With the exploitation of mineral resources of gold, easy to deal with dwindling ore. The gold resources currently difficult to process gold mines account for nearly 60% of the world's gold reserves. The so-called "difficult to handle" means that the gold in the ore cannot be effectively extracted by conventional cyanidation leaching. Bacterial oxidation method for bio-preoxidation of refractory gold ore was first proposed by Pares in France in 1964. In the future work, it has been put into industrial application in South Africa, Brazil, Australia, the United States and other countries. It is known from practice that the recovery rate of gold after bioleaching pretreatment is significantly improved. From a kinetic point of view, the results obtained are less than ideal. Excessive residence time (2 to 5 d) results in excessive operating costs. There is therefore a need to improve bioleaching kinetics. According to the direct mechanism of bioleaching, kinetic improvements should be based on the development of more active bacteria by discovering a new class of bacteria or using genetic control techniques to improve known bacteria. Species commonly used in general mainly room temperature bioleaching bacteria, such as ethylene oxide and iron Thiobacillus thiooxidans, their optimum temperature is 28 ~ 30 ℃. In recent years, research in the metallurgical thermophiles thermolysin also demonstrated the ability to extract metals from various sulfide ore, the ore such as copper, molybdenum, nickel, and promote the recovery of gold and the like; Some results of the study demonstrated tropism The hot bacteria have a faster oxidation rate to the ore than the normal temperature bacteria. Compared with the normal temperature bacteria, the thermophilic bacteria are suitable for the heating reaction system, and the cooling equipment using the medium temperature bacteria can be omitted; the reaction speed can be improved from the viewpoint of kinetics. , shorten the pre-oxidation time. However, there are few studies on the application of thermophiles in China. The purpose of this study is to screen high-efficiency thermophiles, study their morphological characteristics, their ability to oxidize pyrite single minerals, provide basic data for refractory gold pre-oxidation and bioleaching of chalcopyrite, and have important theoretical and practical significance. First, experimental materials and methods (1) ACID mine water Examine geological conditions, geography and climate influence, and in accordance with the proper thermophilic bacteria growth environment, acid mine water taken as a separate sample of coal ore. The experimental use of acid mine pit water is collected from a city in the south. The coal mine is an old mine that has been mined for decades. The gangue minerals in the coal seam are mainly pyrite, and the oxidation of pyrite is clearly seen in the waste mine. The coal mine has an average annual temperature of about 25 ° C, a summer air temperature of up to 40 ° C, and a surface temperature of up to 50 ° C. Therefore, it is possible to separate the target strain from the pit water. The water sample is concentrated and collected by a membrane filtration method, and then separated in a high-speed table centrifuge to obtain an acidic pit water for separation. (2) Separation medium The basic medium composition: (NH 4 ) 2 SO 4 3gL -1 , KCl 0.1 gL -1 , K 2 HPO 4 0.5 gL -1 , MgSO 4 7H 2 O 0.5gL -1 , Ca(NO 3 ) 2 0.01 gL - 1 . During the test, a combination of various energy sources and basic medium was used, and the acid pit water was inoculated to separate the target strain. (3) Pyrite single mineral The pyrite single mineral was cut into a rectangular parallelepiped of 15 mm × 10 mm × 5 mm by a cutter, and one of the surfaces was subjected to preliminary grinding, fine grinding, and polishing to be mirror-finished, and rinsed with distilled water at room temperature for drying. (4) Test methods 1. Method for isolation of strains. The solid medium plate scribing separation method is combined with the liquid medium dilution separation method. The acidic pit water and different media were placed in a multi-dilution plate at different dilutions, placed in a constant temperature biochemical incubator, and the temperature was controlled at 50 ° C for culture. During the culture, the colony growth was observed and counted under a microscope using a hemocytometer. 2. Observation of bacterial morphology. The morphology of the colonies grown in the ore samples was observed by scanning electron microscopy with Hitachi S-570. Strain was observed sample preparation procedure the following steps: (1) Fixed: glutaraldehyde - fixed acid bis hungry, 4H 2.5% glutaraldehyde (or overnight) phosphate buffer three times, each time 15min, 1% hungry Acid (OsO 4 ) was fixed for 2 h, phosphate buffer was washed twice for 15 min each time; (2) Dehydration: ethanol series 30%, 50%, 70%, 85%, 95% ethanol once, each time 15 min, 100% Ethanol twice, each time 15 min; (3) Displacement: isoamyl acetate twice, each time 15 min (or overnight); (4) carbon dioxide critical point drying; (5) ion sputtering gold; (6) Hitachi S-570 scanning electron microscope observation, photography. 3. Observation method of pre-oxidation degree of pyrite. The pyrite single mineral polished piece was placed in a 200 ml flask. The medium was treated with FeS 2 (10 g L -1 ) + yeast (0.02% W/V) as the substrate, the temperature was 52 ° C, and the inoculum was 15 %, the initial pH of the solution was adjusted to 2.0, the shaking speed was 150 r min -1 , and cultured for 5, 8, 12 and 16 days respectively. The samples were taken out and the degree of oxidation of the pyrite monomer minerals was observed by scanning electron microscopy. Second, the morphological characteristics of the target strain The scanning electron micrograph of the target strain is shown in Figure 1. Figure 1 Scanning electron micrograph of the strain Through the series of measures such as sampling, enrichment, separation and purification, an effective method for breeding high-efficiency thermophilic bacteria from nature was established, and finally a thermophilic strain was isolated. According to the molecular biological identification, the strain is a Gram-positive bacteria, a mineralized vegetative bacterium, the cells are rod-shaped, and the cell size is between 0.4 and 2×3 to 6.8 μm, which can be in iron, sulfur, sulfide ore, etc. Growing on different substrates. Obligate aerobic, acidophilic, moderately thermophilic, optimal growth temperature 50 ° C, can survive at 60 ° C. Fe 2+ , sulfide ore is self-supportive for energy growth, yeast is used as energy for heterotrophic growth, iron and yeast are used as energy for mixed vegetative growth; when yeast is present, it can oxidize elemental sulfur. Inorganic substrate is energy self-long health, good cell growth requires enough CO 2, more bacteria grown in the presence of mixed organic nutrient. There are spherical spores formed during cell growth, and the cells are not motility. According to the description of the genus Sulfobacillus in the Berger's Bacterial Identification Manual: Sulfobacillus is present in the acid hot environment rich in iron, sulfur and sulfide ore. It is a Gram-positive, inorganic trophic bacterium, and the cells are rod-shaped. Rod shape, the optimum growth temperature is 52 °C. Comparing the physiological and biochemical characteristics of the selected strains, it is known that it is a moderately thermophilic strain of Sulfobacillus, and the typical species is Sulfobacillus thermosulfidooxidans, which plays an important role in the bioleaching process of metal sulfide ore. Third, the oxidation of pyrite single mineral results Gold has the dual properties of sulphur and pro-iron. In the process of deposit formation, gold is often symbiotic with sulfide minerals; pyrite is the main carrier of gold. Zhang Shibo et al studied the pyrite surface and the Au [HS] 2 - After the solution that effect, the crystal defect surface is pyrite and Au [HS] 2 - effect of the stepped surface and adsorbed thereon and the surface of the torsion The root cause of the folding; Li Jiuling and others believe that the crystal structure of sulfides often has imbalances in electricity prices, vacancies, etc., which provides the possibility that gold replaces one sulfur and shares electrons with another sulfur to enter the pyrite-type structure. Sex. The purpose of refractory gold ore pre-oxidation is to oxidize some or all of the pyrite coated on the outer surface of the gold in order to expose the gold for subsequent cyanide gold extraction. Therefore, it is necessary to study the ability of the selected thermophilic oxidized pyrite monomer minerals. At the end of the oxidation, the results of the oxidation of pyrite were observed by scanning electron microscopy. As shown in Figure 2. Figure 2 Comparison of morphology of pyrite before and after oxidation Before the leaching of pyrite, the mirror surface is smooth, the particles are intact, the edges are neat, and the structure is dense [Fig. 2(a)]. After 5 days of oxidation, the surface of the polished pyrite is slightly eroded [Fig. 2(b)] After 8 days of oxidation, the surface of the polished pyrite was severely eroded [Fig. 2(c)]; as the oxidation time of the bacteria increased, the degree of erosion of pyrite continued to deepen, and after 12 days of erosion, a cavity was formed. [Fig. 2(d)], the erosion formed for 16 d and was accompanied by cracks [Fig. 2(e)]. The voids and cracks formed gradually deepened, and the dense structure of pyrite was destroyed. For refractory gold mines, once the pyrite wrapped in gold single particles is oxidized by bacteria to form voids and cracks, the wrapped gold is exposed, and subsequent direct cyanidation gold extraction becomes very easy. During the oxidation of pyrite, the following reactions occur: It is seen from the reaction formula (1) that acid is generated during the oxidation, which causes the pH value of the solution to continuously decrease, so that the degree of oxidation of the pyrite can be examined from the degree of acid production. As shown in Figure 3. Figure 3 pH versus pyrite oxidation curve It can be seen from Fig. 3 that during the pre-oxidation of pyrite, the pH value of the solution is continuously decreased. After 140 h of pre-oxidation, the pH of the solution reaches 1.2. It can be seen from the test that after 16 days of biological pre-oxidation, the monomeric pyrite is oxidized by more than 60% to achieve the purpose of exposing the monomer gold. Fourth, the conclusion (1) Through a series of measures such as sampling, enrichment, separation and purification, an effective method for breeding high-efficiency thermophilic bacteria from nature was established, and finally a thermophilic strain was isolated and identified as a medium thermophilic strain of Sulfobacillus, a typical species. For Sulfobacillus thermosulfidooxidans, it plays an important role in the bioleaching process of metal sulfide ore. (2) Through the ability of the strain to oxidize pyrite, it is known that the selected thermophilic strain has the ability to pre-oxidize refractory gold ore, and is a highly active and efficient leaching functional bacteria. (3) In order to use the strain for industrial applications, it is necessary to carry out multiple transfer and domestication, and at the same time, it is necessary to domesticate its ability to withstand arsenic and tolerate other heavy metals in the subsequent test. 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