CN103667131A - Method for increasing metal ore leaching rate, and special strain thereof - Google Patents

Method for increasing metal ore leaching rate, and special strain thereof Download PDF

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CN103667131A
CN103667131A CN201310652265.5A CN201310652265A CN103667131A CN 103667131 A CN103667131 A CN 103667131A CN 201310652265 A CN201310652265 A CN 201310652265A CN 103667131 A CN103667131 A CN 103667131A
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ore
alicyclobacillus
genus bacillus
alicyclic acid
metallic ore
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CN103667131B (en
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刘双江
姜成英
彭堂见
郭旭
张明江
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Institute of Microbiology of CAS
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Abstract

The present invention discloses a method for increasing a metal ore leaching rate, and a special strain thereof. According to the present invention, Alicyclobacillus sp. SJ-68 is provided, the preservation number is CGMCC No.7682, and a bacterial agent for leaching the target metal from metal ore is further provided, and comprises Alicyclobacillus sp. SJ-68 described in the claim 1 and Acidithiobacillus caldus SM-1 described in the claim 1; and experiment results prove that the Alicyclobacillus sp. SJ-68CGMCC No.7682 can be separately used for leaching the valuable metal in the sulphide ore or be synergistically used with other strains so as to co-leach the valuable metal in the sulphide ore, can be used for deepening extraction of noble metals or rare metals in sulphide ore concentrates, abandoned mine, lean ore, metallurgical slag and difficultly-treated complex sulphide ores, and has important industrial application prospects in the biological mineral leaching field.

Description

A kind of method and special strain therefore thereof that improves metallic ore leaching rate
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special strain therefore thereof that improves metallic ore leaching rate.
Background technology
Biohydrometallurgy also claims Microorganism Leaching, be the effects such as the oxidation that utilizes certain micro-organisms or its meta-bolites to have some mineral (being mainly sulfide mineral) and element, reduction, dissolving, absorption (absorption), moltenly from ore soak metal or from water, reclaim valuable metal or remove the hydrometallurgy process of poisonous metal.The history of the existing many decades of its development, because the favorable factors such as cost is low, pollution-free, simple to operate are subject to people's attention day by day, become the modern technique of extracting the remarkable economy of having of multiple useful metal and environmental protection interests from low-grade, difficult ore, worldwide obtained popularization, improve and improved.
For a long time, great majority research all concentrates on the focal point of biological metallurgy process to have a liking for sour chemoautotrophic bacteria with application, and in research in recent years, finds also to exist in biological metallurgy environment some facultative heterotrophic bacteria and heterotrophic bacterium.Be separated at present a strain sulfur oxidizing bacterium Acidithiobacillus sp.SM-1, a strain iron-oxidizing bacteria Leptospirillum sp.LJ-1, the facultative heterotrophism iron of strain sulfur oxidizing bacterium Sulfobacillus sp.5-8 (Liu Yanyang., 2010) and the facultative heterotrophism iron-oxidizing bacteria of a strain have a liking for iron alicyclic acid genus bacillus TC-71.Wherein TC-71 is gram-positive microorganism, aerobic, product spore, containing on ferrous solid medium, is producing rust bacterium colony, and growth temperature and pH scope are respectively 22-40 ℃ and 1.0-5.0, and optimum temperuture and pH are respectively 28 ℃ and 2.0, can be with Fe 2+, K 2s 4o 6and pyrite is energy growth.16S rRNA comparison and phylogenetic tree build and show that TC-71 belongs to the novel species of alicyclic acid bacillus (Jiang et al., 2009).
Although about the physiological property of alicyclic acid Bacillus strain and the research of distributional environment existing (Albuquerque et al., 2000; Chen et al., 2004; Deinhard et al., 1987a & 1987b; Goto et al., 2002 & 2003; ), but the report that is not yet used at present biological metallurgy process relevant for alicyclic acid genus bacillus, effect for alicyclic acid genus bacillus in bioleaching process it be unclear that, thereby is necessary alicyclic acid genus bacillus to study in the effect of biological metallurgy process.
Summary of the invention
An object of the present invention is to provide a kind of alicyclic acid genus bacillus (Alicyclobacillus sp.).
Alicyclic acid genus bacillus provided by the invention (Alicyclobacillus sp.) SJ-68, its deposit number is CGMCC No.7682.
Another object of the present invention is to provide a kind of for leach the microbial inoculum of object metal from metallic ore.
Microbial inoculum provided by the invention, is comprised of above-mentioned alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 and happiness warm sour thiobacillus (Acidithiobacillus caldus) SM-1.
In above-mentioned microbial inoculum, the colony forming unit number of described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 and the sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 is than being 1:1.
Above-mentioned alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 or the application of above-mentioned microbial inoculum in leach object metal from metallic ore are also the scope of protection of the invention;
Or the application in object metal leaching rate in improving metallic ore of above-mentioned alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 or above-mentioned microbial inoculum is also the scope of protection of the invention.
In above-mentioned application, described metallic ore is metal sulphide ore deposit;
Described metal sulphide ore deposit is copper-sulphide ores (corresponding object metal is copper), pyrite (corresponding object metal is iron), arsenopyrite, copper glance, nickel sulfide ore, manganese glance, zinc sulfide ore or galena.
The 3rd object of the present invention is to provide a kind of method that leaches object metal from metallic ore.
Method provided by the invention is following 1) or 2):
1) comprise the steps: above-mentioned alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 to be inoculated in the substratum that contains metallic ore, cultivate, obtain free object metal ion or object metal simple-substance;
2) comprise the steps: the alicyclic acid genus bacillus in above-mentioned microbial inoculum (Alicyclobacillus sp.) SJ-68 and happiness warm sour thiobacillus (Acidithiobacillus caldus) SM-1 to like warm sour thiobacillus co-inoculation in the substratum that contains metallic ore, cultivate, obtain free object metal ion or object metal simple-substance.
In aforesaid method,
2), in, the warm sour thiobacillus inoculating cell number of described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 and the sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 happiness is than being 1:1.
In aforesaid method,
Described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 inoculates with alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 nutrient solution form;
The sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 is to like the warm sour thiobacillus nutrient solution form inoculation of warm sour thiobacillus (Acidithiobacillus caldus) SM-1 happiness.
In aforesaid method, described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 nutrient solution is that alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 cultivates the nutrient solution obtaining in autotrophy substratum; Culture medium prescription and cultural method are shown in one or two of embodiment 2;
The sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 nutrient solution is liked warm sour thiobacillus SM-1 for the warm sour thiobacillus of happiness (Acidithiobacillus caldus) and in sulfur-bearing autotrophy substratum, is cultivated the nutrient solution obtaining; Culture medium prescription and cultural method are shown in one or two of embodiment 2.
In aforesaid method, like warm sour thiobacillus (Acidithiobacillus caldus) SM-1 for happiness warm sour thiobacillus (Acidithiobacillus caldus) SM-1CGMCC1.7296.
In aforesaid method,
The described substratum that contains metallic ore is prepared as follows: metallic ore and basic medium are mixed to get to substratum, and the proportioning of described metallic ore and described basic medium is 30-50g:1L; Be specially 50g:1L or 30g:1L;
In aforesaid method, described basic medium is by MgSO 47H 2o, (NH 4) 2sO 4, CaCl 22H 2o, KH 2pO 4, trace element solution A and water forms; MgSO 47H 2the concentration of O in described basic medium is 0.5g/L, (NH 4) 2sO 4concentration in described basic medium is 0.2g/L, CaCl 22H 2o concentration is 0.25g/L, KH 2pO 4concentration in described basic medium is 3.0g/L, and the concentration of trace element solution A in described basic medium is 2ml/L;
Trace element solution A is by CaCl 22H 2o, CuSO 45H 2o, H 3bO 3, MnSO 44H 2o, Na 2moO 42H 2o, CoCl 26H 2o, ZnSO 47H 2o and water form; CaCl 22H 2the concentration of O in trace element solution A is 0.66g/L, CuSO 45H 2the concentration of O in trace element solution A is 0.16g/L, H 3bO 3concentration in trace element solution A is 0.1g/L, MnSO 4h 2the concentration of O in trace element solution A is 0.15g/L, Na 2moO 42H 2the concentration of O in trace element solution A is 0.3g/L, CoCl 26H 2the concentration of O in trace element solution A is 0.18g/L, ZnSO 47H 2the concentration of O in trace element solution A is 0.18g/L.
Described metallic ore is for through the following metallic ore of processing: by metallic ore with acid carry out acidification to acidizing fluid pH value between 1.8-2.0, maintain 24h constant till; The acid that described acidification adopts is that volume ratio is the H of 1:1 2sO 4the aqueous solution;
The particle diameter of described metallic ore is 45 μ m-50 μ m.
In aforesaid method, described metallic ore is metal sulphide ore deposit;
Described metal sulphide ore deposit is specially copper-sulphide ores, pyrite, arsenopyrite, copper glance, nickel sulfide ore, manganese glance, zinc sulfide ore or galena.
Bacterial strain provided by the present invention is alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68, and its patent deposit number is CGMCC No.7682.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 7th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.7682, this strain classification called after (Alicyclobacillus sp.) SJ-68.
In application or method in leach object metal from metallic ore, applicable to various metals ore deposit, if metal sulphide ore deposit is copper-sulphide ores, pyrite, arsenopyrite, copper glance, nickel sulfide ore, manganese glance, zinc sulfide ore or galena, its principle is that bacterial strain of the present invention has accelerated Fe in ore leaching process 2+with the oxidation of reducible sulfur, and then accelerated oxidation dissolution of minerals, make object metal free out.
Of the present invention experimental results show that, the present invention has found that this bacterium of bacterial strain alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682 can grow in low pH value, middle temperature, high salinity media, this bacterium can be worked in coordination with cupric ion in other bacterial strain Chalcopyrite Leachings, and leaching rate is 28.2%; Also can jointly from pyrite (sulphur concentrate), leach iron ion, iron leaching rate is 64.1%.
Alicyclic acid genus bacillus provided by the invention (Alicyclobacillus sp.) SJ-68CGMCC No.7682 has and has a liking for sour growth characteristics, can utilize ferrous iron or reduced sulfur compounds to grow as the energy, also can utilize yeast extract, the organic compound such as glucose are grown, can improve from least two aspects the oxidation efficiency of sulfide mineral: first, this bacterium not only can utilize mineral compound but also can utilize organic compound growth, conform extensively, therefore can in biological metallurgy environment, well grow, can promote the growth of other autotrophys in environment or heterotrophism leaching microbacteria simultaneously, the second, when this bacterium and other ore immersion bacterial cultures exist jointly, due to its stronger oxidation capacity, accelerate ferrous iron oxidation and generate strong oxidizer Fe 3+, for soaking ore deposit process, provide good redox environment, thereby improved, soak ore deposit speed.In addition, the oxidable Sulfur of bacterial strain SM-1CGMCC1.7296 jointly using with SJ-68CGMCC No.7682 in the present invention, in bioleaching process, can assist to eliminate the passivation phenomenon of sulphur, sulphur is oxidized can generate sulfuric acid, be conducive to maintain the sour environment of biological metallurgy, and bacterial strain of the present invention has acid resistance, therefore, in bioleaching process, there is stronger activity, be more conducive to improve leaching efficiency.
In sum, alicyclic acid genus bacillus of the present invention (Alicyclobacillus sp.) SJ-68CGMCC No.7682 can separately or work in coordination with other bacterial classifications and jointly leach the valuable metal in sulphide ores, not only can be for sulphide concentrate, also can be used for abandoned mine, lean ore, mining and metallurgy waste residue, extract with the in-depth of precious metal or rare metal in difficult complicated sulfuration mine, this bacterium has important prospects for commercial application in bioleaching field.
Accompanying drawing explanation
Fig. 1 is SJ-68 scanning (A) and transmission (B) electromicroscopic photograph
Fig. 2 is the growth OD value of SJ-68 when different pH
Fig. 3 is the growth OD value of SJ-68 when differing temps
Fig. 4 is the growth OD value of SJ-68 when different N aCl concentration
Fig. 5 is copper ion concentration typical curve
When Fig. 6 is the warm sour thiobacillus SM-1 leaching copper-sulphide ores of bacterial strain SJ-68 and happiness, in leaching liquid, copper ion concentration changes
Fig. 7 is ferrous iron concentration standard curve
The variation of total concentration of iron when Fig. 8 is the warm sour thiobacillus SM-1 leaching pyrite of bacterial strain SJ-68 and happiness
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The Isolation and Identification of embodiment 1, alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 bacterium
One, separation
Bacterial strain of the present invention is from Tengchong County, Chinese Yunnan Province, to be rich in separation and purification the hot spring periphery muddy water sample of sulphur to obtain, and the separation and purification time is on September 18th, 2012.
The enrichment of microorganism, by add 100mL autotrophy substratum in the shaking flask of 250mL, takes 5 grams of hot spring muddy water samples and adds sterilizing autotrophy substratum, 30 ℃ of enrichment culture.Cultivate after one week, detect under the microscope bacteria growing situation.After enrichment three times, by 10 times of enrichment medium dilutions, the substratum of getting after 0.2mL dilution is coated with plate isolation mono-clonal, adds the Gelrite glue of 7g/L on the basis of solid medium liquid medium within.Streak culture by repeatedly, separation and purification obtains mono-clonal bacterial strain, by this strain number, is SJ-68.
Autotrophy liquid nutrient medium forms: add following material, and water polishing volume: in basic medium, adding final concentration is 13.9g/L Fe 2sO 47H 2o or final concentration are 5g/L Sulfur;
Autotrophy solid medium is that in autotrophy liquid nutrient medium, to add final concentration be 7g/L gelrite glue.
If autotrophy liquid nutrient medium adopts Sulfur, autotrophy solid medium is prepared as follows: in basic medium, adding final concentration is that 10g/L potassium tetrathionate and final concentration are 7g/L gelrite glue.
Basic medium forms: add following material and reach following final concentration, water polishing volume: MgSO wherein 47H 2the concentration of O in basic medium is 0.5g/L, (NH 4) 2sO 4concentration in basic medium is 0.2g/L, CaCl 22H 2the concentration of O in basic medium is 0.25g/L, KH 2pO 4concentration in basic medium is 3.0g/L, and the concentration of trace element solution A in basic medium is 2ml/L; Substratum 1mol/L H 2sO 4adjust pH to 2.5, autoclaving.
Trace element solution A is by CaCl 22H 2o, CuSO 45H 2o, H 3bO 3, MnSO 44H 2o, Na 2moO 42H 2o, CoCl 26H 2o, ZnSO 47H 2o and water form; CaCl 22H 2the concentration of O in trace element solution A is 0.66g/L, CuSO 45H 2the concentration of O in trace element solution A is 0.16g/L, H 3bO 3concentration in trace element solution A is 0.1g/L, MnSO 4h 2the concentration of O in trace element solution A is 0.15g/L, Na 2moO 42H 2the concentration of O in trace element solution A is 0.3g/L, CoCl 26H 2the concentration of O in trace element solution A is 0.18g/L, ZnSO 47H 2the concentration of O in trace element solution A is 0.18g/L.
Two, the evaluation of bacterium
(1) bacterium Morphology and physiology biochemical character is identified
(1) morphologic observation
Colony shape: the dilution spread on solid plate of the SJ-68 bacterium in autotrophy liquid nutrient medium is cultivated to 7d-10d, observe the shape, size, pigment formation, transparency of bacterium colony, moistening, edge and projection.
Result is: on autotrophy substratum solid plate, bacterium colony is rounded, be that rust, surface are more dry, and bacterium colony is complete, big or small 0.3-0.55mm.
Morphological specificity: utilize the form of scanning electronic microscope and transmission electron microscope observation logarithmic phase somatic cells and flagellum raw situation.As shown in Figure 1, SJ-68 scans (A) and SJ-68 transmission (B) electromicroscopic photograph to result, can find out that cell is rod-short (0.5 – 0.6 * 1.0 – 1.3mm), and atrichia forms gemma.
(2) bacterial strain SJ-68 physiological and biochemical property
Growth pH: first determine the pH scope of growth, 250ml triangular flask, every bottle adds 50ml autotrophy substratum, uses H 2sO 4pH is adjusted to 0,1,2,3,4,5,6,7,8,9 respectively with NaOH, inoculum size is 3%(v/v), 30 ℃ of baking ovens are cultivated.Cultivate after three days, with ultraviolet-visible pectrophotometer, measure OD 520, and draw pH curve.Determine the optimal pH of growth, 250ml triangular flask, adds 50ml autotrophy substratum in every bottle again, and within the scope of the pH that can grow at thalline, pH is by 0.5 variation, inoculum size 3%(v/v), 30 ℃ of baking ovens cultivations, measured OD with ultraviolet spectrophotometer after 3 days 520, and draw pH curve.Each pH do three parallel.Cultivate proof: bacterial strain SJ-68 growth pH scope is 2.0-5.5, and the most suitable growth pH value is 2.5, as Fig. 2.
Growth temperature: under best pH condition, carry out growth temperature experiment.In 500ml triangular flask, add 100ml autotrophy substratum, inoculum size 3%(v/v), 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ of cultivations.After 3 days, with ultraviolet-visible pectrophotometer, measure OD 520, observe growing state.Cultivate proof: bacterial strain SJ-68 growth temperature range is 20-40 ℃, and 30 ℃ of optimum growth temperatures, as Fig. 3.
Growth NaCl concentration: 250ml triangular flask, every bottle adds 50ml autotrophy substratum, 0,5,10,20,30,40,50g/L NaCl concentration is respectively:, pH and culture temperature all adopt the optimum condition of experiment gained, inoculum size 3%(v/v), each group all arrange three parallel, separately have an aseptic contrast.Cultivate and after 3 days, with ultraviolet-visible pectrophotometer, measure the OD of bacterium liquid in each bottle 520, observe growing state.Cultivate proof: the NaCl concentration of bacterial strain SJ-68 growth is 0-40g/L, as shown in Figure 4.
Gramstaining: adopt Dussault method, add 20% salt solution on slide glass, after smear is air-dry, cover 5min with 2% acetic acid, fixing and desalination, removes unnecessary acetic acid, dries.Simple stain is directly with luxuriant red or Viola crystallina covering 3min, and washing, dries rear microscopy; Gramstaining dyes 90s by Viola crystallina, washing, and iodine liquid covers, washing, alcohol decolouring 20s, washing, the luxuriant red 90s that redyes, dries microscopy after washing.Experimental results show that bacterial strain SJ-68 Gram-reaction is positive.
Organic substrates utilizes: inoculation 5% autotrophy SJ-68 thalline is in containing the basic medium of corresponding substrate to be measured (2g/L), and pH is 2.5, under optimum temperuture condition, cultivates, and measures OD 520, determine whether growth.Carbon source used (substrate to be measured) is respectively: sucrose, polychrom, L-(+)-pectinose, cellobiose, D-(+)-semi-lactosi, fructose, melizitose, D-Glucose, maltose, N.F,USP MANNITOL, D-MANNOSE, raffinose, inose, lactose, sorbose, fructose, D-(-)-ribose, glycerine, erythritol, Xylitol, extractum carnis, yeast extract and fish peptone etc.
Experimental result shows: bacterial strain SJ-68 can utilize organic substrates extractum carnis, yeast extract and fish peptone, and L-(+)-pectinose, D-(+)-semi-lactosi, D-Glucose, maltose, D-MANNOSE, sorbose, fructose, D-(-)-ribose, the various saccharides such as glycerine and erythritol are energy heterotrophic growth.
Inorganic substrate utilization: K 2o 6s 4, FeSO 47H 2o, Na 2s 2o 35H 2o and elemental sulfur add respectively in basic medium as energy substance, and inoculation 5% autotrophy SJ-68 thalline, cultivates under pH2.0 and optimum temperuture condition, measures OD 520, determine whether growth.
Experimental result shows: SJ-68 can utilize FeSO 47H 2o, elemental sulfur and reduced form sulfide K 2s 4o 6for the growth of energy aerobic autotrophic.
(2) the 16S rRNA gene order of bacterial strain SJ-68 is carried out to Phylogenetic Analysis
Bacterial strain SJ-68 is inoculated in to heterotrophism substratum (formula sees below description), after growing 3 days, centrifugal collection thalline, after damping fluid washing, adopt N,O-Diacetylmuramidase smudge cells, phenol: chloroform extraction method extracts genome DNA and as template, adopts the 16S rRNA gene of bacterium primer 2 7F and 1492R amplification strains A r-4.Forward primer 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', reverse primer 1492R:5'-GGTTACCTTGTTACGACTT-3'.
PCR reaction system (100 μ l): 10 * Taq enzyme Buffer10 μ l, 25mmol/L MgCl 26 μ l, 10mmol/L dNTP2 μ l, Taq DNA enzyme 2.5U, each 2 μ l of 30pmol/L upstream and downstream primer, template DNA 2 μ g, distilled water is mended to 100 μ l.PCR reaction conditions is: 95 ℃ of 5min, 95 ℃ of 1min, 58 ℃ of 5min, 72 ℃ of 1.5min, 30 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Amplified production carries out column purification after agarose gel electrophoresis separation, is connected and is transformed in E.coil DH5 α with T-easy vector, and screening positive clone, extracts plasmid order-checking, and order-checking is completed by Shanghai bio-engineering corporation.
The 16S rRNA gene order length of bacterial strain is 1420bp, as shown in sequence in sequence table 1.SJ-68 and Alicyclobacillus belong to the similarity of bacterial strain between 91.5%-99.5%, and the sequence similarity of A.ferripilum TC-71 is maximum, is 99.5%.Take 16S rRNA DNA homolog as basis, according to Phylogenetic Analysis, show, the bacterial classification that SJ-68 and alicyclic acid genus bacillus Alicyclobacillus belong to is polymerized to one, belongs to the member that Alicyclobacillus belongs to.
To sum up qualification result shows, bacterial strain SJ-68 of the present invention belongs to the bacterial classification of alicyclic acid bacillus (Alicyclobacillus), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 7th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.7682, and the Classification And Nomenclature of this bacterial strain is (Alicyclobacillus sp.) SJ-68.
Three, cultural method
(1) autotrophy is cultivated
Autotrophy substratum forms: in basic medium, add FeSO 47H 2o or elemental sulfur (sulphur) or reduced form sulfide K 2s 4o 6, obtain autotrophy substratum, and water polishing volume; FeSO 47H 2the concentration of O in autotrophy substratum is 13.9g/L, and the concentration of elemental sulfur in autotrophy substratum is 5g/L, reduced form sulfide K 2s 4o 6concentration in autotrophy substratum is 10g/L; Adjust pH is that 2.0-3.5(specifically can be adjusted to 2.5).。
Cultural method: alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682 is inoculated in autotrophy substratum, cultivates under the condition of 30 ℃.
(2) heterotrophism is cultivated
Heterotrophism substratum forms: in basic medium, add yeast extract and glucose, the proportioning of yeast extract and basic medium is 0.2g:1L; The proportioning of glucose and basic medium is 0.5g:1L, and water polishing volume; Adjust pH is that 2.5-3.5(specifically can be adjusted to 3), autoclaving.Wherein, yeast extract and glucose are energy substance.
Cultural method: alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682 is inoculated in heterotrophism substratum, cultivates under 30 ℃ of conditions.
Result: during autotrophy is cultivated, bacteria concentration OD 520reach 0.6 and (be about 8.7 * 10 7cfu/ml), need 5-7 days; During heterotrophism is cultivated, bacteria concentration OD 520reach 0.6 and (be about 8.7 * 10 7cfu/ml), need 3-5 days.The fast growth of bacterium in heterotrophism cultivation.
The application of embodiment 2, alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682
One, SJ-68 combines the application of happiness warm sour thiobacillus (Acidithiobacillus aldus) .SM-1 in dissolving copper-sulphide ores
1, the preparation of raw material and inoculation culture liquid
Yeast extract is purchased from Oxoid Ltd, and catalog number is 1039501.
The ore of copper-sulphide ores forms: Cu0.8%, Fe2.5%, S3%; % represents quality percentage composition.
Before experiment, copper-sulphide ores is crossed to 300 mesh sieves (particle mean size is 48 μ m), then uses the volume ratio of 1:1(acid and water) H 2sO 4acidified aqueous solution, to the acidizing fluid pH value obtaining between 1.8-2.0, maintain 24h constant till; Probably need 3-5 days; Again by the copper-sulphide ores ultraviolet sterilization after acidifying.
For soaking alicyclic acid genus bacillus (Alicyclobacillus sp.) the SJ-68CGMCC No.7682 nutrient solution of ore deposit inoculation, for being cultivated to the nutrient solution obtaining for 5 days 30 ℃ of autotrophy substratum, alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682 (is about 6 * 10 7cfu/ml).
The composition of autotrophy substratum: add FeSO in basic medium 47H 2o, obtains autotrophy substratum, and water polishing volume; FeSO 47H 2the concentration of O in autotrophy substratum is 13.9g/L; The pH value of autotrophy substratum is 2.5.
For soaking the sour thiobacillus of happiness temperature (Acidithiobacillus aldus) SM-1(CGMCC1.7296 of ore deposit inoculation) nutrient solution is for (being about 6.5 * 10 by liking the nutrient solution that warm sour thiobacillus (Acidithiobacillus aldus) SM-1 obtains in 30 ℃ of cultivations of sulfur-bearing autotrophy substratum for 5 days 7cfu/ml).
Sulfur-bearing autotrophy substratum forms (every L substratum contains): 3.0g (NH 4) 2sO 4, 0.5g K 2hPO 43H 2o, 0.5g MgSO 47H 2o, 0.1g KCl, 0.01g Ca (NO 3) 2, 5g sulfur(sulphur), 10ml trace element solution B (filtration sterilization), water is supplied volume; With 1:1 aqueous sulfuric acid, regulate pH to 2.5.
10mL trace element solution B formula is: 11mg FeCl 36H 2o, 0.5mg CuSO 45H 2o, 2.0mg H 3bO 3, 2.0mg MnSO 4h 2o, 0.8mg Na 2moO 42H 2o, 0.6mg CoCl 26H 2o, 0.9mg ZnSO 47H 2o, supplies 10mL with distilled water.
2, dissolve copper-sulphide ores
Experimental group 1:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, (pulp density is the proportioning of copper-sulphide ores and basic medium to add the copper-sulphide ores of ultraviolet sterilization again, be specially 50g:1L), obtain the substratum that contains metallic ore, alicyclic acid genus bacillus (Alicyclobacillus sp.) the SJ-68CGMCC No.7682 nutrient solution that adds again above-mentioned cultivation to obtain afterwards for 5 days, making initial inoculum is 1.0 * 10 7cell/ml(initial medium), 37 ℃ of cultivations, are and soak ore body system; Cu is carried out in sampling every day (being leach liquor) 2+concentration analysis.
Experimental group 2:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, the copper-sulphide ores (proportioning of copper-sulphide ores and basic medium is 50g:1L) that adds again ultraviolet sterilization, obtain the substratum that contains metallic ore, the sour thiobacillus of happiness temperature (Acidithiobacillus caldus) the SM-1CGMCC No.1.7296 nutrient solution that adds again above-mentioned cultivation to obtain afterwards for 5 days, making initial inoculum is 1.0 * 10 7cell/ml(initial medium), 37 ℃ of cultivations, are and soak ore body system; Cu is carried out in sampling every day (being leach liquor) 2+concentration analysis.
Experimental group 3:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, the copper-sulphide ores (proportioning of copper-sulphide ores and basic medium is 50g:1L) that adds again ultraviolet sterilization, obtain the substratum that contains metallic ore, add SJ-68 nutrient solution and the SM-1 nutrient solution cultivated after 5 days, wherein SJ-68 and the SM-1 concentration in basic medium is 0.5 * 10 again 7cell/ml(initial medium); 37 ℃ of cultivations, are and soak ore body system; Cu is carried out in sampling every day (being leach liquor) 2+concentration analysis.
The concentration that leaches cupric ion adopts bisoxalydihydrazone (BCO) spectrophotometry, and copper ion concentration typical curve (mg/L) is shown in Fig. 5.
Control group: except not adding bacterium, all the other are identical with experimental group 3;
Cu in leaching rate=leach liquor 2+concentration (g/L)/(Cu percentage composition % in pulp density (g/L) * ore);
Pulp density in this experiment is 50g/L, and in ore, Cu percentage composition % is 0.8%, and in pulp density (g/L) * ore, Cu percentage composition % is for soaking Cu concentration=50g*0.8%=0.4g/L in ore body system.
3 repetitions are established in experiment, and result is taken the mean.As shown in Figure 6, within the 14th day, ((inoculating and be denoted as the 0th day the same day) the 14th day from inoculating meter on the same day) cultivation results is specific as follows for result:
Bacterial strain SJ-68(is experimental group 1), in leach liquor, the concentration of copper is 64.22mg/L, copper leaching rate is about 16.1%; Bacterial strain SM-1(is experimental group 2), in leach liquor, the concentration of copper is 54.74mg/L, copper leaching rate is 13.7%; In bacterial strain SJ-68 and SM-1 synergy (being experimental group 3) leach liquor, the concentration of copper is 112.91mg/L, and copper leaching rate is 28.2%; And aseptic control group only has acid to do the used time, it is close that the mono-bacterium of copper leaching rate and SJ-68 and SM-1 does the used time, is 13.0% (in leach liquor, the concentration of copper is 51.97mg/L).Result shows, bacterial strain SJ-68 and SM-1 can act synergistically, and improves Cu in copper-sulphide ores 2+leaching yield.
Two, the pyritous ability of the common dissolving of SJ-68 and Acidithiobacillus aldus SM-1
1, the preparation of raw material and inoculation culture liquid
Pyritous ore forms: Fe33.79%, S27.93%, Cu0.012%, As0.008%; % represents quality percentage composition.
Before experiment, pyrite is crossed to 300 mesh sieves (particle mean size is 48 μ m), then use (volume ratio of acid and water) H 2sO 4acidified aqueous solution, to the acidizing fluid pH value obtaining between 1.8-2.0, maintain 24h constant till; Probably need 3-5 days; Again by the pyrite ultraviolet sterilization after acidifying.
For soaking alicyclic acid genus bacillus (Alicyclobacillus sp.) the SJ-68CGMCC No.7682 nutrient solution of ore deposit inoculation, for being cultivated to the nutrient solution obtaining for 5 days 30 ℃ of autotrophy substratum, alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68CGMCC No.7682 (is about 6 * 10 7cfu/ml).
The composition of autotrophy substratum: add FeSO in basic medium 47H 2o, obtains autotrophy substratum, and water polishing volume; FeSO 47H 2the concentration of O in autotrophy substratum is 13.9g/L; The pH value of autotrophy substratum is 2.0.
For soaking the sour thiobacillus of happiness temperature (Acidithiobacillus aldus) SM-1(CGMCC1.7296 of ore deposit inoculation) nutrient solution is for (being about 6.5 * 10 by liking the nutrient solution that warm sour thiobacillus (Acidithiobacillus aldus) SM-1 obtains in 30 ℃ of cultivations of SM-1 substratum for 5 days 7cfu/ml).
Sulfur-bearing autotrophy substratum forms (every L substratum contains): 3.0g (NH 4) 2sO 4, 0.5g K 2hPO 43H 2o, 0.5g MgSO 47H 2o, 0.1g KCl, 0.01g Ca (NO 3) 2, 5g sulfur(sulphur), 10ml trace element solution B (filtration sterilization), water is supplied volume; With 1:1 aqueous sulfuric acid, regulate pH to 2.5.
10mL trace element solution B formula is: 11mg FeCl 36H 2o, 0.5mg CuSO 45H 2o, 2.0mg H 3bO 3, 2.0mg MnSO 4h 2o, 0.8mg Na 2moO 42H 2o, 0.6mg CoCl 26H 2o, 0.9mg ZnSO 47H 2o, supplies 10mL with distilled water.
2, dissolve pyrite
Experimental group 1:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, (pulp density is the proportioning of pyrite and basic medium to add the pyrite of ultraviolet sterilization again, be specially 30g:1L), obtain the substratum that contains metallic ore, alicyclic acid genus bacillus (Alicyclobacillus sp.) the SJ-68CGMCC No.7682 nutrient solution more above-mentioned cultivation being obtained for 5 days is afterwards inoculated into substratum, and making initial inoculum is 1.0 * 10 7cell/ml(initial medium) (initial medium), 37 ℃ of cultivations, are and soak ore body system; Sampling every day (being leach liquor), carries out Fe 2+and Fe 3+concentration analysis.
Experimental group 2:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, the pyrite (proportioning of pyrite and basic medium is 30g:1L) that adds again ultraviolet sterilization, obtain the substratum that contains metallic ore, the sour thiobacillus of happiness temperature (Acidithiobacillus caldus) the SM-1CGMCC No.1.7296 nutrient solution more above-mentioned cultivation being obtained for 5 days is afterwards inoculated into substratum, and making initial inoculum is 1.0 * 10 7cell/ml(initial medium), 37 ℃ of cultivations, are and soak ore body system; Sampling every day (being leach liquor), carries out Fe 2+and Fe 3+concentration analysis.
Experimental group 3:500mL Erlenmeyer flask adds the sterilizing basic medium that 100mL pH value is 2.0, the pyrite (proportioning of pyrite and basic medium is 30g:1L) that adds again ultraviolet sterilization, obtain the substratum that contains metallic ore, SJ-68 nutrient solution and the SM-1 nutrient solution cultivated after 5 days are all inoculated into substratum, wherein the proportioning of SJ-68 and SM-1 and described basic medium is respectively 0.5 * 10 again 7cell/ml(initial medium), 37 ℃ of cultivations, are and soak ore body system; Fe is carried out in sampling every day (being leach liquor) 2+and Fe 3+concentration analysis.
Soak Fe in the process of ore deposit 2+concentration adopts 1,10-spectrophotometric determination o-phenanthroline, Fe 2+concentration standard curve (mg/L) is shown in Fig. 7.
Total iron (Fe in leach liquor 2+and Fe 3+) measure: with oxammonium hydrochloride by Fe wherein 3+reduction generates Fe 2+, then measure by 1,10-nonyl phenol.
Control group 1: except not adding bacterium, all the other are identical with experimental group;
Total concentration of iron (g/L)/(Fe percentage composition in pulp density (g/L) * ore) % in leaching rate=leach liquor;
Pulp density in this experiment is 30g/L, and in ore, Fe percentage composition is 33.79%, and in pulp density (g/L) * ore, Fe percentage composition is Fe concentration=30g*33.79%=10.137g/L in Leaching Systems.
3 repetitions are established in experiment, and result is taken the mean.Result as shown in Figure 8.Specific as follows through 14 days cultivation results (from inoculation meter on the same day (inoculation was denoted as the 0th day the same day) the 14th day):
Experimental group 1: the 14 days, in bacterial strain SJ-68 leach liquor, total concentration of iron is 3.595g/L, iron leaching rate is 35.5%.
Experimental group 2: the 14 days, in bacterial strain SM-1 leach liquor, total concentration of iron is 1.321g/L, iron leaching rate is 13.0%.
Experimental group 3: the 14 days, in the pyritous leach liquor of the collaborative leaching of bacterial strain SJ-68 and SM-1, total concentration of iron is 6.494g/L, iron leaching rate is 64.1%.
Control group is from inoculation meter on the same day (inoculation was denoted as the 0th day the same day), and the 14th day, in leach liquor, total concentration of iron was 1.105g/L, and iron leaching rate is 10.9%.
The above results shows, bacterial strain SJ-68 and SM-1 can act synergistically, and improves pyritous dissolving power.
Figure IDA0000431016790000021

Claims (10)

1. alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68, its deposit number is CGMCC No.7682.
2. for leach a microbial inoculum for object metal from metallic ore, by alicyclic acid genus bacillus claimed in claim 1 (Alicyclobacillus sp.) SJ-68 and happiness warm sour thiobacillus (Acidithiobacillus caldus) SM-1CGMCC1.7296, formed.
3. microbial inoculum according to claim 2, is characterized in that: the colony forming unit number of described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 and the sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1CGMCC1.7296 is than being 1:1.
4. the application of microbial inoculum in leach object metal from metallic ore described in alicyclic acid genus bacillus claimed in claim 1 (Alicyclobacillus sp.) SJ-68 or claim 2 or 3;
Or microbial inoculum application in object metal leaching rate in improving metallic ore described in alicyclic acid genus bacillus claimed in claim 1 (Alicyclobacillus sp.) SJ-68 or claim 2 or 3.
5. application according to claim 4, is characterized in that: described metallic ore is metal sulphide ore deposit;
Described metal sulphide ore deposit is specially copper-sulphide ores, pyrite, arsenopyrite, copper glance, nickel sulfide ore, manganese glance, zinc sulfide ore or galena.
6. from metallic ore, leaching a method for object metal, is following 1) or 2):
1) comprise the steps: alicyclic acid genus bacillus claimed in claim 1 (Alicyclobacillus sp.) SJ-68 to be inoculated in the substratum that contains metallic ore, cultivate, obtain free object metal ion or object metal simple-substance;
2) comprise the steps: the alicyclic acid genus bacillus in microbial inoculum described in claim 2 or 3 (Alicyclobacillus sp.) SJ-68 and like warm sour thiobacillus (Acidithiobacillus caldus) SM-1 co-inoculation in the substratum that contains metallic ore, cultivate, obtain free object metal ion or object metal simple-substance.
7. method according to claim 6, is characterized in that:
2), in, described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 and the sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 inoculating cell number are than being 1:1.
8. according to the method described in claim 6 or 7, it is characterized in that:
Described alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 inoculates with alicyclic acid genus bacillus (Alicyclobacillus sp.) SJ-68 nutrient solution form;
The sour thiobacillus of described happiness temperature (Acidithiobacillus caldus) SM-1 is to like the inoculation of warm sour thiobacillus (Acidithiobacillus caldus) SM-1 nutrient solution form.
9. according to arbitrary described method in claim 6-8, it is characterized in that:
The described substratum that contains metallic ore is prepared as follows: it is 30-50g:1L that metallic ore and basic medium are mixed to get to the proportioning of metallic ore and described basic medium described in substratum
Described metallic ore is for through the following metallic ore of processing: by metallic ore with acid carry out acidification to acidizing fluid pH value between 1.8-2.0, maintain 24h constant till; The acid that described acidification adopts is that volume ratio is the H of 1:1 2sO 4the aqueous solution;
The particle diameter of described metallic ore is 45 μ m-50 μ m.
10. according to arbitrary described method in claim 6-9, it is characterized in that:
Described metallic ore is metal sulphide ore deposit;
Described metal sulphide ore deposit is specially copper-sulphide ores, pyrite, arsenopyrite, copper glance, nickel sulfide ore, manganese glance, zinc sulfide ore or galena.
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CN105779325A (en) * 2014-12-25 2016-07-20 北京有色金属研究总院 Fatty acid bacillus and method for treatment of heavy metal pollution of mine by in situ mineralization of the same
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CN102162029A (en) * 2011-01-21 2011-08-24 天津工业生物技术研究所 Microbiological oxidation and reduction coupling leaching method for valuable metal in manganese oxide ore

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WO2010012030A1 (en) * 2008-07-29 2010-02-04 Bhp Billiton Ssm Development Pty Ltd Process for controlled homogeneous acid leaching
CN101851612A (en) * 2010-04-20 2010-10-06 中国农业科学院饲料研究所 Acid glucanase CELA and gene and application thereof
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CN105779325A (en) * 2014-12-25 2016-07-20 北京有色金属研究总院 Fatty acid bacillus and method for treatment of heavy metal pollution of mine by in situ mineralization of the same
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CN106754490A (en) * 2016-12-03 2017-05-31 青岛漾花湖农业科技有限公司 A kind of culture medium for accelerating Thiobacillus thioxidans propagation
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