The Study On The Speciation And Transformation Of Elemental Sulfur Mediated By Typical Acidophilic Sulfur-oxidizing Microbes

In present work, we chose the typical acidophilic sulfur oxidizing microbes including Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, Sulfobacillus thermosulfidooxidans, Acidianus manzaensis to leach pyrite and chalcopyrite, and Acidithiobacillus ferrooxidans, Acidithiobacillus caldus to oxidize thiosulfate and elemental sulfur powder. The aim of this paper is to obtain some basic data to illustrate the biological oxidation process of inorganic sulfur compounds and the mechanism of bioleaching by investigation: the elemental sulfur speciation transformation mediated by A. ferrooxidans; the screen , identification , the verification of gens and proteins of sulfur activation related extracellular proteins; the sulfur chemical speciation of intracellular and extracellular sulfur globules; growth and surface properties of cells grown on different sulfur-containing energy substrates; the sulfur chemical speciation and transformation on the surface of metal sulfides. The primary subjects of which include six parts as follows:(1) The sulfur K-edge X-ray absorption near edge structure (XANES) spectraIn this part, we investigated the sulfur K-edge XANES spectra of a series of sulfur containing organic and inorganic model compounds with synchrotron radiation X-ray absorption fine structure. And based on these spectra of model compounds, we firsty built a sulfur K-edge XANES spectra gallery in our country. The results showed that sulfur K-edge XANES spectroscopy was a sensitive analytical method. The sulfur chemical speciation of different sulfur containing compounds had exact correlations with its absorption spectra. The defined mixture sample was fitted with model compounds by least square linear combination fitting method. It is possible to analyze and obtain the precise structural information of sulfur in nature sample in situ and the main components of mixture, if the suitable model compounds were involved.(2) Investigation of elemental sulfur speciation transformation mediated by Acidithiobacillus ferrooxidansThe speciation transformation of elemental sulfur mediated by the leaching bacterium, A. ferrooxidans was firstly investigated with the employment of an integrated approach including: SEM, TEM, FT-IR, EDS and XANES. Our results showed that when grown on elemental sulfur powder, A. ferrooxidans ATCC23270 cells were first attached to sulfur particles, and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to linear polysulfides. Up to our knowledge, it is the first time to prove the elemental sulfur must be changed to linear sulfur baseing on the experimental results. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested that these globules consisted of elemental sulfur bound to thiol groups of protein.(3) The screen, identification and functional investigation of extracellular proteins and genes of A. ferrooxidansA set of proteins that changed their levels of synthesis during growth of A. ferrooxidans on elemental sulfur/ferrous iron was characterized by using two dimensional polyacrylamide gel electrophoresis. We identified a total of 18 extracellular proteins with apparent higher abundance grown in element sulfur than in ferrous sulfate. Matrix-assisted laser desorption/ ionization time-of-flight/ time -of-flight mass spectroscopy (MALDI-TOF/MS) analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans. Additionally, six hypothetical proteins (or peptides) of which contain abundant of the cysteine residues. Expression analysis of the corresponding genes by real-time PCR showed the constitutive expression of all those genes was highly upregulated by growth on elmental sulfur compounds (and downregulated by growth on ferrous iron). A gene of AFE₂621 from A. ferrooxidans was cloned and expressed in E. coli. The extracellular proteins of A. ferrooxidans grown on elemental sulfur and the purified AFE₂621 (pinlin) were characterized with sulfur K-edge XANES spectroscopy. The results showed that the thiol-containing amino acid mainly cysteine play an important role in elemental sulfur activation and oxidation.(4) Sulfur species investigation in intra-/extracellular sulfur globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldusFor the first time, the sulfur chemical speciation in intracellular and extracellular sulfur globules of A. ferrooxidans and A. caldus were investigated with the multiple employment of SEM, TEM, EDS and sulfur K-edge XANES. When respectively grown with elemental sulfur (S ) and thiosulfate, A. ferrooxidans could accumulate both linear sulfur and ring sulfur internally, whereas A. caldus could not accumulate intracellular sulfur globules. When grown with thiosulfate, in contrast, the sulfur species in extracellular sulfur globules produced was polymeric sulfur(with unknown) and homocycles(just like artificial made sediment) A. ferrooxidans and A. caldus, respectively.(5) Growth and surface properties of new thermoacidophilic Archaea strain Acidianus manzaensis grown on different substratesThe growth characteristics of Acidianus manzaensis YN-25 have been firstly investigated. The cells were cultured with ferrous sulfate, elemental sulfur, pyrite and chalcopyrite mineral respectively, and the hydrophobicity, electrokinetic behaviour and the surface groups of cells grown under above substrate have been investigated. The results showed that the hydrophobicity and the Zeta-potential of cells grown in ferrous sulfate presented distinctly characteristics compared to the cells cultured in solid substrate. The FT-IR spectra indicated that the sulfur, pyrite and chalcopyrite grown cells contained a higher amount of protein component than the ferrous ion-grown cells on the cells surface.(6) Typical acidophilic sulfur oxidizing microbes leaching pyrite and chalcopyriteThe typical mesophiles A. ferrooxidans,A. thiooxidans, moderate thermophiles S. thermosulfidooxidans and extreme thermophiles Acidianus manzaensis were chosen to leach pyrite and chalcopyrite. For the first time, the characteristics of mineral surface, main components of mineral and the sulfur speciation on the mineral surface were investigated with SEM,XRD and sulfur K-edge XANES spectra. The results showed that there were distinct differences among the sulfur speciation and the main components of leaching residual leached with these pure or mixture of microbes. The extreme thermophiles Acidianus manzaensis had excellent leaching ability with pyrite and chalcopyrite. S. thermosulfidooxidans had better leaching capability on pyrite than chalcopyrite. The mesophiles A. ferrooxidans had better leaching efficiency on chalcopyrite than pyrite. The strains of A. thiooxidans had no oblivious promotion on A. ferrooxidans leaching pyrite and chalcopyrite. During all the leaching experiments, the cells attached to the mineral surfaces firstly. The results of the sulfur speciation and the main components of leaching residual during the Acidianus manzaensis leaching experiments, the crystal of coquimbite and covellite and jarosite were accumulated on the surface of pyrite and chalcopyrite, respectively and the sediments layer of later could negatively affect the dissolution of copper ions. The passivation layer on the surface of metal sulfides were caused by the lowering acidity, in that case, the growth of cells were strongly inhibited. During the leaching of pyrite and chalcopyrite with S. thermosulfidooxidans, the precipitation of jarosite was formed on the surface of metal sulfides. The amount of jarosite produced from the former was much more than the latter, which also form negative barrier between cells and mineral surface. The elemental sulfur sediments were accumulated on the surface of pyrite leached with A. ferrooxidans, and the addition of A. thiooxidans could avoid the accumulation of elemental sulfur. The jarosite sediments on the chalcopyrite were formed with pure A. ferrooxidans and mixture of A. ferrooxidans and A. thiooxidans, which could lead to a gradual passivation of chalcopyrite whereby Cu²⁺ dissolution leveled off. Nevertheless, judging from the leaching curves of pyrite, the precipitation of elemental sulfur could not effected significantly the leaching of iron ions.