CN105368756A - Strain for removing calcium ions and magnesium ions and application of strain - Google Patents
Strain for removing calcium ions and magnesium ions and application of strain Download PDFInfo
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- CN105368756A CN105368756A CN201510932679.2A CN201510932679A CN105368756A CN 105368756 A CN105368756 A CN 105368756A CN 201510932679 A CN201510932679 A CN 201510932679A CN 105368756 A CN105368756 A CN 105368756A
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Abstract
The invention discloses a strain for removing calcium ions and magnesium ions and application of the strain. The strain for removing calcium ions and magnesium ions provided by the invention is bacillus licheniformis SRB3, which is preserved in China General Microbiological Culture Collection Center with preservation number of CGMCC No.10971. The bacillus licheniformis SRB3 CGMCC No.10971 provided by the invention plays a vital role in the settlement of the calcium ions and the magnesium ions, and the strain has a broad application prospect in the aspect of hard water softening.
Description
Technical field
The present invention relates to microorganism field, be specifically related to a strain and remove bacterium of calcium ion and magnesium ion and uses thereof.
Background technology
Use softening water technology the hard water of calcium-magnesium-containing ion can be treated to drinkable water.General water softening can adopt thermal technology and membrane technique to carry out: thermal technology utilizes heat devaporation moisture, thus realizes calcium ions and magnesium ions from the separation water; Then comprise reverse osmosis, membrane distillation and electrodialysis etc. by the membrane technique of electrical energy drive, two kinds of technology all need lot of energy and fund.Nearly ten years, softening water technology was updated, decrease a large amount of monetary losses, but worldwide high energy demand is still and merits attention.Energy expenditure for softening water technology remains a defect, result in high operation cost and water price.Therefore, not only environmental protection is found but also the softening method of economy reduces or the calcium ions and magnesium ions removed in hard water is a very necessary job.
In distributed in nature widely, physiological property is rich and varied for genus bacillus, is one of soil and plant Tiny ecosystem dominant population.It can produce Multiple Classes of Antibiotics, good restraining effect is played to multiple animal and plant and human pathogen bacterium, also there is very strong proteolytic enzyme, lipase, amylase activity, the bacterial strain of bacillus generally has the ability of powerful deposited metal ion simultaneously, the metal ion major part of precipitation is heavy metal and radioactive nuleus element, and therefore genus bacillus is widely used in the industry-by-industries such as medicine, agricultural chemicals, food, feed manufacturing, environmental pollution improvement.
Bacillus licheniformis (Bacilluslicheniformis) is one of bacterial classification more with potential applications in genus bacillus, and it achieves good achievement in research in industries such as medicine, feed manufacturing, agricultural chemicals, and the dead thalline of such as Bacillus licheniformis is to Cr
6+there is good adsorption effect, Bacillus licheniformis R08 dead thalline absorption Pd
2+, adsorptive capacity can reach every gram of thalline absorption 224.8mgPd
2+.Therefore, screen lichem bacillus strain targetedly, prepare water conditioner, effectively to be reduced by artificial inoculation or the calcium ions and magnesium ions removed in hard water is a very necessary job and practicable approach.
Summary of the invention
Technical problem to be solved by this invention how to remove the object that calcium ion and magnesium ion reach water softening.
For solving the problems of the technologies described above, the invention provides the bacterium that a strain can remove calcium ion and magnesium ion.
Bacterium provided by the present invention is Bacillus licheniformis (Bacilluslicheniformis) SRB3, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 06 10th, 2015, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNo.10971.Bacillus licheniformis (Bacilluslicheniformis) SRB3CGMCCNo.10971 is called for short Bacillus licheniformis SRB3.
The present invention also provides a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is Bacillus licheniformis SRB3.
The purposes of described microbial inoculum can be following a1), a2), a3) or a4): a1) remove calcium ion and/or magnesium ion; A2) calcium ion and/or magnesium ion in water is removed; A3) sedimentation calcium ion and/or magnesium ion; A4) calcium ion and/or magnesium ion in sedimentation water.
The preparation method of described microbial inoculum comprises the steps: Bacillus licheniformis SRB3 is seeded to bacteria culture medium and cultivates, and obtains OD
600nmvalue is the bacterium liquid of 0.95, is described microbial inoculum.
Described bacteria culture medium is beef-protein medium or salt culture medium fourth.
The preparation method of described beef-protein medium is specific as follows: extractum carnis 5g, Tryptones 10g and sodium-chlor 5g are dissolved in 1L distilled water, regulates pH to 7.2.
The preparation method of described salt culture medium fourth is specific as follows: by extractum carnis 5g, and Tryptones 10g and sodium-chlor 30g is dissolved in 1L distilled water, regulates pH to 7.6.
In the preparation method of described microbial inoculum, the actual conditions of described cultivation can be: 37 DEG C, 130r/min shaking culture 24h.
Except activeconstituents, described microbial inoculum can also comprise carrier.Described carrier can be solid carrier or liquid vehicle.Described solid carrier can be mineral material, vegetable material or macromolecular compound; Described mineral material can be at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; Described vegetable material can be at least one in Semen Maydis powder, bean powder and starch; Described macromolecular compound can be polyvinyl alcohol and/or polyglycol.Described liquid vehicle can be organic solvent, vegetables oil, mineral oil or water; Described organic solvent can be decane and/or dodecane.In described microbial inoculum, described activeconstituents can with by cultivate viable cell, the fermented liquid of viable cell, the filtrate of cell culture or cell and filtrate the form of mixture exist.The formulation of described composition can be multiple formulation, as liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
As required, tensio-active agent (as polysorbas20, tween 80 etc.), tackiness agent, stablizer (as antioxidant), pH adjusting agent etc. can also be added in described microbial inoculum.
Bacillus licheniformis SRB3 or above-mentioned arbitrary described microbial inoculum also belong to protection scope of the present invention removing the application in calcium ion and/or magnesium ion.
Bacillus licheniformis SRB3 or above-mentioned arbitrary described microbial inoculum also belong to protection scope of the present invention removing the application in calcium ion in water and/or magnesium ion.
Bacillus licheniformis SRB3 or the application of above-mentioned arbitrary described microbial inoculum in sedimentation calcium ion and/or magnesium ion also belong to protection scope of the present invention.
The application in sedimentation water in calcium ion and/or magnesium ion of Bacillus licheniformis SRB3 or above-mentioned arbitrary described microbial inoculum also belongs to protection scope of the present invention.
Described water can be the hard water containing calcium ion and/or magnesium ion.
The existence form of described calcium ion can be Ca
2+.
The existence form of described magnesium ion can be Mg
2+.
The described hard water containing calcium ion and/or magnesium ion can be containing 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+water.
The described hard water containing calcium ion and/or magnesium ion specifically can be containing 0.01mol/LCa
2+with 0.06 ~ 0.12mol/LMg
2+water.
Present invention also offers a kind of method obtaining calcite and/or vaterite.
The method of acquisition calcite provided by the present invention and/or vaterite, comprises the steps: to containing 0.005 ~ 0.015mol/LCa
2+liquid-phase system in add Bacillus licheniformis SRB3.
Present invention also offers a kind of method obtaining monohydrocalcite.
The method of acquisition monohydrocalcite provided by the present invention, comprises the steps: to containing 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+liquid-phase system in add Bacillus licheniformis SRB3.
Present invention also offers a kind of method obtaining nesquehonite.
The method of acquisition nesquehonite provided by the present invention, comprises the steps: to containing 0.10 ~ 0.20mol/LMg
2+liquid-phase system in add Bacillus licheniformis SRB3.
In the method for the method of described acquisition calcite and/or vaterite, the method for described acquisition monohydrocalcite or described acquisition nesquehonite, also comprise the step of carrying out after adding Bacillus licheniformis SRB3 cultivating.
In the method for the method of described acquisition calcite and/or vaterite, the method for described acquisition monohydrocalcite or described acquisition nesquehonite, the condition of described cultivation can be 35 DEG C ~ 39 DEG C, 110 ~ 150r/min cultivates 10 ~ 20 days.
In the method for the method of described acquisition calcite and/or vaterite, the method for described acquisition monohydrocalcite or described acquisition nesquehonite, the condition of described cultivation specifically can be 37 DEG C, 130r/min cultivates 12 days.
Describedly contain 0.005 ~ 0.015mol/LCa
2+liquid-phase system, describedly contain 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+liquid-phase system or describedly contain 0.10 ~ 0.20mol/LMg
2+liquid-phase system in all can contain carbanion and/or bicarbonate ion.
The concentration of described carbanion in above-mentioned arbitrary described liquid-phase system can be 0.01 ~ 0.06mol/L.
The concentration of described carbanion in above-mentioned arbitrary described liquid-phase system specifically can be 0.04mol/L.
The concentration of described bicarbonate ion in above-mentioned arbitrary described liquid-phase system can be 0.01 ~ 0.06mol/L.
The concentration of described bicarbonate ion in above-mentioned arbitrary described liquid-phase system specifically can be 0.03mol/L.
Describedly contain 0.005 ~ 0.015mol/LCa
2+liquid-phase system specifically can be containing 0.01mol/LCa
2+liquid-phase system.
Describedly contain 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+liquid-phase system specifically can be containing 0.01mol/LCa
2+with 0.06 ~ 0.12mol/LMg
2+liquid-phase system.
Describedly contain 0.10 ~ 0.20mol/LMg
2+liquid-phase system specifically can be containing 0.10 ~ 0.12mol/LMg
2+liquid-phase system.
Describedly contain 0.10 ~ 0.20mol/LMg
2+liquid-phase system also can comprise 0.005 ~ 0.015mol/LCa
2+.
Describedly contain 0.10 ~ 0.20mol/LMg
2+liquid-phase system specifically also can comprise 0.01mol/LCa
2+.
Experiment proves, Bacillus licheniformis SRB3 provided by the invention plays vital effect in sedimentation calcium ion and magnesium ion, has broad application prospects in water softening.
Accompanying drawing explanation
Fig. 1 is the form of Bacillus licheniformis SRB3 under high resolution TEM and phylogeny tree graph.
Fig. 2 is growth curve and the pH value change curve of Bacillus licheniformis SRB3 in the substratum of different salt concn.
Fig. 3 is calcium ion changing trend diagram.
Fig. 4 is magnesium ion changing trend diagram.
Fig. 5 is mineral precipitation X-ray diffraction analysis.
Fig. 6 is the scanning electron microscope (SEM) photograph of control group mineral precipitation.
Fig. 7 scanning electron microscope (SEM) photograph that to be experimental group precipitate at calcium-magnesium-containing substratum first Minerals and energy spectrum analysis.
Fig. 8 scanning electron microscope (SEM) photograph that to be experimental group precipitate at calcium-magnesium-containing substratum second Minerals and energy spectrum analysis.
Fig. 9 scanning electron microscope (SEM) photograph that to be experimental group precipitate at calcium-magnesium-containing substratum fourth Minerals and energy spectrum analysis.
Figure 10 is high resolution TEM and the Conjoint Analysis of nano-area electron diffraction of experimental group mineral precipitation.
preservation explanation
Strain name: Bacillus licheniformis
Latin name: (Bacilluslicheniformis)
Strain number: SRB3
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 06 10th, 2015
Register on the books numbering: CGMCCNo.10971 at preservation center
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Substratum used in following embodiment is as follows:
Activation medium: extractum carnis 5g, Tryptones 10g and sodium-chlor 5g are dissolved in 1L distilled water, regulates pH to 7.2, then adds agar 15g.
Extractum carnis Tryptones substratum: extractum carnis 5g, Tryptones 10g and sodium-chlor 5g are dissolved in 1L distilled water, regulates pH to 7.2.
Enrichment medium: solute and concentration thereof are K
2hPO
40.5g/L, NH
4cl1.0g/L, CaCl
20.1g/L, MgSO
47H
2o2.0g/L, yeast leaching powder 1.0g/L, Na
2sO
40.5g/L, Fe (NH
4)
2(SO
4)
20.5g/L, xitix 0.5g/L, L-Cys0.5g/L, the sodium lactate solution 6mL/L of 60%; Solvent is distilled water; PH6.0 ~ 8.0; Wherein Fe (NH
4)
2(SO
4)
2, xitix and L-Cys need first be mixed with mother liquor with sterile distilled water, then use 0.22 μm of membrane filtration degerming, then for the preparation of enrichment medium; The sodium lactate solution of 60% is Chemical Reagent Co., Ltd., Sinopharm Group's product, and catalog number is 30168018.
Pure medium: yeast extract paste 3g, KCl0.25g and agar 15g are dissolved in 1L distilled water, natural pH.
Salt culture medium first: extractum carnis 5g and Tryptones 10g is dissolved in 1L distilled water, regulates pH to 7.6.In salt culture medium first, the concentration of NaCl is 0% (mass percent)
Salt culture medium second: by extractum carnis 5g, Tryptones 10g and sodium-chlor 10g is dissolved in 1L distilled water, regulates pH to 7.6.In salt culture medium second, the concentration of NaCl is 1% (mass percent).
Salt culture medium third: by extractum carnis 5g, Tryptones 10g and sodium-chlor 20g is dissolved in 1L distilled water, regulates pH to 7.6.In salt culture medium third, the concentration of NaCl is 2% (mass percent).
Salt culture medium fourth: by extractum carnis 5g, Tryptones 10g and sodium-chlor 30g is dissolved in 1L distilled water, regulates pH to 7.6.In salt culture medium fourth, the concentration of NaCl is 3% (mass percent).
Salt culture medium penta: by extractum carnis 5g, Tryptones 10g and sodium-chlor 40g is dissolved in 1L distilled water, regulates pH to 7.6.In salt culture medium penta, the concentration of NaCl is 4% (mass percent).
X-ray diffraction analysis uses and turns target X-ray diffractometer, Rigaku Electric company product, and product type is D/Max-RC; Scanning electron microscope analysis uses scanning electron microscope, HIT's product, and product type is HitachiS-4800; Energy spectrum analysis uses GENESIS energy spectrometer, Yi Dakesi company limited of U.S. product; High resolution TEM analysis uses high resolution TEM, and Jeol Ltd.'s Products, product type is JEM-2100.
SrCl
26H
2the O aqueous solution: by 30.4gSrCl
26H
2o is dissolved in 100mL distilled water, the solution obtained.
Embodiment 1, the separation of Bacillus licheniformis (Bacilluslicheniformis) SRB3CGMCCNo.10971, preservation
One, the separation of sulphate reducing bacteria SRB3
1, in 100mL sterile distilled water, add 10g pedotheque (picking up from the mud in ink river bed portion, Chinese Qingdao), stir, static placement 10 minutes, then getting 20mL supernatant liquor adds in 200mL enrichment medium, and with liquid level in sterile liquid paraffin sealing bottles, be placed in 37 DEG C of constant incubator quiescent culture, observation.Be cultured to the 7th day, the blackening of enrichment medium color, the visible sulphate reducing bacteria enrichment success (Fe (NH in enrichment medium
4)
2(SO
4)
2in ferrous ion can with the S in enrichment medium
2-reaction generates the Iron sulfuret of black).
2, after completing steps 1, get supernatant liquor and be seeded to containing 0.5g/LFe (NH
4)
2(SO
4)
2pure medium on, in 37 DEG C of anaerobic culture boxes cultivate, observe.Be cultured to the 7th day, produce black list bacterium colony.Picking list bacterium colony, repeatedly purifying more than 3 times.Be sulphate reducing bacteria SRB3 by the strain of sulfate reduction bacteria Strain Designation screened.
Two, the qualification of sulphate reducing bacteria SRB3
1, Morphological Identification
Sulphate reducing bacteria SRB3 is inoculated on activation medium, within 3 days, observes the form of single bacterium colony afterwards.Result shows, sulphate reducing bacteria SRB3 bacterium colony is circular, diameter 3.0 ~ 5.0mm, and there is fold on surface, and edge is irregular, and color is white (along with incubation time extends, color pulverize is red), and bacterium colony is opaque and dry, does not have wettability.
After sulphate reducing bacteria SRB3 is dyed, be accredited as gram-positive microorganism.Analyze sulphate reducing bacteria SRB3 by high resolution TEM, experimental result is shown in a in Fig. 1.Result shows, the size of sulphate reducing bacteria SRB3 about 1.46 μm × 0.63 μm, bacterium is quarter butt shape, Individual existence.
2,16SrDNA sequence homology analysis
The 16SrDNA of sulphate reducing bacteria SRB3 is as shown in the sequence 1 in sequence table.
By ClustalX software, the sequence 1 in sequence table is compared with the sequence in GenBank, adopt adjacent method (N-J method) phylogenetic tree construction.The experimental result of phylogenetic tree is shown in b in Fig. 1.
Sulphate reducing bacteria SRB3 and Bacillus licheniformis (Bacilluslicheniformis) homology the highest, reach 97%.
Comprehensively each qualification result above-mentioned, sulphate reducing bacteria SRB3 is Bacillus licheniformis (Bacilluslicheniformis).
Three, the preservation of sulphate reducing bacteria SRB3
Bacillus licheniformis (Bacilluslicheniformis) SRB3 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 06 10th, 2015, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCCNo.10971.The full name of sulphate reducing bacteria SRB3 is Bacillus licheniformis (Bacilluslicheniformis) SRB3CGMCCNo.10971, referred to as Bacillus licheniformis SRB3 or SRB3.
Embodiment 2, preparation Bacillus licheniformis SRB3 microbial inoculum
Activated on activation medium by Bacillus licheniformis SRB3, then picking list colony inoculation is in the 250mL Erlenmeyer flask that 100mL beef-protein medium is housed, 37 DEG C, the constant-temperature table of 130r/min is cultivated 1 day, obtains OD
600value is about the Bacillus licheniformis SRB3 microbial inoculum first of 0.95.Bacillus licheniformis SRB3 microbial inoculum first is hereinafter referred to as SRB3 microbial inoculum first.
According to the method described above, except beef-protein medium is replaced with salt culture medium fourth, other step is all identical, obtains OD
600value is about the Bacillus licheniformis SRB3 microbial inoculum second of 0.95.Bacillus licheniformis SRB3 microbial inoculum second is hereinafter referred to as SRB3 microbial inoculum second.
The characteristic of embodiment 3, Bacillus licheniformis SRB3
The present embodiment studies growth curve and the pH change curve of Bacillus licheniformis SRB3 under different salt concn.
Each salt culture medium (salt culture medium first, salt culture medium second, salt culture medium third, salt culture medium fourth or salt culture medium penta) is adopted to proceed as follows respectively:
Get the Erlenmeyer flask of 6 250mL, each Erlenmeyer flask loads 200mL salt culture medium, then 121 DEG C of autoclaving 20min; Be cooled to after room temperature until substratum, 6 Erlenmeyer flasks are divided into experimental group and control group, often organize 3 Erlenmeyer flasks; In the Erlenmeyer flask of experimental group, inoculate SRB3 microbial inoculum first 2mL, in the Erlenmeyer flask of control group, inoculate the aseptic ultrapure water of equal-volume; After inoculation, Erlenmeyer flask is placed in constant-temperature shaking incubator, 37 DEG C, 130r/min cultivation.Sample 4mL when inoculating complete, then every 3 hours sampling 4mL, measure bacteria concentration and pH value.
Carry out three tests, results averaged.
In experimental group, the growth curve of SRB3 is shown in a in Fig. 2.The process of growth of SRB3 in each salt culture medium includes the lag phase 0 ~ 6h of a (in the Fig. 2), the logarithmic phase 6 ~ 12h of a (in the Fig. 2) and the stationary phase 12 ~ 36h of a (in the Fig. 2) three phases, wherein lag phase is the stage of the growing environment of bacterium just in acclimatizing culture medium, logarithmic phase is the stage that bacterium is in active splitting status, stationary phase is because the nutritive substance in substratum is consumed and the accumulation gradually of toxic metabolic products, and the growth velocity of bacterium slows down gradually but stage of still remaining unchanged of total biomass.Result shows, SRB3 grows to 12 hours and namely starts to enter stationary phase in containing the substratum of 4%NaCl, and SRB3 grows to 12 hours and is still in logarithmic phase in the substratum containing 0%, 1%, 2% or 3%NaCl; The concentration that SRB3 grows to 36 hours in containing the substratum of 4%NaCl is 4.70 × 10
8the concentration that cfu/mL, SRB3 grow to 36 hours in the substratum containing 0%, 1%, 2% or 3%NaCl is 8.0 ~ 8.2 × 10
8cfu/mL.Aseptic ultrapure water instead of bacterium liquid due to what inoculate in control group, so the growth curve all without SRB3 in control group.
In experimental group, the pH value change curve of SRB3 is shown in b in Fig. 2.The pH value change of SRB3 in each salt culture medium includes the slow rising stage 0 ~ 6h of b (in the Fig. 2), the decrement phase 6 ~ 12h of b (in the Fig. 2) and the rising stage 12 ~ 60h of b (in the Fig. 2) three phases, wherein slowly rising stage pH value maintains about 7.6, decrement phase pH value is down to about 7.1 from about 7.8, and rising stage pH value continues to rise to about 8.4 from about 7.1.PH value in control group has no significant change.
Embodiment 4, Bacillus licheniformis SRB3 remove the mensuration of calcium ion and magnesium ion ability
1, the making of typical curve
(1) Ca
2+typical curve
Accurately take anhydrous CaCl
21.11g, in 1L volumetric flask, dissolves with sterilized water and is made into Ca
2+concentration is the CaCl of 400mg/L
2mother liquor.The dilution of continuation sterilized water is mixed with Ca
2+concentration is the CaCl of 0,5,10,15,20,25,30 and 35mg/L series
2solution.Atomic absorption spectrophotometer is adopted to measure different concns CaCl
2the light absorption value of solution, repeats for 3 times.With Ca
2+concentration is X-coordinate, and light absorption value is ordinate zou, draws Ca
2+typical curve.
(2) Mg
2+typical curve
Accurately take the pure MgCl of top grade
26H
2o4.1814g, in 500mL volumetric flask, dissolves with sterilized water and is made into Mg
2+concentration is the MgCl of 1000mg/L
2mother liquor.The dilution of continuation sterilized water is mixed with Mg
2+concentration is the MgCl of 0,10,20,30,40,50,75 and 100mg/L series
2solution, to various MgCl
21mLSrCl is added in solution
26H
2the O aqueous solution.Then atomic absorption spectrophotometer is adopted to measure different concns MgCl
2the light absorption value of solution, repeats for 3 times.With Mg
2+concentration is X-coordinate, and light absorption value is ordinate zou, draws Mg
2+typical curve.
2, get salt culture medium fourth, add 1.0mol/LCaCl
2the aqueous solution, makes the Ca in system
2+concentration is 0.01mol/L, obtains calcic substratum.
3, get calcic substratum prepared by step 2, add the MgCl of 2.0mol/L
2the aqueous solution, makes the Mg in system
2+concentration is followed successively by 0mol/L, 0.06mol/L, 0.08mol/L, 0.10mol/L and 0.12mol/L, obtains calcium-magnesium-containing substratum first, calcium-magnesium-containing substratum second, calcium-magnesium-containing substratum third, calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta successively.
4, substratum (obtaining calcium-magnesium-containing substratum first, calcium-magnesium-containing substratum second, calcium-magnesium-containing substratum third, calcium-magnesium-containing substratum fourth or calcium-magnesium-containing substratum penta) is adopted to proceed as follows respectively:
Get the Erlenmeyer flask of 6 250mL, each Erlenmeyer flask loads the substratum of 150mL, 121 DEG C of autoclaving 20min.Be cooled to after room temperature until substratum, in each Erlenmeyer flask, add the 2mol/LNa of 3mL
2cO
3the 1mol/LNaHCO of the aqueous solution and 5mL
3the aqueous solution (Na
2cO
3and NaHCO
3solution aperture be the membrane filtration of 0.22 micron degerming after use again), with NaOH regulate pH to 7.2.
5, after completing steps 4,6 Erlenmeyer flasks are divided into experimental group and control group, often organize 3 Erlenmeyer flasks.In each Erlenmeyer flask of experimental group, inoculate SRB3 microbial inoculum second 1.5mL (inoculative proportion 1:100) prepared by embodiment 2, in each Erlenmeyer flask of control group, inoculate the aseptic ultrapure water of 1.5mL.After inoculation, all Erlenmeyer flasks are placed in constant-temperature shaking incubator, 37 DEG C, 130r/min cultivates 18 days.
In culturing process, the every 48 hours centrifugal 5min of sampling 1.5mL, 10000rpm, collect supernatant liquor, the supernatant liquor sterile distilled water of collection are diluted 10 times, is then the membrane filtration of 0.22 micron with aperture, obtains diluent.The time of sampling first time is after completing steps 4.
6, the light absorption value of diluent is measured with atomic absorption spectrophotometer, according to the Ca that step 1 makes
2+typical curve and Mg
2+typical curve, obtains Ca in diluent
2+and Mg
2+concentration.
In culturing process, the Ca in each substratum
2+variation tendency as shown in Figure 3 (a is control group, and b is experimental group).Experimental result is as follows:
(1) cultivate the 0th day to the 4th day, owing to all adding 2mol/LNa in each substratum
2cO
3the aqueous solution and 1mol/LNaHCO
3the aqueous solution, so the calcium ion concn in each substratum all significantly declines, and create precipitation, but compared with control group, calcium ion concn in experimental group is obviously lower, shows that Bacillus licheniformis (Bacilluslicheniformis) SRB3 has the effect promoting calcium ion sedimentation; Simultaneously along with the increase of Mg/Ca mol ratio in substratum, the concentration remaining calcium ion in nutrient solution becomes large gradually, and visible magnesium ion can hinder the sedimentation of calcium ion.
(2) cultivate the 5th day to the 12nd day, the calcium ion concn downtrending in each substratum is all gradually delayed, and the 13rd day starts, and calcium ion concn is substantially constant.
(3) the 18th day: in experimental group is cultured to, in calcium-magnesium-containing substratum first, calcium-magnesium-containing substratum second and calcium-magnesium-containing substratum third, calcium ion concn is 0 ~ 20mg/L, in calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta, calcium ion concn is between 20 ~ 40mg/L, and visible magnesium ion can hinder the sedimentation of calcium ion; Calcium ion concn in experimental group is starkly lower than control group, visible, and Bacillus licheniformis (Bacilluslicheniformis) SRB3 can promote the sedimentation of calcium ion.
The variation tendency of the magnesium ion in each substratum as shown in Figure 4 (a is control group, and b is experimental group).Experimental result is as follows:
(1) after inoculating SRB3 microbial inoculum second, magnesium ion concentration sharply declines, until cultivate the 14th day still in decline.
(2) be cultured to the 14th day: the magnesium ion concentration in experimental group in calcium-magnesium-containing substratum second, calcium-magnesium-containing substratum third, calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta all between 180 ~ 350mg/L, and in control group magnesium ion concentration all between 350 ~ 550mg/L.Visible, Bacillus licheniformis (Bacilluslicheniformis) SRB3 serves vital effect in the sedimentation of magnesium ion.
The mineral precipitation analysis that embodiment 5, embodiment 4 obtain
The mineral precipitation obtained in embodiment 4 control group and experimental group is further analyzed.
One, X-ray diffraction (X-raydiffraction, XRD) is analyzed
Each substratum bottom settlings of 12 days is cultivated in Example 4 step 5, be placed in 1.5mL centrifuge tube, leave standstill 5min, abandon supernatant liquor, the distilled water then respectively adding 1mL in each centrifuge tube washs, and leaves standstill 5 minutes, abandon supernatant, wash three times, to remove various salt ion, after precipitation seasoning, carry out XRD analysis.XRD scanning angle is 10 °-90 °, and step-length is 0.02, sweep velocity 8 °/min.
Experimental result is shown in that (a is control group to Fig. 5, b is experimental group, and wherein Mg/Ca=0, Mg/Ca=6, Mg/Ca=8, Mg/Ca=10 and Mg/Ca=12 represent the mineral precipitation in calcium-magnesium-containing substratum first, calcium-magnesium-containing substratum second, calcium-magnesium-containing substratum third, calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta respectively) and table 1.Result shows, Bacillus licheniformis (Bacilluslicheniformis) SRB3 has close ties with generation nesquehonite mineral precipitation.In the mineral precipitation that calcium-magnesium-containing substratum fourth in experimental group and experimental group calcium-magnesium-containing substratum penta produce, (004) and (002) crystal face of nesquehonite there occurs preferred orientation phenomenon.The diffraction peak intensity of (400) and (101) crystal face of standard nesquehonite is respectively higher than (004) and (002) crystal face, but under this culture system, the diffraction peak intensity of (004) and (002) crystal face is but respectively higher than (400) and (101) crystal face.Simultaneously, the diffraction peak intensity of the nesquehonite that the calcium-magnesium-containing substratum penta in experimental group produces is higher than the diffraction peak intensity of the nesquehonite that the calcium-magnesium-containing substratum fourth in experimental group produces, and shows that the quantity of the nesquehonite that calcium-magnesium-containing substratum penta produces in experimental group is many or degree of crystallinity is high.
Table 1.XRD assaying precipitation
Two, the scanning electron microscope of mineral precipitation and energy spectrum analysis
Each substratum bottom settlings of 12 days is cultivated in Example 4 step 5, be placed in 1.5mL centrifuge tube, leave standstill 5min, abandon supernatant liquor, the distilled water then respectively adding 1mL in each centrifuge tube washs, and leaves standstill 5 minutes, abandon supernatant, wash three times, to remove various salt ion, the dehydrated alcohol then adding 100 μ L in precipitation suspends.Getting sample segment is placed on Stage microscope, and metal spraying after seasoning is observed under being placed in scanning electron microscope, then carries out energy spectrum analysis.
Experimental result is as follows:
Fig. 6 is the scanning electron microscope (SEM) photograph of the mineral precipitation that control group produces, and wherein a is the surface topography of the mineral precipitation that control group is formed in calcium-magnesium-containing substratum first, and b is the surface topography of the mineral precipitation that control group is formed in calcium-magnesium-containing substratum second.The surface topography of the mineral precipitation that control group is formed in calcium-magnesium-containing substratum third, calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta is identical with the surface topography of the mineral precipitation that control group is formed in calcium-magnesium-containing substratum second.Result shows, the mineral precipitation that control group is formed in each substratum is spherical, and along with the increase of Mg/Ca ratio in substratum, the diameter of the mineral of formation reduces gradually.
Fig. 7 is scanning electron microscope (SEM) photograph and the energy spectrum analysis of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum first.Wherein in Fig. 7, a and b is surface topography, the form of mineral precipitation is spherical and dumbbell shaped, surface ratio is more coarse and containing a large amount of holes, mineral, by the mineral composition of many nano-scale particle shapes, mineral are attached with a large amount of shaft-like thalline being enclosed with fine and close calcium carbonate layer; The diameter of spherical mineral is about 16 μm, and the length of dumbbell shape mineral precipitation is about 16 μm.There is the thalline of two states: a kind of be thalline outside surface wrap up by a large amount of nano-scale particle shape mineral, namely thalline self there occurs mineralising phenomenon, there is the hollow cylindrical mineral shell as shown in c and d in Fig. 7, what the thalline that self mineralising occurs had is attached to mineral surface, have then grown with mineral precipitation together with, become a part (as shown in c white arrow in Fig. 7) for mineral; The second situation is that mineralising does not occur thalline, but the outside surface generation shrinkage of thalline, as shown in d white arrow in Fig. 7.Result shows, in calcium-magnesium-containing substratum first, SRB3 can induce the generation of mineral precipitation, and the volume of the mineral precipitation formed obviously is greater than the volume that control group does not add the mineral precipitation that SRB3 is formed; Can there is mineralising in SRB3 self simultaneously.Carry out energy spectrum analysis to the spherical and dumbbell shape mineral that b white box in Fig. 7 is chosen, result shows that these mineral mainly (in Fig. 7 e), illustrate that the mineral formed are CaCO containing these three kinds of elements of C, O and Ca
3, also containing a small amount of Na element and Si element, (in Fig. 7 e), infer that sodium element may derive from the sodium-chlor in nutrient solution, Si element may derive from Stage microscope to mineral, because the Stage microscope main component of scanning electron microscope is exactly silicon single crystal in addition.
Fig. 8 is scanning electron microscope (SEM) photograph and the energy spectrum analysis of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum second.Scanning electron microscope result shows, the surface topography of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum second there occurs great variety, in peanut shape and spherical (in Fig. 8 a), and the surface growth of peanut shape mineral several laminar mineral and (in Fig. 8 b), is no longer nano-scale particle shape mineral; Peanut shape mineral length is approximately 30 μm, large amount of thin sheets shape mineral growth flocks together and forms a small amount of random mineral (in Fig. 8 c), in mineral precipitation, inlay part thalline (in Fig. 8, the white arrow of d is shown), but do not found that self mineralising occurs thalline.Carry out energy spectrum analysis to the mineral that b white box in Fig. 8 is chosen, result shows that these mineral mainly (in Fig. 8 e), illustrate that the mineral formed are CaCO containing these three kinds of elements of C, O and Ca
3, also containing a small amount of Si element, (, Si element may derive from Stage microscope to mineral in Fig. 8 e), because the Stage microscope main component of scanning electron microscope is exactly silicon single crystal in addition.
The mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum third is substantially identical with energy spectrum analysis with the scanning electron microscope (SEM) photograph of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum second.
Fig. 9 is scanning electron microscope (SEM) photograph and the energy spectrum analysis of the mineral that experimental group is formed in calcium-magnesium-containing substratum fourth.Scanning electron microscope result shows, the surface topography of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum fourth is except oval and random mineral, also there is a large amount of long column shape mineral (a and b) in Fig. 9, the crisp thalline of a large amount of appearance is had to be gathered on mineral surface (a and b) in Fig. 9, when the major axis being attached to the thalline on mineral is just in time parallel to the major axis of long column shape mineral, mineral will produce at the position having thalline to adhere to the depression being parallel to major axis, thalline is then embedded in the position of depression, the existence induction of visible thalline creates long column shape mineral crystal, thalline also can be attached to the surface of crystal simultaneously.Carry out energy spectrum analysis to the mineral that a black box in Fig. 9 is chosen, result shows that these mineral mainly (in Fig. 9 c), illustrate that the mineral formed are CaCO containing these three kinds of elements of C, O and Ca
3, also containing a small amount of Si element and Mg element, (in Fig. 9 c), Mg element may be the Mg due to solution middle and high concentration to mineral in addition
2+be attached to and mineral are formed, Si element may derive from Stage microscope, because the Stage microscope main component of scanning electron microscope is exactly silicon single crystal.Carry out energy spectrum analysis to the long column shape mineral that b black box in Fig. 9 is chosen, these mineral, mainly containing these three kinds of elements of C, O and Mg, illustrate that the mineral formed are MgCO
3, also containing a small amount of Ca and Si element, (in Fig. 9 d), Ca element may be the Ca due to solution middle and high concentration to mineral in addition
2+be attached to that mineral surface causes, Si element may derive from Stage microscope, because the Stage microscope main component of scanning electron microscope is exactly silicon single crystal.Visible, the mineral that experimental group is formed in calcium-magnesium-containing substratum fourth are CaCO
3and MgCO
3.
The mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum penta is substantially identical with energy spectrum analysis with the scanning electron microscope (SEM) photograph of the mineral precipitation that experimental group is formed in calcium-magnesium-containing substratum fourth.
Three, the high resolution TEM (HighResolutionTransmissionElectronMicroscopy, HRTEM) of mineral precipitation and the Conjoint Analysis of nano-area electron diffraction
Each substratum bottom settlings of 12 days is cultivated in Example 4 step 5, be placed in 1.5mL centrifuge tube, leave standstill 5min, abandon supernatant liquor, then the distilled water respectively adding 1mL in each centrifuge tube washs, leave standstill 5 minutes, abandon supernatant, wash three times, to remove various salt ion, then precipitation agate mortar is fully ground, add dehydrated alcohol and suspend, carry out HRTEM and the Conjoint Analysis of nano-area electron diffraction.
Experimental result is as follows:
The interplanar crystal spacing d value of the mineral that experimental group is formed in calcium-magnesium-containing substratum first
be 1.1711,1.3502,1.2938 and 1.4258 (in Figure 10 a), in this experimental result and PDF#24-0027 card data, the spacing 1.1778,1.3553,1.2950 and 1.4168 ten points of calcite is close, therefore the corresponding crystal indices are respectively (0114), (217), (128) and (0012), show that these mineral are calcite.The mineral that experimental group is formed in calcium-magnesium-containing substratum first also have another, the interplanar crystal spacing d value of these mineral
be 3.5036,4.1162 and 5.8041 (in Figure 10 b), in this experimental result and PDF#72-1616 card data, vaterite spacing 3.4966,4.1730 and 5.8147 ten points is close, therefore the corresponding crystal indices are respectively (104), (103) and (101), show that the second mineral are vaterite.Visible, the mineral that experimental group is formed in calcium-magnesium-containing substratum first are calcite and vaterite.
The interplanar crystal spacing d value of the mineral that experimental group is formed in calcium-magnesium-containing substratum second
be 2.4859,2.4220,2.6457 and 2.9145 (in Figure 10 c), in this experimental result and PDF#83-1923 card data, the spacing 2.4905,2.4247,2.6384 and 2.9091 ten points of monohydrocalcite is close, therefore the corresponding crystal indices are respectively (221), (103), (220) and (022), show that these mineral are monohydrocalcites.
The interplanar crystal spacing d value of the mineral that experimental group is formed in calcium-magnesium-containing substratum third
be 2.3728,2.4232,2.5526 and 2.9249 (in Figure 10 d), in this experimental result and PDF#83-1923 card data, the spacing 2.3701,2.4247,2.5476 and 2.9091 ten points of monohydrocalcite is close, therefore the corresponding crystal indices are respectively (032), (103) (212) and (022), show that these mineral are monohydrocalcites.
The interplanar crystal spacing d value of the mineral that experimental group is formed in calcium-magnesium-containing substratum fourth
be 1.7836,2.0819,2.1836 and 2.3737 (in Figure 10 e), in this experimental result and PDF#70-1433 card data, the spacing 1.7833,2.0726,2.1840 and 2.3726 ten points of nesquehonite is close, therefore the corresponding crystal indices are respectively (206), (-222), (-214) and (204), show that these mineral are nesquehonite.The mineral that experimental group is formed in calcium-magnesium-containing substratum fourth also have another, these mineral interplanar crystal spacing d value
be 3.4899,3.9162 and 5.8203 (in Figure 10 f), in this experimental result and PDF#83-1923 card data, monohydrocalcite spacing 3.4869,3.9087 and 5.8183 ten points is close, therefore the corresponding crystal indices are respectively (012), (201) and (101), show that the second mineral are monohydrocalcite.Visible, the mineral that experimental group is formed in calcium magnesium substratum fourth are nesquehonite and monohydrocalcite.
The interplanar crystal spacing d value of the mineral that experimental group is formed in calcium-magnesium-containing substratum penta
be 2.0312,2.0754,2.2003 and 2.2677 (in Figure 10 g), in this experimental result and PDF#70-1433 card data, the spacing 2.0202,2.0726,2.2020 and 2.2726 ten points of nesquehonite is close, therefore the corresponding crystal indices are respectively (006), (-222), (220) and (311), show that these mineral are nesquehonite.Same in the mineral that experimental group is formed in calcium-magnesium-containing substratum penta also have another, these mineral interplanar crystal spacing d value
be 2.8136,3.4478 and 5.2679 (in Figure 10 h), in this experimental result and PDF#83-1923 card data, the spacing 2.8249,3.4544 and 5.2768 ten points of monohydrocalcite is close, therefore the corresponding crystal indices are respectively (031), (120) and (110), show that the second mineral are monohydrocalcite.Visible, the mineral that experimental group is formed in calcium magnesium substratum penta are nesquehonite and monohydrocalcite.
In sum, the mineral that experimental group is formed in calcium-magnesium-containing substratum first, calcium-magnesium-containing substratum second and calcium-magnesium-containing substratum third comprise calcite, vaterite and monohydrocalcite, are CaCO
3, namely the main component of mineral is Ca, C and O, and this conclusion is just in time coincide with EDX analytical results in step one; The mineral that experimental group is formed in calcium-magnesium-containing substratum fourth and calcium-magnesium-containing substratum penta comprise nesquehonite and monohydrocalcite, and namely the main component of mineral is Mg, Ca, C and O, and this conclusion also matches with the analytical results of EDX in step one.
Claims (10)
1. Bacillus licheniformis (Bacilluslicheniformis) SRB3, it is CGMCCNo.10971 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is Bacillus licheniformis according to claim 1 (Bacilluslicheniformis) SRB3CGMCCNo.10971.
3. microbial inoculum described in (Bacilluslicheniformis) SRB3CGMCCNo.10971 of Bacillus licheniformis described in claim 1 or claim 2 is removing the application in calcium ion and/or magnesium ion.
4. microbial inoculum described in (Bacilluslicheniformis) SRB3CGMCCNo.10971 of Bacillus licheniformis described in claim 1 or claim 2 is removing the application in calcium ion in water and/or magnesium ion.
5. the application of microbial inoculum in sedimentation calcium ion and/or magnesium ion described in (Bacilluslicheniformis) SRB3CGMCCNo.10971 of Bacillus licheniformis described in claim 1 or claim 2.
6. the application of microbial inoculum described in (Bacilluslicheniformis) SRB3CGMCCNo.10971 of Bacillus licheniformis described in claim 1 or claim 2 in sedimentation water in calcium ion and/or magnesium ion.
7. obtaining a method for calcite and/or vaterite, comprising the steps: to containing 0.005 ~ 0.015mol/LCa
2+liquid-phase system in add Bacillus licheniformis described in claim 1 (Bacilluslicheniformis) SRB3CGMCCNo.10971.
8. obtaining a method for monohydrocalcite, comprising the steps: to containing 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+liquid-phase system in add Bacillus licheniformis described in claim 1 (Bacilluslicheniformis) SRB3CGMCCNo.10971.
9. obtaining a method for nesquehonite, comprising the steps: to containing 0.10 ~ 0.20mol/LMg
2+liquid-phase system in add Bacillus licheniformis described in claim 1 (Bacilluslicheniformis) SRB3CGMCCNo.10971.
10. the method as described in as arbitrary in claim 7 to 9, is characterized in that:
Describedly contain 0.005 ~ 0.015mol/LCa
2+liquid-phase system, describedly contain 0.005 ~ 0.015mol/LCa
2+with 0.05 ~ 0.20mol/LMg
2+liquid-phase system or describedly contain 0.10 ~ 0.20mol/LMg
2+liquid-phase system in all containing carbanion and/or bicarbonate ion.
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