CN106754458B - Fluoride-resistant mineral leaching bacteria combination and engineering continuous expansion culture method and application thereof - Google Patents
Fluoride-resistant mineral leaching bacteria combination and engineering continuous expansion culture method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorine-resistant mineral leaching bacteria combination and an engineering continuous expansion culture method and application thereof, wherein the fluorine-resistant mineral leaching bacteria combination is composed of Acidithiobacillus ferrooxidans Retech KF-I, Acidithiobacillus ferrooxidans Retech KF-II, Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans Retech Co-culture KF-III. The bacterium combination can adapt to the growth of acidic fluorine-containing conditions and can rapidly oxidize Fe2+Is Fe3+The ability of the cell to perform. The engineering continuous enlarging culture equipment can be used for carrying out the scale culture of the bacteria combination to produce the leaching bacteria and Fe3+The bacterial strain cultivated by the method has stable activity and good adaptability, and can be widely applied in the field of biological metallurgy.
Description
Technical Field
The invention relates to the technical field of biological metallurgy, in particular to a fluorine-resistant leaching ore mixed bacterium combination, and an engineering continuous expansion culture method and application thereof.
Background
The biological metallurgy technology has the characteristics of cleanness, short flow and low cost, and is commonly used for extracting valuable metals such as copper, uranium, molybdenum, vanadium, cobalt, zinc and the like in ores. At present, ore leaching bacteria are about twenty kinds, and most of the ore leaching bacteria belong to chemoautotrophic bacteria. The most commonly used leaching bacteria are Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum. The bacteria all live by various inorganic salts in the growth and reproduction processes, can obtain energy required by the life process by oxidizing ferrous iron, elemental sulfur, reduced compounds and the like, and does not need any organic nutrient substances. Most ore leaching strains have the property of growing under an acidic condition and have different growth temperatures which are favored, such as the medium temperature of 20-35 ℃, the medium thermophilic bacteria of 40-55 ℃ and the extreme thermophilic bacteria of 55-85 ℃.
Fluorine-containing ores such as fluorite, fluorapatite and the like are often accompanied by copper and uranium ores, and fluorine is easily dissolved under acidic conditions. Fluorine has a strong inhibitory effect on bacterial activity. In order to improve the leaching efficiency of valuable metals in fluorine-containing ores, strains with strong tolerance to fluorine need to be obtained by adopting a biological metallurgy technology. In particular, in engineering applications, strains with stable bacterial oxidative activity are required.
After the strains cultured and domesticated in a laboratory are in a new environment for engineering application, the problems of long adaptation time, low bacterial activity and the like often exist. The conventional method can adjust and control the temperature, pH, ventilation amount, inoculation amount and the like to obtain the optimal culture effect. Although the growth external conditions of bacteria are improved, the slow period of the growth of bacteria can be shortened to a certain extent, and the activity of bacteria is still limited. Furthermore, the strains introduced in the biological metallurgical process are often not continuously produced, which has a certain influence on the maintenance of bacterial activity.
Disclosure of Invention
The invention aims to provide a fluorine-resistant mineral-leaching bacteria combination which can rapidly oxidize Fe2+Is Fe3+And can endure an environment with a total fluorine concentration of 3.0g/L, exhibit a stable bacterial oxidation activity, and is suitable for ore metallurgy with accompanying contents of fluorite, fluorapatite, and the like.
Another object of the present invention is to provide a culture medium and a culture method for domesticating and/or culturing fluoride-leaching-resistant bacteria in a laboratory.
The invention also aims to provide a method and equipment for engineering continuous expansion culture of the fluorine-leaching-resistant mixed bacteria combination.
The fourth purpose of the invention is to provide the application of the fluorine-leaching-resistant mixed bacteria combination.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a fluorine-resistant mineral leaching bacteria combination, which is prepared from Retech KF-I, KF-II and Retech KF-III according to the proportion of 1:1:1 in proportion;
wherein, the classification name of the strain of Retech KF-I is as follows: acidithiobacillus ferrivorans Retech KF-I, the preservation unit is: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 7, month 1 in 2013, the preservation number is: CGMCC No. 7835;
the classification name of the strain of Retech KF-II is as follows: acidithiobacillus ferrooxidans Retech KF-II, the preservation unit is: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 7, month 1 in 2013, the preservation number is: CGMCC No. 7836;
retech KF-III is a mixed culture of Acidithiobacillus ferrooxidans and Leptospira ferriphila, with the preservation units: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 11/14/2014, with a deposit number of: CGMCC No. 9998.
Still further, the fluoride-leaching resistant bacteria combination has a total fluoride tolerance concentration of 3.0 g/L.
The invention also provides a culture medium for domesticating and/or culturing the fluorine-resistant leaching-ore bacteria combination in a laboratory stage, which comprises the following components: ca (NO)3)2 0.01g/L～0.10g/L，MgSO4·7H2O0.50g/L～20.0g/L，K2HPO4·3H2O 0.10g/L～1.0g/L，Al2(SO4)3·18H2O10.0g/L～50.0g/L，Fe2(SO4)30.0g/L～1.90g/L，NH4F1.60-10.0 g/L and FeSO4·7H2O14.70g/L～44.20g/L。
The invention also provides a method for domesticating and/or culturing the fluorine-resistant mineral-leaching bacteria combination on the engineering application site, which is characterized in that fluorine-containing pit water or tail liquid is used for inoculating the fluorine-resistant mineral-leaching bacteria combination, the pH value is 1.5-2.5, the temperature is 30-35 ℃, and the air filling amount is 0.5-5.0 m3/(m2H), the inoculation amount of the mature bacterial liquid is 5-30%, and Fe is controlled2+The concentration range is 0.5 g/L-5.0 g/L.
The invention also provides a process for engineering expanded culture of the fluorine-resistant leaching ore bacteria combination, which comprises the following steps:
1) preparing a culture solution: adopting fluorine-containing mine pit water or tail liquid in a proportioning tank to control Fe in the solution2+Preparing culture solution required by the combined growth of the fluoride-leaching-resistant bacteria with the concentration range of 0.5 g/L-5.0 g/L;
2) inoculating bacteria: inoculating a bacterial liquid which is a combination of the culture solution prepared in the step 1) and the fluorine-resistant leaching bacteria into an inoculation groove, wherein the inoculation volume of the bacterial liquid is 5-30% of the volume of the culture solution;
3) and (3) rapid oxidation growth: the oxidation-reduction potential of the solution reaches more than 350mV, which indicates that the fluoride-resistant leaching-ore bacteria combination passes through the adaptation period, and then the fluoride-resistant leaching-ore bacteria combination enters an oxidation tank for rapid oxidation growth until the concentration of the bacteria liquid reaches 108The oxidation-reduction potential of the solution reaches more than 450mV when the strain/mL is more than one, which indicates that the bacterial liquid grows into a stable period;
4) mature bacterial liquid use and return inoculation: enabling the stationary-phase bacterial liquid to enter a finished product tank, wherein 5% -30% of mature bacterial liquid returns to the inoculation tank, and the rest is subjected to ore leaching operation;
wherein, the inoculation tank, the oxidation tank and the finished product tank control the culture conditions: pH1.5-2.5, growth temperature 30-35 deg.C, and air filling amount 0.5-5.0 m3/(m2·h)。
The invention also provides a system for industrial continuous enlarged culture of leaching bacteria, which comprises: a batching tank, a inoculation tank, an oxidation tank and a finished product tank;
wherein, the proportioning tank, the inoculation tank, the oxidation tank and the finished product tank are all provided with a pH monitoring device and an oxidation-reduction potential monitoring device, the tanks are connected in sequence through connecting pipes, and each connecting pipe is provided with a valve for flow control; the inoculation tank, the oxidation tank and the finished product tank are all provided with a temperature rising device, a temperature monitoring device, an air flow meter and a microporous aerator; the inoculation groove and the oxidation groove are both provided with sewage outlets;
a stirring device is arranged in the batching tank and is used for mixing the components of the culture medium; a connecting pipe is positioned at the upper part of the batching tank and is used for inputting culture medium components;
the inoculation groove is also connected with a bacteria liquid return pipe for receiving partial bacteria liquid returned by the finished product groove
The finished product tank is connected with a bacteria liquid discharge pipe, an acid-resistant pump and a fluid meter are arranged on the bacteria liquid discharge pipe, part of the measured bacteria liquid is output for ore leaching treatment, and the other part of the measured bacteria liquid returns to the inoculation tank through a bacteria liquid return pipe.
Furthermore, the pH monitoring device is an acidimeter, the oxidation-reduction potential monitoring device is an oxidation-reduction potentiometer, the heating device is a heating rod, the temperature monitoring device is a temperature sensor, and the temperature control range is 10-90 ℃.
The equipment has the function of continuously culturing mineral leaching bacteria, can culture mesophilic bacteria, moderate thermophilic bacteria and extreme thermophilic bacteria, and can produce finished bacteria liquid of 1.0m every day3～50.0m3Bacteria concentration up to 107～109One strain per mL, and the strain return inoculation amount is 5-30%.
The invention also provides application of the fluorine-resistant mineral leaching bacteria combination in biological metallurgy of fluorine-containing mines.
The fluorine-resistant mineral leaching bacteria combination used by the invention is obtained by adopting the following method:
obtaining of fluoride-resistant mineral leaching bacteria
The fluorine-resistant leaching ore strains used by the invention are respectively enriched in fluorine-containing ore areas. The fluorine-resistant domestication of the strain adopts asynchronous domestication, and the tolerance of the strain is improved by gradually increasing the concentration of fluorine.
1. Laboratory stage acclimation/culture:
in the domestication stage in the laboratory, the low-fluorine domestication and the high-fluorine domestication are respectively carried out on each bacterium in the fluoride-leaching-resistant bacterium combination, so that the total fluorine concentration of each bacterium in the fluoride-leaching-resistant bacterium combination is 3.0 g/L.
1) Acclimation at low fluorine concentration:
addition of soluble fluoride to F in 9K Medium-Adjusting the pH value to 2.0-2.5 at a concentration of 0.05g/L, inoculating strains, placing in an air bath constant temperature oscillation incubator at a temperature of 33 ℃, a shaker rotation speed of 160rpm, counting the growth of bacteria by a microscope until the logarithmic phase is reached, and adding Fe in the culture solution2+Culturing for 5-10 times when the concentration is less than 0.2g/L and the fluorine ion resistance concentration is 0.05g/L, and oxidizing Fe by strain2+To Fe3+The time of (2) is shortened to be within 2d, and the concentration of bacteria reaches 106More than one cell/mL, and increase F when the fluorine-resistant growth of the bacteria is stable-Concentration, F-Increasing the concentration by 100-200 mg/L each time, repeating the acclimatization step until the strain pair F-The tolerance of (A) is up to 1.0 g/L.
Wherein the components and the concentrations of the 9K culture medium are respectively as follows: (NH)4)2SO4 3.0g/L，KCl 0.1g/L，K2HPO4 0.5g/L，MgSO4·7H2O 0.5g/L，Ca(NO3)2 0.01g/L，FeSO4·7H2O44.20 g/L, ammonium fluoride (or other substances which can be dissolved in water and generate free fluorine, such as sodium fluoride, potassium fluoride and the like) is taken as a fluorine source, and F is prepared-The concentration is 0.05g/L to 1.0g/L, each domestication interval is determined according to domestication conditions, the total fluorine concentration is monitored, and domestication under high fluorine concentration is carried out when the fluorine resistance of the strain reaches 1.0 g/L.
2) Acclimation at high fluorine concentration:
preparing a high-fluorine culture medium: the culture medium takes the components of the wastewater produced in the fluorine-containing mining area as reference values, and the components are as follows: ca (NO)3)2 0.01g/L～0.10g/L，MgSO4·7H2O 0.50g/L～20.0g/L，K2HPO4·3H2O0.10g/L～1.0g/L，Al2(SO4)3·18H2O 10.0g/L～50.0g/L，Fe2(SO4)3 0.0g/L～1.90g/L，NH4F1.60-10.0 g/L and FeSO4·7H2O 14.70g/L～44.20g/L。
Adjusting the pH value of the high-fluorine culture medium to 2.0-2.5, inoculating the strain domesticated with low fluorine concentration, putting the strain into an air bath constant-temperature oscillation incubator, keeping the temperature at 33 ℃, rotating the shaker at 160rpm, counting the bacteria growth by a microscope until the strain oxidizes Fe2+To Fe3+The time of (2) is shortened to be within 2d, and the bacterial concentration reaches 106More than one cell/mL, and increase F when the fluorine-resistant growth of the bacteria is stable-Concentration, F-Increasing the concentration by 200-500 mg/L each time, repeating the acclimatization step until the strain pair F-Finally, the tolerance is 3.0 g/L.
The step further improves the fluorine resistance and adaptability of the mineral leaching bacteria.
2. Acclimatization and culture in engineering field
Mixing the fluoride-leaching-resistant bacteria domesticated in a laboratory according to a proportion to form a fluoride-leaching-resistant bacteria combination, and domesticating and culturing on an engineering site.
1) Acclimatization of engineering application site
Inoculating a fluorine-resistant mineral leaching bacteria combination subjected to fluorine-resistant domestication in a laboratory into fluorine-containing pit water or tail liquid, controlling the pH to be 1.5-2.5, controlling the temperature to be 30-35 ℃, and controlling the amount of air to be filled to be 0.5-5.0 m3/(m2H) controlling the concentration of fluorine in the solution to be 0.5-5.0 g/L and controlling Fe2+The concentration range is 0.5 g/L-5.0 g/L, when the bacterial oxidation activity reaches more than 0.5 g/(L.h), the acclimatization is completed.
The engineering field domestication of the strain can adapt to the growth of industrial heap leaching field production wastewater, so that the bacterial activity is adapted and kept improved, and simultaneously, the water resource can be saved, thereby being beneficial to environmental protection.
The culture process can produce mature bacteria liquid continuously, maintain stable bacteria activity and culture bacteria in enlarged scale.
2) Engineering continuous scale-up culture
A system for engineering continuous enlarging culture of mineral leaching bacteria is used for continuous enlarging culture of fluorine-resistant mineral leaching bacteria combination.
Preparing the nutrient solution required by the combined growth of the fluorine-resistant leaching-ore bacteria in a batching tank, specifically fluorine-containing mine water or tail solution, controlling the concentration range of fluorine in the solution to be 0.5-5.0 g/L, and controlling Fe2+The concentration is 0.5 g/L-5.0 g/L; inoculating a bacterial liquid which is a combination of the prepared culture solution and the fluorine-resistant leaching-ore bacteria into an inoculation groove, wherein the inoculation volume of the bacterial liquid is 5-30% of the volume of the culture solution; the oxidation-reduction potential of the solution reaches more than 350mV, which indicates that the fluoride-resistant leaching-ore bacteria combination passes through the adaptation period, and then the fluoride-resistant leaching-ore bacteria combination enters an oxidation tank for rapid oxidation growth until the concentration of the bacteria liquid reaches 108The oxidation-reduction potential of the solution reaches more than 450mV when the strain/mL is more than one, which indicates that the bacterial liquid grows into a stable period; enabling the stationary-phase bacterial liquid to enter a finished product tank, wherein 5% -30% of mature bacterial liquid returns to the inoculation tank, and the rest is subjected to ore leaching operation;
wherein, inoculation groove, oxidation groove, finished product groove are installed pH monitoring device, redox potential monitoring device, rising temperature device, temperature monitoring device, air flowmeter and micropore aerator for control culture condition is: the pH value is 1.5-2.5, the growth temperature is 30-35 ℃, and the aeration rate of the air is 0.5-5.0 m3/(m2H) wherein the product tank is run for 0.5m3/(m2H) micro-aeration to prevent bacterial decay.
The system and the process divide functional areas according to the growth cycle (divided into a lag phase, a logarithmic growth phase, a stationary phase and a decline phase) of the ore leaching bacteria, and reasonably control: (1) preparing a culture solution area, preparing nutrient substances required by the growth of ore leaching bacteria, and uniformly mixing the materials by matching with a stirring device; (2) inoculating a bacterial area, inoculating ore leaching strains and nutrient solution required by the growth of the ore leaching strains, and matching with an aeration device and a heating device; (3) the rapid oxidation growth area is matched with an aeration device and a heating device, and the volume of the rapid oxidation growth area can be adjusted according to the production requirement; (4) and (5) outputting mature bacterial liquid and returning to inoculate. The culture conditions are as follows: pH of 1.5-2.5, temperature of 30-35 deg.C, and air charging amount of 0.5-5.0 m3/(m2H), the inoculation amount of the mature bacterial liquid is 5-30%. The fluorine-resistant mineral-leaching bacteria are preferably selectedThe expanding culture process comprises the following steps: pH2.1, temperature 33 deg.C, and aeration air amount 2.0m3/(m2H), the mature bacterial liquid is returned to the inoculum size of 10%.
The cultured product contains fluoride-resistant mineral-leaching bacteria and Fe3+The solution of (4) has an oxidation effect on the reduced metal minerals.
The Acidithiobacillus ferrooxidans Retech KF-I, the Acidithiobacillus ferrooxidans Retech KF-II, and the mixed culture of Acidithiobacillus ferrooxidans and Leptospirillum Retech KF-III exist in acid mine water generally, are common leaching bacteria, and are non-toxic and harmless to the environment and people.
The invention has the beneficial effects that:
the fluorine-resistant mineral-leaching-bacteria combination provided by the invention has the advantages that each strain in the combination has strong fluorine resistance and high bacterial oxidation activity. The engineering continuous enlarging culture method effectively maintains the oxidation activity of the strain, conveniently and flexibly provides fresh bacterial liquid, restrains the loss of bacteria caused by entering the decay period, prevents the generation of precipitates such as iron vitriol and the like, and has good biological metallurgy application effect.
Drawings
FIG. 1 is a schematic diagram of a continuous enlarged culture system for fluoride-leaching-resistant bacteria combined engineering provided by the invention.
FIG. 2 shows the situation of maintaining the iron oxidation activity of bacteria in the fluorine-resistant leaching-ore bacteria combination and the engineering continuous amplification culture process thereof.
Reference numerals:
1: a batching tank; 2: a seed receiving tank; 3: an oxidation tank; 4: a finished product groove; 5: a stirring device; 6: a microporous aerator; 7: a heating rod; 8: a temperature sensor; 9: pH acidity meter: 10: an oxidation-reduction potentiometer; 11: an air flow meter; 12: a liquid flow meter; 13: an acid-resistant pump; 14: a connecting pipe; 15: a sewage draining outlet; 16: an injection pipe; 17: a bacteria liquid access pipe; 18: a bacteria liquid return pipe; 19: a bacteria liquid output pipe; 20: air.
Detailed Description
The present invention will be described in detail with reference to examples. The embodiments are for better understanding of the present invention and are not intended to limit the scope of the present invention, and any equivalent or known modifications are included in the scope of the present invention.
As shown in FIG. 1, the process of continuous scale-up culture using the engineered continuous scale-up culture system provided by the present invention comprises: putting inorganic chemical components or production wastewater required by the growth of ore leaching bacteria into a batching tank 1 through an injection pipe 16, uniformly mixing by using a stirring device 5, adjusting the pH value of the solution to a range suitable for the growth of the bacteria by using a pH acidimeter 9, and controlling the oxidation-reduction potential to be in the range suitable for the growth of the bacteria by using an oxidation-reduction potentiometer 10 to prepare a culture medium; a culture medium enters the inoculation groove 2 through a connecting pipe 14, a bacterium liquid enters the inoculation groove 2 through an access pipe 17, aeration is carried out through a microporous aerator 6, the entering amount of air 20 is controlled by an air flow meter 11, the temperature is adjusted to be suitable for the growth of bacteria through a temperature control device (such as a heating rod 7 and a temperature sensor 8), and the pH and the oxidation-reduction potential in the inoculation groove 2 are controlled by a pH acidity meter 9 and an oxidation-reduction potentiometer 10; after the bacteria degree adaptation period, the bacteria enter the oxidation tank 3 through the connecting pipe 14, the micropore aerator 6, the pH acidity meter 9, the oxidation-reduction potentiometer 10 and the temperature control device are used for regulating and controlling the growth conditions of the bacteria, mature bacteria liquid is finally cultured and enters the finished product tank 4, the finished product bacteria liquid is classified through the acid-proof pump 13 and the fluid meter 12, part of the bacteria liquid is introduced through the bacteria liquid output pipe 19, and part of the bacteria liquid is connected to the bacteria liquid access pipe 17 through the bacteria liquid return pipe 18 and returns to the inoculation tank 2, and meanwhile, a culture cycle is completed.
Example 1 fluoride-tolerant acclimatization and expansion culture of fluoride-tolerant mineral-leaching bacteria combinations
1) Collecting:
collecting leaching bacteria liquid from pit water and ore of fluorine-containing mine, inoculating the leaching bacteria liquid in 9K culture medium, adjusting pH to 2.0, and placing in air bath constant temperature oscillation incubator at 33 deg.C and shaking table rotation speed of 160 rpm. Counting the growth of bacteria to logarithmic phase by microscope and Fe in culture solution2+When the concentration is lower than 0.2g/L, concentrating the leaching bacteria liquid, and inoculating into a new 9K culture medium for culture. The operation is carried out until the mineral leaching bacteria grow for a weekThe period is shortened to less than 2d and tends to be stable.
Placing the cultured mineral leaching bacteria liquid into an Eppendorf Centrifuge 5804R type refrigerated Centrifuge, centrifuging the bacteria liquid at a low speed of 1000rpm for 5min to remove solid precipitates such as jarosite and the like, then centrifuging at a high speed of 11000rpm for 5min to obtain bacteria, and then performing fluorine-resistant domestication on the three bacteria respectively.
2) Acclimation at low fluorine concentration:
preparing 9K culture medium, adding ammonium fluoride until the fluorine ion concentration is 0.05g/L, adjusting pH to 2.0, inoculating mineral leaching bacteria, placing into air bath constant temperature shaking incubator at 33 deg.C, shaking table rotation number of 160rpm, and counting bacteria growth to logarithmic phase with microscope, and Fe in culture solution2+When the concentration is lower than 0.2g/L, the culture with the fluorine ion resistant concentration of 0.05g/L is carried out for more than 5 times, the operation is carried out until the growth cycle of the mineral leaching bacteria is shortened to be within 2d, and the bacteria concentration reaches 106More than one cell/mL, when the fluorine-resistant growth of the thalli is stable; increase F-Concentration, each time increasing F-The domestication operation is repeated according to the level with the concentration of 100mg/L until the fluoride ion resistant concentration of the ore leaching bacteria reaches 1.0 g/L.
3) Acclimation at high fluorine concentration:
changing a high-fluorine culture medium for domestication, wherein the specific culture medium is as follows: ca (NO)3)20.01g/L，MgSO4·7H2O12.54g/L，K2HPO4·3H2O 0.50g/L，Al2(SO4)3·18H2O 40.0g/L，Fe2(SO4)3 1.90g/L，NH4F5.85 g/L and FeSO4·7H2O 44.20g/L。
Adjusting the pH value of the high-fluorine culture medium to 2.0-2.5, inoculating the strain domesticated with low fluorine concentration, putting the strain into an air bath constant-temperature oscillation incubator, keeping the temperature at 33 ℃, rotating the shaker at 160rpm, counting the bacteria growth by a microscope until the strain oxidizes Fe2+To Fe3+The time of (2) is shortened to be within 2d, and the bacterial concentration reaches 106More than one cell/mL, and increase F when the fluorine-resistant growth of the bacteria is stable-Concentration, F-Increasing the concentration by 500mg/L each time, repeating the acclimatization step untilStrain pair F-Finally, the tolerance is 3.0 g/L.
The step further improves the fluorine resistance and adaptability of the mineral leaching bacteria.
4) Engineering field domestication:
mixing three domesticated bacteria according to the proportion of 1:1:1, and culturing by using fluorine-containing pit water or tail liquid under the culture conditions that: the pH value is 2.1, the growth temperature is 33 ℃, and the air filling amount is 2.0m3/(m2H), the bacterial ferrous oxide activity reaches more than 0.5g/(L h), which indicates the completion of acclimatization.
The method is carried out in an industrial production field, and the fluorine resistance of the leaching strain is improved and the method is adaptive to the field environment.
Example 2 Industrial scale-up culture
The continuous enlarged culture system provided by the invention is utilized, as shown in figure 1, uranium extraction tail liquid is added into a proportioning tank 1, and 22.10Kg/m of industrial ferrous sulfate is supplemented3To supplement Fe2+. Mixing uniformly by a stirring device 5, adjusting the pH to 2.1 +/-0.05, introducing into an inoculation tank 2, inoculating bacteria, inoculating fluorine-resistant mineral leaching bacteria with the total amount of 10%, and aerating at 2.0m3/(m2H), temperature 33. + -. 1 ℃; after the bacteria is in the adaptation period, the bacteria enters an oxidation tank 3 with the aeration rate of 2.0m3/(m2H), the temperature is 33 +/-1 ℃, and a rapid growth stage is started; finally, the mixture enters a finished product tank 4 for micro-aeration with the aeration rate of 0.5m3/(m2H) to prevent bacterial decay. Finally, the seed returning and introduction operation is performed by the acid-proof pump 13. Continuously producing finished bacterial liquid 1.2m every day3The concentration of bacteria reaches 10 by microscopic examination8More than one/mL, can meet the introduction requirement.
As shown in fig. 2, the scale is 1.20m3Continuous operation of the engineering amplification culture for one month, and measuring the bacterial oxidation Fe every day2 +Is Fe3+The highest rate of the bacterial oxidation activity reaches about 1.4 g/(L.h), and the bacterial oxidation activity is relatively stable.
Example 3: heap bioleaching of uraninite containing fluorine
The biological heap leaching of a certain uranium ore has the heap construction scale of 4500t, the fluorine content in the ore is 2.2 percent, and the uranium grade is 0.2 percent.
The fluorine-resistant leaching ore bacteria provided by the invention are used for the biological heap leaching test of the uranium ore after being subjected to engineering continuous amplification culture. The conventional leaching rate of uranium is improved to more than 90% from about 85%, the leaching time is shortened by 30-50 days compared with the prior art, and the consumption of sulfuric acid is reduced to about 4% from about 6% originally.
The invention has the advantages that: the fluorine-resistant mineral leaching bacteria combination provided by the invention can quickly oxidize Fe after engineering continuous amplification culture2+Is Fe3+Provides fresh strains for biological heap leaching, can obviously improve leaching efficiency and reduce production cost. The strain is cultured by using the waste water, thereby realizing the recycling of the waste liquid and reducing the environmental protection pressure.
Claims (5)
1. The fluorine-resistant mineral leaching bacteria combination is characterized in that the bacteria combination is prepared by mixing Retech KF-I, Retech KF-II and Retech KF-III according to the proportion of 1:1:1 in proportion;
wherein, the classification name of the strain of Retech KF-I is as follows:Acidithiobacillus ferrivorans retech KF-I, the preservation unit is: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 7, month 1 in 2013, the preservation number is: CGMCC number 7835;
the classification name of the strain of Retech KF-II is as follows:Acidithiobacillus ferrooxidansretech KF-II, the preservation unit is: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 7, month 1 in 2013, the preservation number is: CGMCC number 7836;
retech KF-III is a mixed culture of ferrous sulfate oxide bacillus and leptospirillum ferriphilum, and the preservation unit is as follows: china general microbiological culture Collection center, addresses are: the preservation date of No. 3 Xilu Beijing Xiyang district, China academy of sciences, microbial research institute is as follows: 11/14/2014, with a deposit number of: CGMCC number 9998;
wherein the total fluorine tolerance concentration of the fluorine-resistant mineral bacteria combination is more than 3.0 g/L.
2. A method for domesticating and/or culturing the fluorine-resistant mineral bacteria combination according to claim 1 in an engineering application site, which is characterized in that the fluorine-resistant mineral bacteria combination is inoculated in fluorine-containing pit water or tail liquid, the inoculation amount is 5-30%, and Fe2+The concentration range is 0.5 g/L-5.0 g/L, and the culture conditions are as follows: pH of 1.5-2.5, temperature of 30-35 deg.C, and air charging amount of 0.5-5.0 m3/(m2∙ h), when the bacterial oxidation activity reaches more than 0.5g/(L ∙ h), the acclimatization is completed.
3. A process for engineering expanded culture of the fluoride-leaching-resistant bacteria combination of claim 1, comprising the steps of:
1) preparing a culture solution: controlling Fe by adopting fluorine-containing pit water or tail liquid in proportioning tank2+Preparing culture solution required by the combined growth of the fluoride-leaching-resistant bacteria with the concentration range of 0.5 g/L-5.0 g/L;
2) inoculating bacteria: inoculating a bacterial liquid which is a combination of the culture solution prepared in the step 1) and the fluorine-resistant leaching bacteria into an inoculation groove, wherein the inoculation volume of the bacterial liquid is 5-30% of the volume of the culture solution;
3) and (3) rapid oxidation growth: the oxidation-reduction potential of the solution reaches more than 350mV, which indicates that the fluoride-resistant leaching-ore bacteria combination passes through the adaptation period, and then the fluoride-resistant leaching-ore bacteria combination enters an oxidation tank for rapid oxidation growth until the concentration of the bacteria liquid reaches 108The oxidation-reduction potential of the solution reaches more than 450mV when the strain/mL is more than one, which indicates that the bacterial liquid grows into a stable period;
4) mature bacterial liquid use and return inoculation: the bacteria liquid in the stabilization phase enters a finished product tank to become mature bacteria liquid, the inoculation amount of the mature bacteria liquid is 5-30%, and the rest is subjected to ore leaching operation;
wherein the culture conditions of the inoculation tank, the oxidation tank and the finished product tank are controlled to be pH1.5-2.5, the growth temperature is 30-35 ℃, and the amount of the air is 0.5-5.0 m3/(m2∙h)；
The system for engineering continuous enlarged culture of leaching bacteria adopted by the process comprises: a batching tank, a inoculation tank, an oxidation tank and a finished product tank;
wherein, the proportioning tank, the inoculation tank, the oxidation tank and the finished product tank are all provided with a pH monitoring device and an oxidation-reduction potential monitoring device, the tanks are connected in sequence through connecting pipes, and each connecting pipe is provided with a valve for flow control; the inoculation tank, the oxidation tank and the finished product tank are all provided with a temperature rising device, a temperature monitoring device, an air flow meter and a microporous aerator; the inoculation groove and the oxidation groove are both provided with sewage outlets;
a stirring device is arranged in the batching tank and is used for mixing the components of the culture medium; the batching tank is connected with an injection pipe for inputting the components of the culture medium;
the inoculation groove is also connected with a bacteria liquid injection pipe and is used for receiving partial bacteria liquid returned by the finished product groove through a bacteria liquid return pipe;
the finished product tank is connected with a bacteria liquid output pipe, an acid-proof pump and a fluid meter are arranged on the bacteria liquid output pipe, part of the bacteria liquid after measurement is output for ore leaching treatment, and the other part of the bacteria liquid returns to the inoculation tank through a bacteria liquid return pipe and a bacteria liquid access pipe;
wherein the culture conditions of the inoculation tank, the oxidation tank and the finished product tank are controlled to be pH2.1, temperature 33 deg.C, and air volume of 2.0m3/(m2∙h)；
The amount of the mature bacterial liquid returned to inoculation is 10%.
4. The process of claim 3, wherein the pH monitoring device is an acidimeter, the oxidation-reduction potential monitoring device is an oxidation-reduction potentiometer, the temperature raising device is a heating rod, the temperature monitoring device is a temperature sensor, and the temperature control range is 10 ℃ to 90 ℃.
5. Use of the fluoride-leaching-resistant bacteria combination according to claim 1 in the biometally of a fluoride-containing mine.
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