CN108795801A - Culture method of acidophilic thiobacillus ferrooxidans - Google Patents
Culture method of acidophilic thiobacillus ferrooxidans Download PDFInfo
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- CN108795801A CN108795801A CN201810563734.9A CN201810563734A CN108795801A CN 108795801 A CN108795801 A CN 108795801A CN 201810563734 A CN201810563734 A CN 201810563734A CN 108795801 A CN108795801 A CN 108795801A
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- 241000605222 Acidithiobacillus ferrooxidans Species 0.000 title claims abstract description 66
- 238000012136 culture method Methods 0.000 title 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims abstract description 55
- 230000003647 oxidation Effects 0.000 claims abstract description 35
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 29
- 230000000694 effects Effects 0.000 claims abstract description 20
- 230000000638 stimulation Effects 0.000 claims abstract description 14
- 230000008859 change Effects 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 79
- 239000000243 solution Substances 0.000 claims description 62
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 60
- 238000011081 inoculation Methods 0.000 claims description 28
- 235000015097 nutrients Nutrition 0.000 claims description 27
- 239000012530 fluid Substances 0.000 claims description 25
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 24
- 230000033116 oxidation-reduction process Effects 0.000 claims description 22
- 241000605118 Thiobacillus Species 0.000 claims description 15
- 230000002906 microbiologic effect Effects 0.000 claims description 11
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000446 fuel Substances 0.000 claims description 8
- 238000005424 photoluminescence Methods 0.000 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000013589 supplement Substances 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- 229910052564 epsomite Inorganic materials 0.000 claims description 4
- 239000005864 Sulphur Substances 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 230000005611 electricity Effects 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 3
- 238000005070 sampling Methods 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 2
- 239000007788 liquid Substances 0.000 abstract 1
- 238000012544 monitoring process Methods 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 10
- 229910052742 iron Inorganic materials 0.000 description 9
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 8
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 7
- 238000002798 spectrophotometry method Methods 0.000 description 7
- 238000006213 oxygenation reaction Methods 0.000 description 6
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000004936 stimulating effect Effects 0.000 description 5
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 4
- 238000006477 desulfuration reaction Methods 0.000 description 4
- 230000023556 desulfurization Effects 0.000 description 4
- 229910001385 heavy metal Inorganic materials 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000010802 sludge Substances 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003245 coal Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- NIFIFKQPDTWWGU-UHFFFAOYSA-N pyrite Chemical compound [Fe+2].[S-][S-] NIFIFKQPDTWWGU-UHFFFAOYSA-N 0.000 description 2
- 229910052683 pyrite Inorganic materials 0.000 description 2
- 239000011028 pyrite Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 206010022971 Iron Deficiencies Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- -1 iron ion Chemical class 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 229910052935 jarosite Inorganic materials 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052976 metal sulfide Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for culturing acidophilic thiobacillus ferrooxidans, which comprises the steps of obtaining high-activity high-concentration acidophilic thiobacillus ferrooxidans through common shake culture and continuous passage in the first stage, continuously culturing under the synergistic action of applied potential in the second stage, monitoring the change of cathode potential in a culture system during the period, regularly sampling and measuring the ferrous oxidation rate, the bacterial concentration and the redox potential, and carrying out transfer passage on the acidophilic thiobacillus ferrooxidans liquid subjected to electrochemical stimulation to obtain high-density and high-ferrous oxidation rate strains.
Description
Technical field
The present invention relates to a kind of cultural methods of Acidithiobacillus ferrooxidans, belong to biotechnology.
Background technology
Prokaryota, chemotrophy prokaryotes door, bacterium guiding principle, sulphur in Acidithiobacillus ferrooxidans microorganism belonging to genus
Change bacterium section, Thiobacillus.Single bacterium is in rod-short, and length 1 to a few micrometers is about 0.5 micron wide, two sections of blunt circles, amphitrichous,
It can vivaciously move, belong to Gram-negative bacteria, mainly utilize CO2For carbon source, and the inorganic nutrients such as absorbed nitrogen, phosphorus synthesize itself
Cell.Acidithiobacillus ferrooxidans have been foster chemosynthetic bacterias, its most suitable growth pH is 1.5~2.5, most adaptability
Long temperature is 28~35 DEG C.
With the research that deepens continuously to Acidithiobacillus ferrooxidans physio-biochemical characteristics, it has been found that such micro- life
Object suffers from important application prospect in metallurgical industry and environmental protection etc..Microorganism can be through a variety of ways to mineral
It is acted on, the ion converted the valuable element in mineral in solution.In the environment of metal sulfide mine, acidophilia oxygen
Change ferrous Thiobacillus to play an important roll the oxygenolysis of sulfide.Know that there are many oxidation of pyrite bacterium at present, they can
In the case of aerobic, energy is obtained by oxidation of elemental sulfur, pyrite, iron ion etc., and by fixed carbon or other is had
Machine nutrients and grow, wherein Acidithiobacillus ferrooxidans be still considered as in acidic environment soak mine leading strain.This
Outside, Acidithiobacillus ferrooxidans can be used for handling the heavy metal in sludge, utilize Acidithiobacillus ferrooxidans pair
The oxidation-adsorption of heavy metal acts on, and using bioleaching, can not only reduce acid consumption, but also heavy metal removing rate is high.Most
It is new and to import external source inorganic iron studies have shown that by adding Acidithiobacillus ferrooxidans endogenous in rich sludge, to a huge sum of money
The sludge for belonging to pollution carries out bioleaching, can effectively remove heavy metal, and can be made into after sludge dewatering iron-holder be up to 6%~
8% biological organic state iron fertilizer, can be used as calacareous soil iron deficiency rescues agent.Since the pollution of current atmospheric sulfur dioxide is tight
Weight, Acidithiobacillus ferrooxidans can also be applied to desulfurization technology, reduce the discharge of sulfur dioxide.Coal desulfurization master at present
Chemical method is relied on, although desulfurization degree is higher, reaction needs hot conditions, cost higher.It is aoxidized using acidophilia
The biological desulfurization of ferrous Thiobacillus, sulphur that can be in effective place to go coal, and reaction condition is mild, and processing cost is low.
Although Acidithiobacillus ferrooxidans have good application prospect in environmental project, till now absolutely
Most of researchs are still in the laboratory research stage.Main cause is as follows：Being of Acidithiobacillus ferrooxidans can be certainly first
Bacteria, growth and breeding speed is slow, Oxidation of Fe2+Speed is low, is the major obstacle for limiting engineer application.Secondly, acidophilia oxidation is sub-
The activity of iron Thiobacillus is easy effected by environmental factors, such as is easily influenced by temperature, pH, above or below optimum growh
When temperature (30~35 DEG C), the growth of bacterium almost stops；For another example when pH value is less than 1.5, bacterial growth just by strong inhibition,
But when more than 2.5, Fe3+Hydrolysis generates a large amount of precipitations, other substances such as As in reaction solution3+、Cu2+Deng presence also can be different
Inhibit to degree the growth of bacterium.Finally, work as Fe2+It can inhibit the activity of Acidithiobacillus ferrooxidans when excessively high so that sub-
Iron oxidation rate is slack-off, and conversion ratio is also relatively low, this is to the Fe for preparing high concentration3+Reaction solution causes difficulty, and the Fe of high concentration3+
Be that prepare bio-polymeric ferric sulfate, biological desulphurization necessary, at the same be also to speed up biology leach it is necessary.
Invention content
Present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of culture sides of Acidithiobacillus ferrooxidans
Method, the cultural method can obtain the Acidithiobacillus ferrooxidans of high density and high ferrous oxidation rate.
Technical solution
A kind of cultural method of Acidithiobacillus ferrooxidans, includes the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, be inoculated with acidophilia ferrous oxide
Then the initial bacterium solution of Thiobacillus is cultivated in 28~32 DEG C, the constant-temperature table of 170~190r/min, culture to bacterium solution
Rufous is deep to by light green color change and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：Seed liquor made from step (1) is taken, Fe containing 4.5g/L is inoculated into2+9K fluid nutrient mediums in,
Then it is cultivated in 28~32 DEG C, the constant-temperature table of 170~190r/min, obtains bacterium solution；
(3) collection of thalline：When the oxidation-reduction potential of the bacterium solution of step (2) reaches 560mV, filtered immediately,
Filtered filtrate is centrifuged, high activity Acidithiobacillus ferrooxidans thalline is obtained；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode
Room is separated between cathode chamber and anode chamber with proton exchange membrane, and anode working electrode is added into anode chamber and is free of Fe2+9K
Cathode working electrode, magnetic rotor and Fe containing 4.5g/L are added into cathode chamber for culture medium2+9K culture mediums, toward contain 4.5g/L
Fe2+9K inoculation of medium step (3) high activity Acidithiobacillus ferrooxidans thalline, after inoculation, 28~32 DEG C,
It is cultivated under conditions of 170~190r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By the anode and cathode of data collector respectively with the moon
Pole working electrode is connected with anode working electrode, the external resistor for 50 Ω that connect between two working electrodes, data acquisition
After device is powered, the oxidation-reduction potential of cathode chamber bacterium solution is measured every 12h, it is micro- by dual chamber after current potential reaches 540~560mV
Biological fuel cell injection port supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is separately connected cathode work
Electrode and reference electrode are stimulated under the outer plus constant potential of 0.1~0.6V, and stimulation time is 50~60h, is obtained additional
Bacterium solution after electro photoluminescence；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+
9K fluid nutrient mediums in, secondary culture is carried out in 28~32 DEG C, the constant-temperature table of 170~190r/min to get high density
With the Acidithiobacillus ferrooxidans of high ferrous oxidation rate.
In step (1), the inoculum concentration of the initial bacterium solution of Acidithiobacillus ferrooxidans is 8~10v%.
In step (1), (2), (4) and (6), the Fe containing 4.5g/L2+9K fluid nutrient mediums formula：3g/L
(NH4)2SO4, 0.5g/L MgSO4·7H2O, 0.5g/L K2HPO4, 0.1g/L KCl, 0.01g/LCa (NO3)2, 4.5g/L
FeSO4·7H2O, it is 1.8 to adjust pH.
In step (2), inoculum concentration is 8~10v%.
In step (3), the rotating speed of centrifugation is 8000~12000r/min, and the time is 10~20min.
It is described to be free of Fe in step (4)2+9K culture mediums formula：3g/L(NH4)2SO4, 0.5g/L MgSO4·
7H2O, 0.5g/L K2HPO4, 0.1g/L KCl, 0.01g/LCa (NO3)2, it is 1.8 to adjust pH.
In step (4), inoculum concentration 1v%.
In step (6), inoculum concentration is 8~10v%.
Advantageous effect：The present invention is by Acidithiobacillus ferrooxidans after the stimulation of additional constant potential so that bacterium is dense
Degree and ferrous oxidation rate improve, and the method for the present invention is simple for process, and investment is low with operating cost, have higher scientific research valence
Value and market economy benefit.
Description of the drawings
Fig. 1 is the oxidation-reduction potential that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials；
Fig. 2 is the potential that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials；
Fig. 3 is the concentration that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials；
Fig. 4 is embodiment 1-4 stimulates the ferrous iron in lower cathode chamber bacterium solution to be metabolized effect with comparative example 1-2 difference applying electrical potentials
Rate.
Specific implementation mode
The present invention is further described with case study on implementation below in conjunction with the accompanying drawings.
Remarks：In following embodiments, the Fe containing 4.5g/L2+9K fluid nutrient mediums formula：
3g/L(NH4)2SO4, 0.5g/L MgSO4·7H2O, 0.5g/L K2HPO4, 0.1g/L KCl, 0.01g/LCa
(NO3)2, 4.5g/L FeSO4·7H2O, it is 1.8 to adjust pH.
It is described to be free of Fe2+9K culture mediums preparation method：
3g/L(NH4)2SO4, 0.5g/L MgSO4·7H2O, 0.5g/L K2HPO4, 0.1g/L KCl, 0.01g/LCa
(NO3)2, it is 1.8 to adjust pH.
Embodiment 1
A kind of cultural method of Acidithiobacillus ferrooxidans, includes the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, take 1 250mL to sterilize
The sterilized Fe containing 4.5g/L of 90mL are added in conical flask2+9K fluid nutrient mediums (pH=1.8), inoculation 10mL acidophilias oxidation
The initial bacterium solution of ferrous Thiobacillus moves into 30 DEG C, is cultivated in the constant-temperature table of 180r/min, and culture is to bacterium solution by light green color
Change is deep to rufous and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：4 500mL conical flasks to sterilize are taken, it is each that the sterilized Fe containing 4.5g/L of 225mL are added2+
9K fluid nutrient mediums (pH=1.8), be inoculated with the above-mentioned seed liquors of 25mL, trained in 30 DEG C, the constant-temperature table of 180r/min
It supports, after 60h, expands culture and complete, obtain bacterium solution；
(3) collection of thalline：Wait for the oxidation-reduction potential of the bacterium solution of step (2) reaching 560mV that (PHS-3C precision pH meters are straight
Connect measurement), it is filtered immediately, filtered filtrate 10000r/min is centrifuged into 15min, it is sub- to obtain the oxidation of high activity acidophilia
Iron Thiobacillus thalline；It is dense through blood counting chamber method calculating bacterium, final concentration of 1.7 × 109A/mL；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode
Room is separated between cathode chamber and anode chamber with proton exchange membrane, and anode working electrode is added into anode chamber and 300mL is free of
Fe2+9K culture mediums, cathode working electrode, magnetic rotor and 300mL Fe containing 4.5g/L are added into cathode chamber2+9K culture
Base, toward Fe containing 4.5g/L2+9K inoculation of medium 3mL steps (3) high activity Acidithiobacillus ferrooxidans thalline,
After inoculation, cultivated under conditions of 30 DEG C, 180r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By 2700MUL TIMETER/DATE
The anode and cathode of ACQUISITION SYSTEM type data collectors is connect with cathode working electrode and anode working electrode respectively, and two
After data collector is powered, cathode chamber bacterium solution is measured every 12h for the external resistor of one 50 Ω of series connection between a working electrode
Oxidation-reduction potential after current potential reaches 560mV, supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is distinguished
Cathode working electrode and reference electrode are connected, is stimulated outside 0.2V plus under constant potential, is fired by double-chamber microbiological per 4h
Material battery sample tap takes a sample, sample volume 2mL to continue to acquire cathode potential data, after stimulating 50h, obtains outer power-up thorn
Bacterium solution after swashing；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+
9K fluid nutrient mediums in, secondary culture is carried out in 30 DEG C, the constant-temperature table of 180r/min, measures ferrous 95% oxygenation efficiency institute
The time (phenanthroline spectrophotometry (HJ/T 345-2007)) of consumption finds afterwards, after the stimulation of 0.2V applying electrical potentials, acidophilus
Property the ferrous time consumed of Thiobacillus Ferrooxidans'Oxidation 95% be 20h, it was demonstrated that this method has obtained high density and height is ferrous
The Acidithiobacillus ferrooxidans of oxidation rate.
Embodiment 2
A kind of cultural method of Acidithiobacillus ferrooxidans, includes the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, take 1 250mL to sterilize
The sterilized Fe containing 4.5g/L of 90mL are added in conical flask2+9K fluid nutrient mediums (pH=1.8), inoculation 10mL acidophilias oxidation
The initial bacterium solution of ferrous Thiobacillus moves into 30 DEG C, is cultivated in the constant-temperature table of 180r/min, and culture is to bacterium solution by light green color
Change is deep to rufous and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：4 500mL conical flasks to sterilize are taken, it is each that the sterilized Fe containing 4.5g/L of 225mL are added2+
9K fluid nutrient mediums (pH=1.8), be inoculated with the above-mentioned seed liquors of 25mL, trained in 30 DEG C, the constant-temperature table of 180r/min
It supports, after 60h, expands culture and complete, obtain bacterium solution；
(3) collection of thalline：Wait for the oxidation-reduction potential of the bacterium solution of step (2) reaching 560mV that (PHS-3C precision pH meters are straight
Connect measurement), it is filtered immediately, filtered filtrate 10000r/min is centrifuged into 15min, it is sub- to obtain the oxidation of high activity acidophilia
Iron Thiobacillus thalline；It is dense through blood counting chamber method calculating bacterium, final concentration of 1.7 × 109A/mL；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode
Room is separated between cathode chamber and anode chamber with proton exchange membrane, and anode working electrode is added into anode chamber and 300mL is free of
Fe2+9K culture mediums, cathode working electrode, magnetic rotor and 300mL Fe containing 4.5g/L are added into cathode chamber2+9K culture
Base, toward Fe containing 4.5g/L2+9K inoculation of medium 3mL steps (3) high activity Acidithiobacillus ferrooxidans thalline,
After inoculation, cultivated under conditions of 30 DEG C, 180r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By 2700MUL TIMETER/DATE
The anode and cathode of ACQUISITION SYSTEM type data collectors is connect with cathode working electrode and anode working electrode respectively, and two
After data collector is powered, cathode chamber bacterium solution is measured every 12h for the external resistor of one 50 Ω of series connection between a working electrode
Oxidation-reduction potential after current potential reaches 560mV, supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is distinguished
Cathode working electrode and reference electrode are connected, is stimulated outside 0.4V plus under constant potential, is fired by double-chamber microbiological per 4h
Material battery sample tap takes a sample, sample volume 2mL to continue to acquire cathode potential data, after stimulating 50h, obtains outer power-up thorn
Bacterium solution after swashing；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+
9K fluid nutrient mediums in, secondary culture is carried out in 30 DEG C, the constant-temperature table of 180r/min, measures ferrous 95% oxygenation efficiency institute
The time (phenanthroline spectrophotometry (HJ/T 345-2007)) of consumption finds afterwards, after the stimulation of 0.4V applying electrical potentials, acidophilus
Property the ferrous time consumed of Thiobacillus Ferrooxidans'Oxidation 95% be 20h, it was demonstrated that this method has obtained high density and height is ferrous
The Acidithiobacillus ferrooxidans of oxidation rate.
Embodiment 3
A kind of cultural method of Acidithiobacillus ferrooxidans, includes the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, take 1 250mL to sterilize
The sterilized Fe containing 4.5g/L of 90mL are added in conical flask2+9K fluid nutrient mediums (pH=1.8), inoculation 10mL acidophilias oxidation
The initial bacterium solution of ferrous Thiobacillus moves into 30 DEG C, is cultivated in the constant-temperature table of 180r/min, and culture is to bacterium solution by light green color
Change is deep to rufous and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：4 500mL conical flasks to sterilize are taken, it is each that the sterilized Fe containing 4.5g/L of 225mL are added2+
9K fluid nutrient mediums (pH=1.8), be inoculated with the above-mentioned seed liquors of 25mL, trained in 30 DEG C, the constant-temperature table of 180r/min
It supports, after 60h, expands culture and complete, obtain bacterium solution；
(3) collection of thalline：Wait for the oxidation-reduction potential of the bacterium solution of step (2) reaching 560mV that (PHS-3C precision pH meters are straight
Connect measurement), it is filtered immediately, filtered filtrate 10000r/min is centrifuged into 15min, it is sub- to obtain the oxidation of high activity acidophilia
Iron Thiobacillus thalline；It is dense through blood counting chamber method calculating bacterium, final concentration of 1.7 × 109A/mL；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode
Room is separated between cathode chamber and anode chamber with proton exchange membrane, and anode working electrode is added into anode chamber and 300mL is free of
Fe2+9K culture mediums, cathode working electrode, magnetic rotor and 300mL Fe containing 4.5g/L are added into cathode chamber2+9K culture
Base, toward Fe containing 4.5g/L2+9K inoculation of medium 3mL steps (3) high activity Acidithiobacillus ferrooxidans thalline,
After inoculation, cultivated under conditions of 30 DEG C, 180r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By 2700MUL TIMETER/DATE
The anode and cathode of ACQUISITION SYSTEM type data collectors is connect with cathode working electrode and anode working electrode respectively, and two
After data collector is powered, cathode chamber bacterium solution is measured every 12h for the external resistor of one 50 Ω of series connection between a working electrode
Oxidation-reduction potential after current potential reaches 560mV, supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is distinguished
Cathode working electrode and reference electrode are connected, is stimulated outside 0.5V plus under constant potential, is fired by double-chamber microbiological per 4h
Material battery sample tap takes a sample, sample volume 2mL to continue to acquire cathode potential data, after stimulating 50h, obtains outer power-up thorn
Bacterium solution after swashing；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+
9K fluid nutrient mediums in, secondary culture is carried out in 30 DEG C, the constant-temperature table of 180r/min, measures ferrous 95% oxygenation efficiency institute
The time (phenanthroline spectrophotometry (HJ/T 345-2007)) of consumption finds afterwards, after the stimulation of 0.5V applying electrical potentials, acidophilus
Property the ferrous time consumed of Thiobacillus Ferrooxidans'Oxidation 95% be for 24 hours, it was demonstrated that this method has obtained high density and height is ferrous
The Acidithiobacillus ferrooxidans of oxidation rate.
Embodiment 4
A kind of cultural method of Acidithiobacillus ferrooxidans, includes the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, take 1 250mL to sterilize
The sterilized Fe containing 4.5g/L of 90mL are added in conical flask2+9K fluid nutrient mediums (pH=1.8), inoculation 10mL acidophilias oxidation
The initial bacterium solution of ferrous Thiobacillus moves into 30 DEG C, is cultivated in the constant-temperature table of 180r/min, and culture is to bacterium solution by light green color
Change is deep to rufous and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：4 500mL conical flasks to sterilize are taken, it is each that the sterilized Fe containing 4.5g/L of 225mL are added2+
9K fluid nutrient mediums (pH=1.8), be inoculated with the above-mentioned seed liquors of 25mL, trained in 30 DEG C, the constant-temperature table of 180r/min
It supports, after 60h, expands culture and complete, obtain bacterium solution；
(3) collection of thalline：Wait for the oxidation-reduction potential of the bacterium solution of step (2) reaching 560mV that (PHS-3C precision pH meters are straight
Connect measurement), it is filtered immediately, filtered filtrate 10000r/min is centrifuged into 15min, it is sub- to obtain the oxidation of high activity acidophilia
Iron Thiobacillus thalline；It is dense through blood counting chamber method calculating bacterium, final concentration of 1.7 × 109A/mL；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode
Room is separated between cathode chamber and anode chamber with proton exchange membrane, and anode working electrode is added into anode chamber and 300mL is free of
Fe2+9K culture mediums, cathode working electrode, magnetic rotor and 300mL Fe containing 4.5g/L are added into cathode chamber2+9K culture
Base, toward Fe containing 4.5g/L2+9K inoculation of medium 3mL steps (3) high activity Acidithiobacillus ferrooxidans thalline,
After inoculation, cultivated under conditions of 30 DEG C, 180r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By 2700MUL TIMETER/DATE
The anode and cathode of ACQUISITION SYSTEM type data collectors is connect with cathode working electrode and anode working electrode respectively, and two
After data collector is powered, cathode chamber bacterium solution is measured every 12h for the external resistor of one 50 Ω of series connection between a working electrode
Oxidation-reduction potential after current potential reaches 560mV, supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is distinguished
Cathode working electrode and reference electrode are connected, is stimulated outside 0.6V plus under constant potential, is fired by double-chamber microbiological per 4h
Material battery sample tap takes a sample, sample volume 2mL to continue to acquire cathode potential data, after stimulating 50h, obtains outer power-up thorn
Bacterium solution after swashing；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+
9K fluid nutrient mediums in, secondary culture is carried out in 30 DEG C, the constant-temperature table of 180r/min, measures ferrous 95% oxygenation efficiency institute
The time (phenanthroline spectrophotometry (HJ/T 345-2007)) of consumption finds afterwards, after the stimulation of 0.6V applying electrical potentials, acidophilus
Property the ferrous time consumed of Thiobacillus Ferrooxidans'Oxidation 95% be for 24 hours, it was demonstrated that this method has obtained high density and height is ferrous
The Acidithiobacillus ferrooxidans of oxidation rate.
Comparative example 1
The additional constant potential of step (5) is changed to 0.7V, remaining is same as Example 1.
Experiment is found, after stimulating 50h, cathode potential changes unobvious, and ferrous iron does not aoxidize substantially, and bacterium is dense not to improve.
When secondary culture, the time (phenanthroline spectrophotometry (HJ/T that ferrous 95% oxygenation efficiency is consumed is measured
It 345-2007)) finds afterwards, after the stimulation of 0.7V applying electrical potentials, Acidithiobacillus ferrooxidans growth metabolism is suppressed, very
To death, ferrous iron does not aoxidize substantially.
Comparative example 2
The additional constant potential of step (5) is changed to 0V, i.e., does not add applying electrical potential stimulation, remaining is same as Example 1.
When secondary culture, the time (phenanthroline spectrophotometry (HJ/T that ferrous 95% oxygenation efficiency is consumed is measured
345-2007)) find afterwards, not after applying electrical potential stimulation, Acidithiobacillus ferrooxidans oxidation 95% is ferrous consumed when
Between be 60h.
Fig. 1 is the oxidation-reduction potential that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials；
It is that per 4h, sampling directly measures by PHS-3C precisions pH meter, as shown in Figure 1, after the stimulation of different potentials, cathode oxidation reduction
The rate that current potential reaches 560mV is different, and potential is lower, and the rate reached is faster, but only in a certain range, and potential is more than 0.6V
Afterwards, applying electrical potential has inhibiting effect to the growth of acidophilia ferrous oxide；And the oxidation-reduction potential of 110h is higher, illustrates ferrous iron
What is aoxidized is more thorough.
Fig. 2 is the potential that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials, is to pass through
2700MUL TIMETER/DATE ACQUISITION SYSTEM type data collector the real time measures, the result is by cathode internal
Outer source potential two parts that the potential and controllable programming power supply that Acidithiobacillus ferrooxidans ferrous oxide generates provide are common
Effect gained.As seen from Figure 2, when external source addition potential is 0.2V, 0.4V, Acidithiobacillus ferrooxidans ferrous iron oxygen
Change thoroughly, show to form precipitation as jarosite passivating film in electrode, reduces cathode potential, and 0.4V is more than 0.2V, therefore electricity
Gesture is also higher；When external source addition potential is 0.5V, 0.6V, ferrous oxidation is incomplete, and potential is relatively low, and 0.6V is more than 0.4V,
Therefore potential is also higher；When outer source potential is 0V, 0.7V, the former does not form passivating film, potential highest, the latter's ferrous iron in electrode
Substantially it does not aoxidize, potential is minimum.
Fig. 3 is the concentration that embodiment 1-4 stimulates lower cathode chamber bacterium solution with comparative example 1-2 difference applying electrical potentials, is by 16
The blood counting chamber of 25 lattice of lattice * counts gained, and calculation formula is：The small lattice inner cell number/100*400* of cell number/mL=100
104* extension rate.As shown in Figure 3, the bacterium of Acidithiobacillus ferrooxidans is dense when applying electrical potential is 0V, in cathode is
1.2*108A/mL, the bacterium of Acidithiobacillus ferrooxidans is dense when applying electrical potential is 0.2~0.6V, in cathode reaches
1.45*108~1.62*108A/mL, it is opposite to improve 21%~35%, and when applying electrical potential is 0.7V, acidophilia oxygen in cathode
Change ferrous Thiobacillus not grow substantially.
Fig. 4 is embodiment 1-4 stimulates the ferrous iron in lower cathode chamber bacterium solution to be metabolized effect with comparative example 1-2 difference applying electrical potentials
Rate, ferrous iron concentration are measured by phenanthroline spectrophotometry (HJ/T 345-2007), as shown in Figure 4, are stimulated in applying electrical potential
In 10h, very fast when 0.2V~0.6V ferrous oxidations rate is with respect to 0V, 0.7V is slower, with the progress of time, 0.2V, 0.4V
Ferrous oxidation is thorough in cathode, 0.5V, 0.6V ferrous iron 0.5~1g/L of residue, and 0.7V ferrous oxidations are less.
In conclusion after obtaining high activity high concentration Acidithiobacillus ferrooxidans body, add outside 0.2V~0.6V
The acidophilia ferrous oxide bacterial strain of the available high ferrous oxidation rate of high density under the stimulation of potential, and applying electrical potential be 0.2V when
Best results, bacterium concentrated phase without applying electrical potential stimulation to improving 35%；Using 95% ferrous oxidation rate as standard, it is opposite without
Applying electrical potential improves 300%.Acidithiobacillus ferrooxidans growth is suppressed when applying electrical potential is 0.7V, even
It is dead.
Claims (8)
1. a kind of cultural method of Acidithiobacillus ferrooxidans, which is characterized in that include the following steps：
(1) prepared by seed liquor：Prepare pH1.8, Fe containing 4.5g/L2+9K fluid nutrient mediums, be inoculated with acidophilia ferrous oxide sulphur bar
Then the initial bacterium solution of bacterium is cultivated in 28~32 DEG C, the constant-temperature table of 170~190r/min, culture is to bacterium solution by shallow
Green change is deep to rufous and oxidation-reduction potential reaches 560mV, obtains seed liquor；
(2) expand culture：Seed liquor made from step (1) is taken, Fe containing 4.5g/L is inoculated into2+9K fluid nutrient mediums in, then
It is cultivated in 28~32 DEG C, the constant-temperature table of 170~190r/min, obtains bacterium solution；
(3) collection of thalline：When the oxidation-reduction potential of the bacterium solution of step (2) reaches 560mV, is filtered, will be taken out immediately
Filtrate after filter is centrifuged, and high activity Acidithiobacillus ferrooxidans thalline is obtained；
(4) inoculation and culture of thalline：Using double-chamber microbiological fuel cell equipment, which includes cathode chamber and anode chamber,
It is separated with proton exchange membrane between cathode chamber and anode chamber, anode working electrode is added into anode chamber and is free of Fe2+9K training
Base is supported, cathode working electrode, magnetic rotor and Fe containing 4.5g/L are added into cathode chamber2+9K culture mediums, toward contain 4.5g/L
Fe2+9K inoculation of medium step (3) high activity Acidithiobacillus ferrooxidans thalline, after inoculation, 28~32 DEG C,
It is cultivated under conditions of 170~190r/min；
(5) additional constant potential stimulates Acidithiobacillus ferrooxidans：By the anode and cathode of data collector respectively with cathode work
Make electrode to connect with anode working electrode, the external resistor for 50 Ω that connect between two working electrodes, data collector is logical
After electricity, every the oxidation-reduction potential that 12h measures cathode chamber bacterium solution, after current potential reaches 540~560mV, pass through double-chamber microbiological
Fuel cell injection port supplements the Fe of 4.5g/L again2+, the positive and negative electrode of controllable programming power supply is separately connected cathode working electrode
And reference electrode, it is stimulated under the outer plus constant potential of 0.1~0.6V, stimulation time is 50~60h, obtains outer power-up thorn
Bacterium solution after swashing；
(6) secondary culture：Using the bacterium solution after additional electro photoluminescence as inoculation bacterium solution, it is inoculated into pH1.8, Fe containing 4.5g/L2+9K
In fluid nutrient medium, secondary culture is carried out in 28~32 DEG C, the constant-temperature table of 170~190r/min to get high density and height
The Acidithiobacillus ferrooxidans of ferrous oxidation rate.
2. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that thermophilic in step (1)
The inoculum concentration of the initial bacterium solution of acidic oxidation ferrous iron Thiobacillus is 8~10v%.
3. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that step (1), (2),
(4) and in (6), the Fe containing 4.5g/L2+9K fluid nutrient mediums formula：3g/L(NH4)2SO4, 0.5g/L MgSO4·
7H2O, 0.5g/L K2HPO4, 0.1g/L KCl, 0.01g/LCa (NO3)2, 4.5g/L FeSO4·7H2O, it is 1.8 to adjust pH.
4. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that in step (2), connect
Kind amount is 8~10v%.
5. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that in step (3), from
The rotating speed of the heart is 8000~12000r/min, and the time is 10~20min.
6. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that in step (4), institute
It states and is free of Fe2+9K culture mediums formula：3g/L(NH4)2SO4, 0.5g/L MgSO4·7H2O, 0.5g/L K2HPO4, 0.1g/L
KCl, 0.01g/LCa (NO3)2, it is 1.8 to adjust pH.
7. the cultural method of Acidithiobacillus ferrooxidans as described in claim 1, which is characterized in that in step (4), connect
Kind amount is 1v%.
8. the cultural method of Acidithiobacillus ferrooxidans as described in any one of claim 1 to 7, which is characterized in that step
Suddenly in (6), inoculum concentration is 8~10v%.
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