroscopy（TEM-EDS）.SEM results showed that in the process of V（Ⅴ）reduction,the strain would produce oxidative damage leading to cell damage,and EDS analysis confirmed vanadium precipitation around the bacteria.XPS analysis showed that E.kobei reduced V（Ⅴ）to V（Ⅳ）and precipitated it.The results of FTIR showed that hydroxyl group,carbonyl group and amide group were involved in bioadsorption and transformation.TEM-EDS confirmed that V（Ⅴ）can be reduced and precipitated both inside and outside bacteria.The electron transport mechanism of V（Ⅴ）bioreduction by Enterobacter kobei was investigated by adding different electron transport inhibitors and electron shuttler.The results showed that capsaicin,quinacrine and Cu Cl₂ could inhibit the reduction of V（Ⅴ）,but the membrane reductase inhibitors rotenone,N-acetyl-L methionine and antimycin A had no effect on the reduction.The V（Ⅴ）bioreduction was mainly achieved in the cell of the strain.When the riboflavin concentration was 10μmol/L,the redox potential of the solution reached-382 m V after 48 h continuous culture.The voltammetry curve（CV curve）of microbial reduction V（Ⅴ）solution showed that E.kobei could produce redox active substances,and V（Ⅴ）and riboflavin enhanced the electrochemical activity of the solution.Riboflavin reduced the effect of capsaicin,quinacline and Cu Cl₂ on V（Ⅴ）bioreduction.Chlorella pyrenoidosa and Scenedesmus obliquus were used as model aquatic organisms to study the toxicity changes of V（Ⅴ）microorganisms before and after reduction and the effects on algae growth.The results showed that V（Ⅴ）inhibited the growth of C.pyrenoidosa and S.obliquus in different degrees.However,after V（Ⅴ）bioreduction,the growth stress of C.pyrenoidosa and S.obliquus decreased,and the algal density content of the two algal species increased significantly after 96 h growth.The chlorophyll a content of C.pyrenoidosa and S.obliquus were increased to 17.42 and 15.81 mg/L,respectively.