Interaction Of Clay Minerals With Pseudomonas Aeruginosa And Its Influence Mechanism On The Biological Reduction Of Cr(?)

Many species of microbes can exhibit Cr(VI)-reducing capacity in the Cr(VI)-polluted soil.The ability of those bacteria to reduce Cr(VI)can be influenced by a variety of soilgeochemical factors.Especially,clay mineral,an important component of soil,can interact with the Cr(VI)-reducing microbes to form clay minerals/microbes complex with high specific surface area and active interface reaction,which can play a significant role in controlling and regulating the environmental geochemical behaviors of Cr(VI)such as bioreduction,species distribution,transportation and transformation.In this paper,clay minerals and a commonly occurring soil microorganism Pseudomonas aeruginosa(P.aeruginosa)were selected as the objects of study.Firstly,the influence of soil-geochemical factors on Cr(VI)-reducing ability of growing cells of P.aeruginosa were analyzed.Secondly,the ability of resting cells of P.aeruginosa in nutrient-poor medium was also studied.And then,the difference of Cr(VI)-reducing behaviors between the growing cells and the resting cells were compared.Last but not the least,the effect of clay minerals on the Cr(VI)-reducing capacity of P.aeruginosa was investigated in order to preliminarily illustrate the influence mechanism of the interaction between clay mineral and P.aeruginosa on the biological reduction of Cr(VI),which can provide a theoretical basis for the bioremediation of Cr(VI)-contaminated soils.The research work mentioned above has achieved the following results:(1)The toxicity of Cr(VI)towards P.aeruginosa was closely related to the physical and chemical properties of medium.The minimum inhibitory concentration(400 mg/L)in the liquid nutrient broth(NB)medium was higher than that in the solid nutrient agar medium(150 mg/L).The resistance of P.aeruginosa towards many heavy metals ions in the NB medium varied from kind to kind,following the order(from strong to weak): Cr(VI)>Pb(II)>Cu(II)?Ni(II)?Cd(II)>Co(II)>Hg(II).A variety of factors,including pH,temperature,salinity,initial Cr(VI)concentration,electron donors and co-existed ions,had a vital influence on Cr(VI)reduction by growing cells of P.aeruginosa in the nutrient broth medium.The optimum conditions for Cr(VI)reduction by P.aeruginosa were pH of 7,temperature of 37 oC,and salinity concentration of 0.5 %(w/w).P.aeruginosa was able to use a variety of organic compounds including glucose,lactose,sucrose,oxalic acid,citric acid,sodium acetate,glycerin and butyric alcohol as electron donors,with glucose exhibiting the most significant promoting effect.The Cr(VI)-reducing capacity of P.aeruginosa was inhibited by low concentrations(20 mg/L)of heavy metal ions(Pb(II),Co(II),and Cd(II)),10 mM of oxyanions(MoO42-,NO3-,SO42-,and PO43-),and by various concentrations of sodium azide ranging from 1 to 20 mM.However,20 mg/L of Ni improved the efficiency of Cr(VI)reduction.While the efficiency of Cr(VI)reduction decreased with an increase in the initial concentrations of Cr(VI),Cr(VI)reduction rate firstly increased with increasing concentrations of Cr(VI)and then decreased at higher concentrations under the optimum Cr(VI)-reducing conditions.Cr(VI)was efficiently reduced by P.aeruginosa over an oxidation-redox potential ranging from +150 to 360 mV within 24 h,showing Cr(VI)reduction could occur in the presence of higer redox potential.The total Cr content in solution declined slightly,during Cr(VI)reduction,but overall it remained almost constant.The resultant Cr(III)existed mainly in the NB medium in the form of soluble Cr(III)end products,whereas a small amount of Cr(III)interacted with carboxyl and amino groups on the surface of P.aeruginosa,leading to the immobilization of Cr(III)by cells.(2)The optimum conditions for Cr(VI)reduction by resting cells of P.aeruginosa in Tris-HCl buffer solution were pH of 7.0,temperature of 37 oC,and sodium acetate concentration of 5 g/L.Under the above-mentioned conditions,the efficiency of Cr(VI)reduction increased with an increase in cell density ranging from 0 to 3.2 g/L(dry weight),and 3.2 g/L of resting cells of this strain reduced 10 mg/L of Cr(VI)by 86 % within 24 h.The pseudo first-order kinetic model accurately described the reduction process at each initial Cr(VI)concentration.The Cr(VI)-reducing efficiency and the rate coefficients for Cr(VI)reduction gradually declined when initial Cr(VI)concentration increased from 5 to 40 mg/L,whereas the specific Cr(VI)reduction rates increased firstly and then decreased with an increase in initial Cr(VI)concentration.After 24 h,while remnant Cr(VI)existed only in the supernatant,the content of resultant Cr(III)in supernatant,on cell surface and inside cells was 2.62,1.06,and 5.07 mg/L,respectively,which was an indicative of extracellular and intracellular reduction of chromate.Extracellular reduction product Cr(III)not only combined with Tris in the solution,but also interacted with carboxyl,amino,and phosphate groups leading to the immobilization of Cr(III)on bacterial surface;the intracellular reduction product Cr(III)coordinated with proteins or nucleic acid distributed in the cytoplasm and formed electron-dense opaque particles containing Cr(III)resulting in the accumulation of Cr(III)inside resting cells of P.aeruginosa.The bacterial cells themselves played an dominant role in Cr(III)immobilization.(3)Both the soluble protease and the membrane-associated protease resulting from cells of P.aeruginosa were Cr(VI)reductase,with the former exhibiting high level Cr(VI)-reducing potential.Moreover,the two kinds of Cr(VI)reductases mentioned above are not inducible enzymes but constitutive reductases.Both the Cr(VI)-reducing behavior of P.aeruginosa and the environmental fate of Cr were influenced by medium types,kinds of electron donors and metabolic activities of P.aeruginosa.After 24 h of Cr(VI)reduction,the binding ratio of Total Cr by bacterial cells of P.aeruginosa in different types of medium systems followed the order: Tris-HCl buffer solution containing sodium acetate(~ 65 %)> NB medium containing sodium acetate(~ 36 %)> NB medium containing glucose(~ 7 %).In the glucose-amended NB medium,the soluble protease secreted during the process of the bacterial metabolism and membrane-bound protease as well as glucose's by-products were responsible for Cr(VI)reduction,and Cr(VI)reduction and Total Cr immobilization occurred mainly in the solution.In the sodium acetatesupplemented NB medium,both the soluble protease secreted through the metabolism of this strain and membrane-bound protease were involved during the process of Cr(VI)reduction,and Cr(VI)reduction and Total Cr accumulation happened in the solution and on the bacterial surface.In the Tris-HCl buffer solution containing sodium acetate,both the soluble protease distributed inside cells and the membrane-associated protease co-participated in the process of Cr(VI)reduction,and both the bacterial interior and exterior surfaces of cells were sites for Cr(VI)reduction and Cr(III)immobilization.(4)The synergistic effect was observed between clay minerals and P.aeruginosa during the process of Cr(VI)bio-reduction in the glucose-supplemented NB medium.Cr(VI)bioreduction by both free cells and clay minerals-amended cells followed the pseudo-first-order kinetic model,with the latter one fitting better.The addition of clay minerals(e.g.vermiculite)could decrease the surface roughness of Cr(VI)-laden cells and change the cell morphology and dimension.Moreover,the active sites of clay minerals could interact with aliphatic species and proteins which mainly resulted from microbial exudate or cell wall components to form clay minerals-cell complex.In the complex,clay minerals,i.e.kaolinite and vermiculite,significantly enhanced the Cr(VI)-reducing activity of P.aeruginosa by improving the consumption rate of glucose,acting as growth-supporting materials for the bacterial metabolism,providing a micro-environment to protect cells from the toxicity of higher Cr(VI)concentration,enhancing the tolerance of P.aeruginosa towards Cr(VI),and by stimulating the growth and propagation of cells of this strain and.The adsorption of total chromium on clay minerals-bacteria complex was small,and Cr(VI)bio-removal was not mainly due to the adsorption of Cr(VI)onto cells or clay minerals or clay minerals-cells complex but mainly due to the Cr(VI)reduction capacity of Pseudomonas aeruginosa.