| The sulfur/ferrous iron-oxidizing microorganisms contribute to the transformation of sulfur and other intermediary sulfur compounds to sulfuric acid, regenerating protons and Fe3+for the bioleaching process. The microbial ferrous oxidation model has been described clearly, but the sulfur oxidation mechanism remains unclear because of the complexity of the sulfur bio-oxidation process. To clarify the molecular mechanism of microbial sulfur activation and oxidation in the acidophilic sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans, techniques of microbiology, biochemistry and molecular biology, and the in situ synchrotron radiation X-Ray fluorescence analysis were used. The main contents and results are as follows.1. Establishment of the experimental methods was mentioned. By choosing surfactant Tween-80as the stabilizing agent, the barium sulfate turbidimetry for determining sulfate concentration in the complex bioleaching samples was improved. Compared with the national standard method GB/T5750.5-2006, it is faster and easier for operation, indicating its wide applicability. Extracellular, outer membrane and periplasimic proteins of A. ferrooxidans were separated by an innovative continuous treatment using hot water bathe followed by a one-step Triton X-114phase-partitioning. The two dimensional polyacrylamide gel electrophoresis for the extracted compartment-specific proteins was also optimized.2. Isolation and identification of three species of acidophilic sulfur-oxidizing bacteria were described and their sulfur oxidation related physiological characteristics were compared. Ultrastructure of the three strains Acidithiobacillus albertensis BY-05, A. ferrooxidans DXS and Acidithiobacillus thiooxidans BY-02showed that inside the cells there were highly refractile sulfur granules, which may play the role of energy storage. The new strain A. albertensis BY-05had high sulfur oxidation capacity, and could enhance the leaching capacity of chalcopyrite and sphalerite when mixed with A. ferrooxidans. 3. The relevant sulfur speciation on the surface of chalcopyrite leached by three typical mesophilic bacteria A. ferrooxidans, Leptospirillum ferriphilum and A. thiooxidans and their mixture was investigated using X-ray diffraction (XRD) and X-ray absorption near edge structure (XANES). The results showed that the mixed culture had a higher sulfur/iron oxidation activity than the pure cultures of the bacteria. The fitting results of XANES spectra indicated that the mixed culture restrained the sulfur and jarosite passivation layer obviously. Additionally, the dissolution rate of chalcopyrite was higher when the redox potential was less than400mV (vs SCE), and above540mV (vs SCE) the rate decreased apparently.4. Comparative proteomics strategy was used to screen the sulfur activation and oxidation relative proteins from specific cellular compartments of A. ferrooxidans ATCC23270,39expression upregulated proteins (including13extracellular proteins,9outer membrane proteins and17periplasmic proteins) selected in the2-DE profiles were identified by MALDI-TOF MS/MS and verified at transcriptional level by RT-qPCR. About half of the selected proteins were function-unknown, others were annotated to categories of energy metabolism, transport and binding, cell structure, cellular processes, etc.. Bioinformatics prediction showed that70%of them contain cysteine residues in sequence. Eight proteins which contain abundant of the cysteine residues even have one or two functional motifs such as-CXXC-The thiol groups on the A. ferrooxidans cell surface were selectively marked by Ca2+and observed by in situ micro synchrotron radiation X-Ray Fluorescence (Micro-SR-XRF) mapping analysis, the result revealed that the number of the thiols on the surface of the cells grown on elemental sulfur was about five times as that grown on ferrous substrate. It indicates that the thiol-rich proteins played important roles in sulfur activation and oxidation process.5. Effects of the surfactant Tween-80on the growth, sulfur oxidation, and expression of selected typical sulfur metabolism relevant genes of A. ferrooxidans ATCC23270were investigated. The results showed that in the presence of10-2g/L Tween-80the growth of A. ferrooxidans and its metabolism on the insoluble substrate S0and CuFeS2was promoted. After24days of bioleaching, the copper extraction yield of chalcopyrite at10-2g/L of Tween-80increased by16%compared to the bioleaching experiment without Tween-80. FT-IR spectra analysis revealed that this was probably caused by the extracellular polymeric substances whose composition could be changed by the surfactant addition. RT-qPCR was used to analyze the differential expressions of17selected sulfur metabolism relevant genes in response to the addition of Tween-80. Down-regulation of the extracellular protein genes indicated the influence of Tween-80on bacteria-sulfur adsorption. Variation of the expression level of the enzymes provided a supplement to sulfur metabolism investigation.6. The differential utilization of α-and μ-sulfur by A. ferrooxidans was also investigated in this thesis. The growth and sulfur oxidation of A. ferrooxidans on μ-S showed shorter lag phase. The initial adsorption capacity of the cells was higher on μ-S than that on α-S. The results of SEM, DRIFTS and XRD analyses indicate that the surface of sulfur was modified by cells and the orthorhombic α-S was destroyed by A. ferrooxidans from special region of the crystal. Differential expression of11selected sulfur adsorption-activation and metabolism relevant genes was detected by RT-qPCR. The results show that the expression of the hydrophobic substrate transport proteins and the sulfur metabolism related proteins was up-regulated, and the adsorption and activation related proteins were down-regulated when the cells were grown on μ-S. |
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