| This thesis compares the different energy metabolism of A.ferrooxidans Thiobacillus acidophilus bacteria and A.thiobacillus bacteria on a separate sphalerite and mixed leaching.A higher bioleaching ratio(61%) was reached when using Acidithiobacillus ferriooxidans mixed bacteria compared to that using Acidithiobacillus thiooxidans(20%) after bioleaching for 18 days.What's more,the bioleaching ratio and rate were reached 97%and 0.3g/L.day when using mixed bacteria,respectively.The bioleaching rates were only reached 0.17 g/L.day for Acidithiobacillus ferriooxidans and 0.06g/L.day for Acidithiobacillus thiooxidans.Elemental sulfur was overlayed on the surface of solid residues from system with sterile or Acidithiobacillus ferriooxidans.However,there was not detected about elemental sulfur on residues surface from system with Acidithiobacillus thiooxidans due to its oxidizing-sulfur property.The elemental sulfur may inhibit the bioleaching when considering the different bioleaching rates.A high redox potential can be maintained by Acidithiobacillus ferriooxidans which can oxidize ferrious iron to ferric iron.Thus,ferric iron can be continuously generated,as a result,a continuous leaching be maintained.The role of A.thiooxidans is to oxidize and dissolve the sulfur layer(passivation) formed on mineral surface which is benifical to enhance the chemical leaching.Also,acid leaching can be improve and jarosite be inhibited due to the oxidization of elemental sulfur to sulfuric acid.The toxicity of Cu2+ was reflected in the inhibition of bacterial respiration.However,the inhibition becomes obvious only after Cu2+ was added into medium for 30 minters.When bacterias are grown in sulfur medium containing copper ions,copper ions inhibit the activities of sulfite oxidase and APS reductase.Little inhibition is happen to other involved enzymes in sulfur metabolism.Further experiments show that copper ions have an indirect influence on enzymatic activities.The cellular location of enzymes was determined by measuring enzymatic activities under the condition of different pH and cellular components.As was testified by the treatment of protease and valinomycin on bacteria,Acidithiobacillus caldus can maintain the low concentration of copper ions in cytoplasm by the transportation of membrane protein from inside cell to outside cell.The membrane protein is similar to ATP-pump associated with the consumpation of ATP.The analysis on enzymatic activities showed that a gene can encode the sulfide dehydrogenase which can oxidize sulfide to elemental sulfur and transport the electron from sulfide to cytochrome c.Its enzymatic activity is highest at about pH 7 when measued in different pH and different cellular components,this suggests that the protein should be located in cytoplasm.A ribosome-binding site(GGAG) is detected at 7 bp upstream of start code from the above gene.The -10 and -35 domains,however,are not found in FCSD.Similar to other homogenous proteins,there are some high conserved amino acid sequences binding FAD in sulfide dehydrogenase.In addition,two cysteamine can form disulfide bond which is in relation to sulfide binding to protein.There are high homogenous(39%) in amino acid sequences between SQR and FCSD. There are also conserved FAD-binding domains,βαβdomain and disulfide bond site in the amino acid sequences of both SQR and FCSD.When bacteria are grown in medium with ferrious irons as growth energy,the mRNA expression levels of FCSD and SQR are higher than that of bacteria grown in medium with sulfur and thiosulfate.However, their enzymatic activites are highest in sulfur medium,and lowest in thiosulfate.This indicates that the expression levels of FCSD and SQR are regulated on transcribe and translation,especially,the latter are strongly affect the expression of FCSD and SQR.Based on the analysis on expression levels,FCSD and SQR perform the function of sulfur metabolism.It is analysed and compared about FCSD,SQR and corresponding bacterial 16srDNA uploaded in NCBI from various species.The results show that SQR and FCSD are evolutionary in ancient epoch and extentive distribution from archaea to bacteria.FCSD are more important than SQR to sulfur metabolism and sulfur cycles on ancient earth due to the higher expression level and more extentive distribution of FCSD.A preliminary model for the FCSD protein was constructed using the approach of comparative protein modeling on the basis of the sequences of the FCSD protein from Chromatium vinosum.Cyc 158 and cyc 331 can form the disulfide bond.The formation and disconnection of disulfide bond have a very important influence on binding sulfide to protein and catalyzing the sulfide oxidization.In addition,Gly298, Ser299,Phe330 and Phe332 perform the electron transfer between protein and FAD.Further,Glul64 is in relation to accepting electron from FAD.We constructed the mutant expression plasmids of $299A and G164A residues using site directed mutagenesis.Mutant proteins were expressed in E.coli and purified.Enzymatic activities of mutation S299A and G164A are very lower than wild protein.This reveals that ratiocination based on model are true and these residues play key roles to enzymatic activity.FCSD was successfully assembled on a gold electrode by means of nano-gold and cyctemise.The Cyclic voltammmetric response of the modified gold electrode was investigated under the condition of 0.02mol/L PBS buffer(pH 7.4).A pair of stable and quasi-reversible redox peak was observed from cyclic voltammograms.Epc and Epa are 0.13 and-0.034v,respectively.The form potential is 63my.Epa increase and Epc decrease when Na2S is added into electrolyte.This indicates that the immobilized FCSD can electrochemically catalyze the oxidization of sulfide.The i-t experiment shows that the modified enzymatic gold electrode is very sensitive to response to sulfide.The response time is about 4s when attaining a stable current of 95%.The response current is linely proportionable to the concentration of sulfide at the range of sulfide from 2×10-4 to 2.5×10-3 mol/L.Tthe equation is ip=0.4819+2.64c with a correlation coefficient of 0.992.The response sensitiablity to sulfide is 1.1×10-5 mol/L and kmapp is 4.5mmol/L.To estimate the stability and repetition of modified electrode, electrochemical behavior was investigated after some reagents bein g added into electrolyte such as Fe3+(1.5×10-3mol/L),K+(1.5×10-3mo 1/L),NO3-(1.5×10-3mol/L),SO42-(1.5×10-3mol/L),Citric acid(1.5×10-3mol/L),Glucose(1.5×10-3mol/L).The results show that these reag ents have not influence on response to sulfide.In addition,the mo dified electrodes show also high stability and repetition(RSD＜5%) about the electrodes from various constructed time.
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