Isolation And Identification Of Strain W-3-1 From Acid Mine Environment Of Tongling,Anhui Province And Sulfur Oxidation With W-3-1

The samples collected from acid mine drainage and sediments in Tongling,Anhui province were cultured by selective media of FeT?FeS?Waksman and Starky,and a strain of sulfur-oxidizing bacterium W-3-1 was isolated from acid mine drainage.A detailed study on the biological properties and identification of W-3-1 was carried out in this paper.Firstly,the colony of W-3-1 on FeT solid medium was white and transparent with neat edge.After three days of growth of w-3-1 in Waksman liquid medium,the medium became turbid and elemental sulfur would sink to the bottom of the medium.Secondly,the strain was short rod-shaped.The size(length x width)of the bacteria was 0.6?m-1?m × 0.4?m-0.6?m.It was able to move without endospore structure and was Gram-negative.The bacterium did not contain fexirubin pigment.Thirdly,W-3-1 was a chemolithoautotrophic bacterium,mainly using carbon dioxide in the air as a carbon source,and it couldn't use common carbon sources such as glucose,lactose,maltose,mannose and so on.W-3-1 could utilize phenylalanine and urea as nitrogen sources,but not lucosamine and nitrate.W-3-1 did not possess the activity of ornithine decarboxylase,lysine decarboxylase,and arginine decarboxylase amino acid decarboxylase,and had the ability to produce hydrogen sulfide.Fourthly,The optimum growth pH of W-3-1 was 4,the optimum growth temperature was 30?,the optimum growth salinity was 0%,and the logarithmic growth phase was 2-8 days.Fifthly,the 16S rDNA sequencing and phylogenetic analysis of W-3-1 showed that it had the highest similarity with that of Acidithiobacillus thiooxidans,which was 97%.It was initially determined that W-3-1 was the genus of Acidithiobacillus.The sulfur oxidation ability of W-3-1 was investigated.It was found that when elemental sulfur,Na2S2O3,and K2S4O6 were used as energy substrates,the oxidation of elemental sulfur by W-3-1 was best.When the concentration of elemental sulfur in the medium was 10 g/L,W-3-1 has the strongest growth and acid production ability.Adding a small amount of Tween 80 to the medium would promote the growth of W-3-1.When the concentration of Tween 80 was 0.5 g/L,the growth of W-3-1 and the oxidation of elemental sulfur were the strongest.Excess Tween 80 would inhibit the growth of W-3-1.The results showed that only intact W-3-1 living cells could oxidize elemental sulfur,and the direct contact of W-3-1 with the surface of elemental sulfur was a prerequisite for its oxidation reaction.In addition,after the reaction of elemental sulfur and W-3-1,the surface morphology of elemental sulfur also changed,many pit marks appeared on it,and the roughness increased greatly.Also,a large number of long rod-shaped holes appeared on the surface of elemental sulfur,similar to the shape of the bacteria,indicating that W-3-1 had oxidized the elemental sulfur.W-3-1 was inoculated into the medium supplemented with pyrite.It was found that W-3-1 could be grown using pyrite as an energy substrate,but its growth was not as good as when elemental sulfur was used as a substrate.At the same time,it could be concluded from the chemical changes in the solution of the bacteria group and the control group that the oxidation of pyrite by W-3-1 was stronger than the natural oxidation of the pyrite.From the scanning electron microscopy and energy spectrum analysis results of pyrite,it could be seen that when the W-3-1 interacts with pyrite for 30 days,a large number of pit marks appear on the surface of the pyrite,and a lot of irregular sheet-like structures are accumulated.The secondary sediments were presumed to be jarosite.The pyrite was further oxidized for 50 days,the surface collapsed and the secondary precipitates increased,indicating that the presence of W-3-1 could promote pyrite Oxidation.In addition,the influencing factors of the interaction between W-3-1 and pyrite showed that the finer the pyrite particles,the better the growth of W-3-1 and the higher the degree of oxidation of pyrite;within a certain range(0-15 g/L),the greater the pyrite concentration,the better the W-3-1 growth and the stronger the oxidation of pyrite.