| The two strains of iron-oxidizing bacteria W-1-2 and W-5-3 studied in this paper were isolated from acid mine drainage and sediment samples in the acid mine environment of Tongling,Anhui Province.Systematic biological identification and iron oxidation and pyrite leaching were studied for these two strains.The 16S rRNA gene sequencing results and Blast comparison of the two strains showed that the sequence similarity between the strain W-1-2 and Thiomanas sp.CB2 was 99%,which demonstrated that strain W-1-2 could be identified as Thiomanas sp..The sequence similarity of strain W-5-3 and Pandoraea thiooxydans was 99%,which demonstrated that strain W-5-3 could be identified as Pandoraea sp..The biological characteristics of the two strains were studied.The results showed that the colonies of strain W-5-3 and strain W-1-2 on FeT solid medium were yellow-brown and nearly round in shape.The colony edge of strain W-1-2 was more uniform,and the colony edge of strain W-5-3 was more irregular.The cell shape of both strains was rod.Both strains are gram-negative aerobic bacteria with no endospore structure and the cells of both strains can move.Strain W-1-2 can utilize maltose,glucose,bile esculin,acetate,malonate and other energy substances,and the utilization of hydrogen sulfide and urea is not obvious.Sucrose,lactose,mannose,arabinose,sorbose,rhamnose,melezitose,glucosamine,sodium pyruvate,phenylalanine,mannitol,salicyl,etc.cannot be used as energy substances.Strain W-5-3 can use maltose,glucose,bile esculin,glucosamine,malonate,dextrin and other energy substances,and the utilization of sodium pyruvate,hydrogen sulfide,and urea is not obvious.Sucrose,lactose,mannose,xylose,arabinose,sorbose,rhamnose,melezitose,phenylalanine,mannitol,salicyl,tartrate,acetate,etc.cannot be used as energy substances.The enzymatic properties of W-1-2 and W-5-3 are as follows:ornithine decarboxylase,arginine dihydrolase,arginine decarboxylase are active,and the valine decarboxylase is inactive.The optimum pH of strain W-1-2 is 5.5,and the optimum pH of strain W-5-3 is 5.0.W-5-3 is more adaptable to lower pH than W-1-2.The optimum temperature for both strains is 30? and they both grow better in FeT liquid medium without NaCl.W-5-3 grows faster than W-1-2 and has a higher maximum bacterial concentration.Through the research on the oxidation of Fe2+ by W-1-2 and W-5-3,it was found that the growth of the two strains was best in a culture medium with an initial Fe2+concentration of 5 mM,and the addition of 100mL of liquid medium in a 250 mL conical flask was more conducive to the growth of both strains.Both strains cannot oxidize Fe2+ only by extracellular secretions,inactivated bacteria,and active enzymes in cells.Only complete living cells can oxidize Fe2+ to Fe3+.The leaching effect of two strains on pyrite was investigated with and without Fe2+ addition.The changes of pyrite surface were studied by scanning electron microscope and energy spectrum analysis.The leaching of pyrite was analyzed by determining the concentration of Fe3+ in the solution.The results showed that W-1-2 and W-5-3 had low leaching degree to pyrite by direct oxidation under the condition of no additional Fe2+,while adding a small amount of Fe2+ in the medium could significantly increase leaching rate of pyrite.It showed that the mechanism of leaching of pyrite was the synergistic mechanism when Fe2+ was added,which meant there were both the direct oxidation by the strains and the chemical oxidation by Fe3+.Different inoculum volume also influenced the leaching rate of pyrite.Controlling the initial inoculum volume of the two strains could increase the leaching degree of pyrite.The study on the oxidation of Fe2+ and the leaching effect on pyrite by two strains have important practical significance for the prevention,source treatment and bioremediation of acid mine drainage. |
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