CN105368756B - The bacterium and application thereof of one plant of removing calcium ion and magnesium ion - Google Patents

The bacterium and application thereof of one plant of removing calcium ion and magnesium ion Download PDF

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CN105368756B
CN105368756B CN201510932679.2A CN201510932679A CN105368756B CN 105368756 B CN105368756 B CN 105368756B CN 201510932679 A CN201510932679 A CN 201510932679A CN 105368756 B CN105368756 B CN 105368756B
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bacillus licheniformis
srb3
culture medium
magnesium
calcium
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CN201510932679.2A
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CN105368756A (en
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韩作振
闫华晓
赵辉
韩梅
赵延洋
孙彬
孟瑞瑞
庄定祥
陆凌雪
李雯君
秦志远
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山东科技大学
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RPROCESSES USING MICROORGANISMS
    • C12R1/00Processes using microorganisms
    • C12R1/01Processes using microorganisms using bacteria or actinomycetales
    • C12R1/07Bacillus
    • C12R1/10Bacillus licheniformis
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00Preparation of elements or inorganic compounds except carbon dioxide

Abstract

The invention discloses the bacteriums and application thereof of one plant of removing calcium ion and magnesium ion.The bacterium of removing calcium ion and magnesium ion provided by the present invention is bacillus licheniformis (Bacillus licheniformis) SRB3, is CGMCC No.10971 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Bacillus licheniformis (Bacillus licheniformis) SRB3CGMCC No.10971 provided by the invention serve vital in sedimentation calcium ion and magnesium ion, have broad application prospects in terms of water softening.

Description

The bacterium and application thereof of one plant of removing calcium ion and magnesium ion

Technical field

The present invention relates to microorganism fields, and in particular to the bacterium and application thereof of one plant of removing calcium ion and magnesium ion.

Background technology

Can be drinkable water by the processing of the hard water of calcic magnesium ion with softened water technology.Hot skill can be used in general water softening Art and membrane technology carry out:Thermal technology utilizes heat devaporation moisture, to realize calcium ions and magnesium ions from the separation in water;By electric energy The membrane technology of driving includes then reverse osmosis, membrane distillation and electrodialysis etc., and two kinds of technologies are required to consume big energy and fund. Last decade, softened water technology are continuously improved, and reduce a large amount of monetary losses, but worldwide high energy demand is still It merits attention.Energy expenditure for softened water technology is still a defect, results in high operation cost and water price.Cause This, it is a very necessary work to find the softening method reduction of environment friendliness saving or the calcium ions and magnesium ions in removing hard water Make.

Bacillus is very extensive in distributed in nature, and physiological property is rich and varied, is soil and plant microecology advantage One of population.It can generate Multiple Classes of Antibiotics, play good inhibiting effect to a variety of animal and plant and human pathogen bacterium, also have Have very strong protease, lipase, an amylase activity, at the same the bacterial strain of bacillus generally have powerful deposited metal from The metal ion of the ability of son, precipitation is largely heavy metal and radioactive nucleus element, therefore bacillus is widely used in The industry-by-industries such as medicine, pesticide, food, feed processing, environmental pollution improvement.

Bacillus licheniformis (Bacillus licheniformis) be strain more with potential applications in bacillus it One, achieve preferable achievement in research, such as the dead thalline pair of bacillus licheniformis in industries such as medicine, feed processing, pesticides Cr6+With good adsorption effect, the dead thalline of bacillus licheniformis R08 adsorb Pd2+, adsorbance can reach every gram of thalline and inhale Attached 224.8mg Pd2+.Therefore, lichem bacillus strain is targetedly screened, water treatment agent is prepared, is had by artificial infection The calcium ions and magnesium ions that effect is reduced or removed in hard water are a very necessary job and practicable approach.

Invention content

The technical problem to be solved by the present invention is to how remove calcium ion and magnesium ion to achieve the purpose that water softening.

In order to solve the above technical problems, the present invention provides the bacteriums that one plant can remove calcium ion and magnesium ion.

Bacterium provided by the present invention is bacillus licheniformis (Bacillus licheniformis) SRB3, and the bacterial strain is It was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, ground on 06 10th, 2015 Location is:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.10971.Bacillus licheniformis (Bacillus licheniformis) SRB3CGMCC No.10971 abbreviation bacillus licheniformis SRB3.

The present invention also provides a kind of microbial inoculum, the active constituent of the microbial inoculum is bacillus licheniformis SRB3.

The purposes of the microbial inoculum can be following a1), a2), a3) or a4):A1 calcium ion and/or magnesium ion) are removed;A2 it) takes off Calcium ion and/or magnesium ion in water removal;A3 calcium ion and/or magnesium ion) are settled;A4) settle in water calcium ion and/or magnesium from Son.

The preparation method of the microbial inoculum includes the following steps:Bacillus licheniformis SRB3 is seeded to bacteria culture media to go forward side by side Row culture, obtains OD600nmValue is 0.95 bacterium solution, the as described microbial inoculum.

The bacteria culture media is beef-protein medium or salt culture medium fourth.

The preparation method of the beef-protein medium is specific as follows:By beef extract 5g, tryptone 10g and chlorination Sodium 5g is dissolved in 1L distilled water, adjusts pH to 7.2.

The preparation method of the salt culture medium fourth is specific as follows:Beef extract 5g, tryptone 10g and sodium chloride 30g is molten In 1L distilled water, pH to 7.6 is adjusted.

In the preparation method of the microbial inoculum, the actual conditions of the culture can be:37 DEG C, 130r/min shaken cultivations for 24 hours.

In addition to active constituent, the microbial inoculum can also include carrier.The carrier can be solid carrier or liquid-carrier.Institute It can be mineral material, vegetable material or high-molecular compound to state solid carrier;The mineral material can be clay, talcum, kaolinite At least one of soil, montmorillonite, white carbon, zeolite, silica and diatomite;The vegetable material can be corn flour, bean powder and shallow lake At least one of powder;The high-molecular compound can be polyvinyl alcohol and/or polyglycols.The liquid-carrier can be organic molten Agent, vegetable oil, mineral oil or water;The organic solvent can be decane and/or dodecane.In the microbial inoculum, the active constituent It can be with the shape of the zymotic fluid of the living cells, living cells that are cultured, the filtrate of cell culture or cell and the mixture of filtrate Formula exists.The dosage form of the composition can be a variety of dosage forms, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder Or water dispersible granules.

As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum Agent (such as antioxidant), pH adjusting agent.

Application of the bacillus licheniformis SRB3 or any of the above-described microbial inoculums in removing calcium ion and/or magnesium ion also belongs to In protection scope of the present invention.

Application of the bacillus licheniformis SRB3 or any of the above-described microbial inoculums in removing water in calcium ion and/or magnesium ion Also belong to protection scope of the present invention.

Application of the bacillus licheniformis SRB3 or any of the above-described microbial inoculums in sedimentation calcium ion and/or magnesium ion also belongs to In protection scope of the present invention.

Application of the bacillus licheniformis SRB3 or any of the above-described microbial inoculums in settling water in calcium ion and/or magnesium ion Also belong to protection scope of the present invention.

The water can be the hard water containing calcium ion and/or magnesium ion.

The existence form of the calcium ion can be Ca2+

The existence form of the magnesium ion can be Mg2+

The hard water containing calcium ion and/or magnesium ion can be to contain 0.005~0.015mol/L Ca2+With 0.05~ 0.20mol/L Mg2+Water.

The hard water containing calcium ion and/or magnesium ion concretely contains 0.01mol/L Ca2+With 0.06~ 0.12mol/L Mg2+Water.

The present invention also provides a kind of methods of acquisition calcite and/or vaterite.

The method provided by the present invention for obtaining calcite and/or vaterite, includes the following steps:To containing 0.005~ 0.015mol/L Ca2+Liquid-phase system in bacillus licheniformis SRB3 is added.

The present invention also provides a kind of methods obtaining monohydrocalcite.

The method provided by the present invention for obtaining monohydrocalcite, includes the following steps:To containing 0.005~ 0.015mol/L Ca2+With 0.05~0.20mol/L Mg2+Liquid-phase system in bacillus licheniformis SRB3 is added.

The present invention also provides a kind of methods obtaining nesquehonite.

The method provided by the present invention for obtaining nesquehonite, includes the following steps:To containing 0.10~0.20mol/ LMg2+Liquid-phase system in bacillus licheniformis SRB3 is added.

The method for obtaining calcite and/or vaterite, the method for obtaining monohydrocalcite or the acquisition three Further include the steps that being cultivated after bacillus licheniformis SRB3 is added in the method for hydromagnesite.

The method for obtaining calcite and/or vaterite, the method for obtaining monohydrocalcite or the acquisition three In the method for hydromagnesite, the condition of the culture can cultivate 10~20 days for 35 DEG C~39 DEG C, 110~150r/min.

The method for obtaining calcite and/or vaterite, the method for obtaining monohydrocalcite or the acquisition three In the method for hydromagnesite, concretely 37 DEG C of the condition of the culture, 130r/min are cultivated 12 days.

It is described to contain 0.005~0.015mol/L Ca2+Liquid-phase system, described contain 0.005~0.015mol/LCa2+ With 0.05~0.20mol/L Mg2+Liquid-phase system or described contain 0.10~0.20mol/L Mg2+Liquid-phase system in Contain carbanion and/or bicarbonate ion.

Concentration of the carbanion in any of the above-described liquid-phase system can be 0.01~0.06mol/L.

Concentration of the carbanion in any of the above-described liquid-phase system concretely 0.04mol/L.

Concentration of the bicarbonate ion in any of the above-described liquid-phase system can be 0.01~0.06mol/L.

Concentration of the bicarbonate ion in any of the above-described liquid-phase system concretely 0.03mol/L.

It is described to contain 0.005~0.015mol/L Ca2+Liquid-phase system concretely contain 0.01mol/L Ca2+Liquid Phase system.

It is described to contain 0.005~0.015mol/L Ca2+With 0.05~0.20mol/L Mg2+Liquid-phase system concretely Contain 0.01mol/LCa2+With 0.06~0.12mol/L Mg2+Liquid-phase system.

It is described to contain 0.10~0.20mol/L Mg2+Liquid-phase system concretely contain 0.10~0.12mol/L Mg2+ Liquid-phase system.

It is described to contain 0.10~0.20mol/L Mg2+Liquid-phase system may also include 0.005~0.015mol/L Ca2+

It is described to contain 0.10~0.20mol/L Mg2+Liquid-phase system specifically may also include 0.01mol/L Ca2+

It is demonstrated experimentally that bacillus licheniformis SRB3 provided by the invention sedimentation calcium ion and magnesium ion in rise it is most important Effect, have broad application prospects in terms of water softening.

Description of the drawings

Fig. 1 is forms and systematic growth tree graph of the bacillus licheniformis SRB3 under high resolution TEM.

Fig. 2 be different salinity culture medium in bacillus licheniformis SRB3 growth curve and pH value change curve.

Fig. 3 is calcium ion trend chart.

Fig. 4 is magnesium ion trend chart.

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 is the scanning electron microscope (SEM) photograph and energy spectrum analysis that experimental group is precipitated in calcium-magnesium-containing culture medium first Minerals.

Fig. 8 is the scanning electron microscope (SEM) photograph and energy spectrum analysis that experimental group is precipitated in calcium-magnesium-containing culture medium second Minerals.

Fig. 9 is the scanning electron microscope (SEM) photograph and energy spectrum analysis that experimental group is precipitated in calcium-magnesium-containing culture medium fourth Minerals.

Figure 10 is high resolution TEM and the nano-area electronic diffraction Conjoint Analysis of experimental group mineral precipitation.

Preservation explanation

Strain name:Bacillus licheniformis

Latin name:(Bacillus licheniformis)

Strain number:SRB3

Preservation mechanism:China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation mechanism is referred to as:CGMCC

Address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3

Preservation date:On 06 10th, 2015

Collection is registered on the books number:CGMCC No.10971

Specific implementation mode

The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Culture medium as used in the following examples is as follows:

Activation medium:Beef extract 5g, tryptone 10g and sodium chloride 5g are dissolved in 1L distilled water, adjust pH to 7.2, Then agar 15g is added.

Beef extract tryptone culture medium:Beef extract 5g, tryptone 10g and sodium chloride 5g are dissolved in 1L distilled water, adjusted Save pH to 7.2.

Enriched medium:Solute and its a concentration of K2HPO40.5g/L, NH4Cl 1.0g/L, CaCl20.1g/L, MgSO4· 7H2O 2.0g/L, yeast extract 1.0g/L, Na2SO40.5g/L, Fe (NH4)2(SO4)20.5g/L, ascorbic acid 0.5g/L, L- Cys 0.5g/L, 60% sodium lactate solution 6mL/L;Solvent is distilled water;PH6.0~8.0;Wherein Fe (NH4)2(SO4)2, it is anti- Bad hematic acid and L-Cys need first to be configured to mother liquor with sterile distilled water, then use 0.22 μm of membrane filtration degerming, are subsequently used for preparing richness Collect culture medium;60% sodium lactate solution is Sinopharm Chemical Reagent Co., Ltd.'s product, catalog number 30168018.

Pure medium:Yeast extract 3g, KCl 0.25g and agar 15g are dissolved in 1L distilled water, natural pH.

Salt culture medium first:Beef extract 5g and tryptone 10g are dissolved in 1L distilled water, adjust pH to 7.6.Salt culture medium first In, a concentration of 0% (mass percent) of NaCl

Salt culture medium second:Beef extract 5g, tryptone 10g and sodium chloride 10g are dissolved in 1L distilled water, adjust pH to 7.6. In salt culture medium second, a concentration of 1% (mass percent) of NaCl.

Salt culture medium third:Beef extract 5g, tryptone 10g and sodium chloride 20g are dissolved in 1L distilled water, adjust pH to 7.6. In salt culture medium third, a concentration of 2% (mass percent) of NaCl.

Salt culture medium fourth:Beef extract 5g, tryptone 10g and sodium chloride 30g are dissolved in 1L distilled water, adjust pH to 7.6. In salt culture medium fourth, a concentration of 3% (mass percent) of NaCl.

Salt culture medium penta:Beef extract 5g, tryptone 10g and sodium chloride 40g are dissolved in 1L distilled water, adjust pH to 7.6. In salt culture medium penta, a concentration of 4% (mass percent) of NaCl.

X-ray diffraction analysis use turns target X-ray diffractometer, Rigaku Electric company product, product type D/ Max-RC;It is Hitachi S-4800 that scanning electron microscope analysis, which uses scanning electron microscope, Hitachi, Japan product, product type,;Energy Spectrum analysis uses GENESIS energy disperse spectroscopies, Yi Dakesi Co., Ltds of U.S. product;High resolution TEM analysis uses high score Resolution transmission electron microscope, Jeol Ltd.'s Products, product type JEM-2100.

SrCl2·6H2O aqueous solutions:By 30.4g SrCl2·6H2O is dissolved in 100mL distilled water, obtained solution.

Embodiment 1, bacillus licheniformis (Bacillus licheniformis) SRB3CGMCC No.10971 separation, Identification and preservation

One, the separation of sulfate reducing bacteria SRB3

1,10g pedotheques (sludge for picking up from Chinese Qingdao ink river bed portion) are added in 100mL sterile distilled waters, stir It mixes uniformly, it is static to place 10 minutes, it then takes 20mL supernatants to be added in 200mL enriched mediums, sterile liquid paraffin is used in combination Liquid level in sealing bottles is placed in stationary culture in 37 DEG C of constant incubators, observation.To the 7th day, enriched medium color became for culture It is black, it is seen that sulfate reducing bacteria is enriched with the successfully (Fe (NH in enriched medium4)2(SO4)2In ferrous ion can with enrichment train Support the S in base2-Reaction generates the ferrous sulfide of black).

2, after completing step 1, supernatant is taken to be seeded to (the NH of Fe containing 0.5g/L4)2(SO4)2Pure medium on, 37 DEG C It cultivates, observe in anaerobic culture box.Culture generated black single bacterium colony to the 7th day.Picking single bacterium colony, purify 3 times repeatedly or more. It is sulfate reducing bacteria SRB3 by the strain of sulfate reduction bacteria Strain Designation screened.

Two, the identification of sulfate reducing bacteria SRB3

1, Morphological Identification

Sulfate reducing bacteria SRB3 is inoculated on activation medium, the form of single bacterium colony is observed after 3 days.The result shows that Sulfate reducing bacteria SRB3 bacterium colonies are round, and there is fold on 3.0~5.0mm of diameter, surface, and edge is irregular, color be white (with Incubation time extends, and color becomes pink), bacterium colony is opaque and dry, without wettability.

After sulfate reducing bacteria SRB3 is dyed, it is accredited as gram-positive bacteria.It is analyzed by high resolution TEM Sulfate reducing bacteria SRB3, experimental result is shown in a in Fig. 1.The result shows that about 1.46 μ m of the size of sulfate reducing bacteria SRB3 0.63 μm, bacterium is quarter butt shape, individualism.

2,16S rDNA sequence homology analysis

The 16S rDNA of sulfate reducing bacteria SRB3 are as shown in the sequence 1 in sequence table.

The sequence 1 in sequence table is compared with the sequence in GenBank by Clustal X softwares, using adjoining Method (N-J methods) phylogenetic tree construction.Phylogenetic tree experimental result is shown in b in Fig. 1.

Sulfate reducing bacteria SRB3 and bacillus licheniformis (Bacillus licheniformis) homology highest, reach 97%.

In summary each qualification result, sulfate reducing bacteria SRB3 are bacillus licheniformis (Bacillus licheniformis)。

Three, the preservation of sulfate reducing bacteria SRB3

Bacillus licheniformis (Bacillus licheniformis) SRB3 was preserved in China on 06 10th, 2015 (abbreviation CGMCC, address are Microbiological Culture Collection administration committee common micro-organisms center:BeiChen West Road, Chaoyang District, BeiJing City 1 Number institute 3), deposit number is CGMCC No.10971.The full name of sulfate reducing bacteria SRB3 is bacillus licheniformis (Bacillus licheniformis) SRB3CGMCC No.10971, referred to as bacillus licheniformis SRB3 or SRB3.

Embodiment 2 prepares bacillus licheniformis SRB3 microbial inoculums

Bacillus licheniformis SRB3 is activated on activation medium, then picking single bacterium colony is inoculated in equipped with 100mL oxen In the 250mL conical flasks of meat extract peptone culture medium, 37 DEG C, cultivate 1 day on the constant-temperature table of 130r/min, OD is obtained600Value is about For 0.95 bacillus licheniformis SRB3 microbial inoculum first.Bacillus licheniformis SRB3 microbial inoculum first is hereinafter simply 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 all sames obtain To OD600The bacillus licheniformis SRB3 microbial inoculum second of value about 0.95.Bacillus licheniformis SRB3 microbial inoculum second is hereinafter simply referred to as SRB3 microbial inoculum second.

The characteristic of embodiment 3, bacillus licheniformis SRB3

The present embodiment studies the growth curve of bacillus licheniformis SRB3 and pH change curves under different salinity.

Each salt culture medium (salt culture medium first, salt culture medium second, salt culture medium third, salt culture medium fourth or salt is respectively adopted Culture medium penta) it proceeds as follows:

The conical flask of 6 250mL, each conical flask is taken to be packed into 200mL salt culture mediums, then 121 DEG C of high pressure sterilizations 20min;After culture medium is cooled to room temperature, 6 conical flasks are divided into experimental group and control group, every group of 3 conical flasks;To experiment It is inoculated with SRB3 microbial inoculum first 2mL in the conical flask of group, isometric sterile ultra-pure water is inoculated with into the conical flask of control group;Inoculation finishes Afterwards, conical flask is placed in constant-temperature shaking incubator, 37 DEG C, 130r/min cultures.Inoculation samples 4mL when finishing, then every 3 hours 4mL is sampled, bacteria concentration and pH value are measured.

It is tested three times, results are averaged.

The growth curve of SRB3 is shown in a in Fig. 2 in experimental group.Growth courses of the SRB3 in each salt culture medium includes prolonging Demurrage (0~6h of a in Fig. 2), logarithmic phase (6~12h of a in Fig. 2) and stationary phase (12~36h of a in Fig. 2) three phases, Wherein lag phase is the stage of growing environment of the bacterium just in acclimatizing culture medium, and logarithmic phase is that bacterium is in active divided The stage of state, stationary phase are the bacterium since the nutriment in culture medium is consumed and the gradual accumulation of toxic metabolic products Growth rate slow down gradually but stage that total biomass still remains unchanged.The result shows that SRB3 is in the training containing 4%NaCl It supports to grow in base and initially enters within 12 hours stationary phase, and SRB3 is raw in the culture medium containing 0%, 1%, 2% or 3%NaCl Length is to 12 hours still in logarithmic phase;SRB3 grown in the culture medium containing 4%NaCl 36 hours a concentration of 4.70 × 108The concentration that cfu/mL, SRB3 grow to 36 hours in the culture medium containing 0%, 1%, 2% or 3%NaCl is 8.0~ 8.2×108cfu/mL.Since what is be inoculated in control group is sterile ultra-pure water rather than bacterium solution, so without SRB3 in control group Growth curve.

The pH value change curve of SRB3 is shown in b in Fig. 2 in experimental group.PH value variations of the SRB3 in each salt culture medium is wrapped It includes the slow rising stage (0~6h of b in Fig. 2), decline phase (6~12h of b in Fig. 2) and rising stage (12~60h of b in Fig. 2) three A stage declines phase pH value and is down to 7.1 or so from 7.8 or so wherein slow rising stage pH value maintains 7.6 or so, the rising stage PH value persistently rises to 8.4 or so from 7.1 or so.PH value in control group has no significant change.

The measurement of embodiment 4, bacillus licheniformis SRB3 removing calcium ion and magnesium ion ability

1, the making of standard curve

(1)Ca2+Standard curve

Accurately weigh anhydrous CaCl21.11g is made into Ca in 1L volumetric flasks, with sterile water dissolution2+A concentration of 400mg/L's CaCl2Mother liquor.Continue to be configured to Ca with sterile water dilution2+A concentration of 0,5,10,15,20,25,30 and 35mg/L series CaCl2Solution.Various concentration CaCl is measured using atomic absorption spectrophotometer2The light absorption value of solution, 3 repetitions.With Ca2+It is dense Degree is abscissa, and light absorption value is ordinate, draws Ca2+Standard curve.

(2)Mg2+Standard curve

Accurately weigh top pure grade MgCl2·6H2O 4.1814g are made into Mg in 500mL volumetric flasks, with sterile water dissolution2+ The MgCl of a concentration of 1000mg/L2Mother liquor.Continue to be configured to Mg with sterile water dilution2+A concentration of 0,10,20,30,40,50,75 With the MgCl of 100mg/L series2Solution, to various MgCl21mL SrCl are added in solution2·6H2O aqueous solutions.Then original is used Sub- absorption spectrophotometer measures various concentration MgCl2The light absorption value of solution, 3 repetitions.With Mg2+A concentration of abscissa, extinction Value is ordinate, draws Mg2+Standard curve.

2, salt culture medium fourth is taken, 1.0mol/L CaCl are added2Aqueous solution makes the Ca in system2+A concentration of 0.01mol/L, Obtain calcic culture medium.

3, the calcic culture medium for taking step 2 to prepare, is added the MgCl of 2.0mol/L2Aqueous solution makes the Mg in system2+Concentration Be followed successively by 0mol/L, 0.06mol/L, 0.08mol/L, 0.10mol/L and 0.12mol/L, obtain successively calcium-magnesium-containing culture medium first, Calcium-magnesium-containing culture medium second, calcium-magnesium-containing culture medium third, calcium-magnesium-containing culture medium fourth and calcium-magnesium-containing culture medium penta.

4, culture medium is respectively adopted (to obtain calcium-magnesium-containing culture medium first, calcium-magnesium-containing culture medium second, calcium-magnesium-containing culture medium third, contain Calcium and magnesium culture medium fourth or calcium-magnesium-containing culture medium penta) it proceeds as follows:

The conical flask of 6 250mL, each conical flask is taken to be packed into the culture medium of 150mL, 121 DEG C of high pressure sterilization 20min.It waits for After culture medium is cooled to room temperature, the 2mol/L Na of 3mL are added in each conical flask2CO3The 1mol/L of aqueous solution and 5mL NaHCO3Aqueous solution (Na2CO3And NaHCO3Used again after the membrane filtration degerming that solution is 0.22 micron with aperture), with NaOH tune Save pH to 7.2.

5, after completing step 4,6 conical flasks are divided into experimental group and control group, every group of 3 conical flasks.To experimental group The SRB3 microbial inoculum second 1.5mL (inoculative proportions 1 that in each conical flask prepared by inoculation embodiment 2:100), to each cone of control group The sterile ultra-pure water of 1.5mL is inoculated in shape bottle.After inoculation, all conical flasks are placed in constant-temperature shaking incubator, 37 DEG C, 130r/min is cultivated 18 days.

In incubation, sampling 1.5mL, 10000rpm centrifuge 5min within every 48 hours, supernatant are collected, by the supernatant of collection Liquid dilutes 10 times with sterile distilled water, and it is 0.22 micron of membrane filtration then to use aperture, obtains dilution.First sub-sampling Time is after completing step 4.

6, the light absorption value that dilution is measured with atomic absorption spectrophotometer, the Ca made according to step 12+Standard curve and Mg2+Standard curve obtains Ca in dilution2+And Mg2+Concentration.

In incubation, the Ca in each culture medium2+Variation tendency as shown in Figure 3 (a is control group, and b is experimental group). Experimental result is as follows:

(1) it cultivates the 0th day to the 4th day, due to adding 2mol/L Na in each culture medium2CO3Aqueous solution and 1mol/L NaHCO3Aqueous solution, so the calcium ion concentration in each culture medium is remarkably decreased, and produces precipitation, but with control group phase Than the calcium ion concentration in experimental group is considerably lower, shows bacillus licheniformis (Bacillus licheniformis) SRB3 Play the role of promoting calcium ion sedimentation;Simultaneously with the increase of Mg/Ca molar ratios in culture medium, remaining calcium ion in culture solution Concentration becomes larger, it is seen that magnesium ion can hinder the sedimentation of calcium ion.

(2) it cultivates the 5th day to the 12nd day, the calcium ion concentration downward trend in each culture medium is gradually delayed, and is opened within the 13rd day Begin, calcium ion concentration is basically unchanged.

(3) culture was to the 18th day:In experimental group, calcium-magnesium-containing culture medium first, calcium-magnesium-containing culture medium second and calcium-magnesium-containing culture medium Calcium ion concentration is 0~20mg/L in third, in calcium-magnesium-containing culture medium fourth and calcium-magnesium-containing culture medium penta calcium ion concentration 20~ Between 40mg/L, it is seen that magnesium ion can hinder the sedimentation of calcium ion;Calcium ion concentration in experimental group is significantly lower than control group, can See, bacillus licheniformis (Bacillus licheniformis) SRB3 can promote the sedimentation of calcium ion.

The variation tendency of magnesium ion in each culture medium is as shown in Figure 4 (a is control group, and b is experimental group).Experimental result It is as follows:

(1) after being inoculated with SRB3 microbial inoculum second, magnesium ion concentration drastically declines, until culture is still declining on the 14th day.

(2) culture was to the 14th day:Calcium-magnesium-containing culture medium second, calcium-magnesium-containing culture medium third, calcium-magnesium-containing culture medium fourth in experimental group And the magnesium ion concentration in calcium-magnesium-containing culture medium penta is between 180~350mg/L, and magnesium ion concentration exists in control group Between 350~550mg/L.As it can be seen that sedimentations of bacillus licheniformis (Bacillus licheniformis) SRB3 in magnesium ion In play the role of it is vital.

The mineral precipitation analysis that embodiment 5, embodiment 4 obtain

The mineral precipitation obtained in 4 control group of embodiment and experimental group is further analyzed.

One, X-ray diffraction (X-ray diffraction, XRD) is analyzed

Each culture medium bottom precipitation that 12 days are cultivated in 4 step 5 of Example is placed in 1.5mL centrifuge tubes, stands 5min, Supernatant is abandoned, the distilled water that 1mL is then respectively added in each centrifuge tube is washed, and stands 5 minutes, abandons supernatant, is washed three times, To remove various salt ions, precipitation carries out XRD analysis after spontaneously drying.XRD scanning angles are 10 ° -90 °, and step-length 0.02 is swept Retouch 8 °/min of speed.

Experimental result is shown in Fig. 5, (a is control group, and b is experimental group, wherein Mg/Ca=0, Mg/Ca=6, Mg/Ca=8, Mg/ Ca=10 and Mg/Ca=12 respectively represents calcium-magnesium-containing culture medium first, calcium-magnesium-containing culture medium second, calcium-magnesium-containing culture medium third, calcium-magnesium-containing Mineral precipitation in culture medium fourth and calcium-magnesium-containing culture medium penta) and table 1.The result shows that bacillus licheniformis (Bacillus Licheniformis) SRB3 has close ties with nesquehonite mineral precipitation is generated.Calcium-magnesium-containing culture medium in experimental group In the mineral precipitation that fourth and experimental group calcium-magnesium-containing culture medium penta generate, (004) and (002) crystal face of nesquehonite is selected Excellent orientation phenomenon.It is brilliant that the diffraction peak intensity of (400) and (101) crystal face of standard nesquehonite is respectively higher than (004) and (002) Face, but under the cultivating system, the diffraction peak intensity of (004) and (002) crystal face is but respectively higher than (400) and (101) crystal face.Together When, the diffraction peak intensity for the nesquehonite that the calcium-magnesium-containing culture medium penta in experimental group generates is higher than the calcium-magnesium-containing in experimental group The diffraction peak intensity for the nesquehonite that culture medium fourth generates shows three hydromagnesites that calcium-magnesium-containing culture medium penta generates in experimental group The quantity of mine is more or crystallinity is high.

Table 1.XRD assayings precipitate

Two, the scanning electron microscope of mineral precipitation and energy spectrum analysis

Each culture medium bottom precipitation that 12 days are cultivated in 4 step 5 of Example is placed in 1.5mL centrifuge tubes, stands 5min, Supernatant is abandoned, the distilled water that 1mL is then respectively added in each centrifuge tube is washed, and stands 5 minutes, abandons supernatant, is washed three times, To remove various salt ions, the absolute ethyl alcohol that 100 μ L are then added into precipitation suspends.Sample segment is taken to be placed in objective table On, metal spraying after natural drying is placed under scanning electron microscope and observes, and then carries out energy spectrum analysis.

Experimental result is as follows:

Fig. 6 is the scanning electron microscope (SEM) photograph for the mineral precipitation that control group generates, and wherein a is control group in calcium-magnesium-containing culture medium first The surface topography of the mineral precipitation of formation, b are the surface topography for the mineral precipitation that control group is formed in calcium-magnesium-containing culture medium second. The surface shape for the mineral precipitation that control group is formed in calcium-magnesium-containing culture medium third, calcium-magnesium-containing culture medium fourth and calcium-magnesium-containing culture medium penta Looks are identical as the surface topography of mineral precipitation that control group is formed in calcium-magnesium-containing culture medium second.The result shows that control group is each The mineral precipitation formed in culture medium is spherical shape, and in culture medium Mg/Ca than increase, the diameter of the mineral of formation It is gradually reduced.

Fig. 7 is scanning electron microscope (SEM) photograph and the energy spectrum analysis for the mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium first.Its A and b is surface topography in middle Fig. 7, and the form of mineral precipitation is spherical and dumbbell shaped, and surface is relatively rough and contains a large amount of hole Gap, mineral are attached on mineral and are largely enclosed with the rod-shaped of fine and close calcium carbonate layer by many nano-scale particle shape mineral compositions Thalline;The diameter of spherical mineral is about 16 μm, and the length of dumbbell shape mineral precipitation is about 16 μm.There is the bacterium of two states Body:One is thalline outer surfaces to be wrapped up by a large amount of nano-scale particle shape mineral, i.e., mineralising phenomenon has occurred in thalline itself, goes out The hollow cylindrical mineral shell as shown in c in Fig. 7 and d is showed, the thalline some that itself mineralising occurs is attached to mineral surfaces, has Then with mineral precipitation growth together, become a part for mineral (as shown in c white arrows in Fig. 7);Second of feelings Condition is that mineralising does not occur for thalline, but shrinkage occurs for the outer surface of thalline, as shown in d white arrows in Fig. 7.The result shows that containing SRB3 can induce the generation of mineral precipitation in calcium and magnesium culture medium first, and the volume of the mineral precipitation formed is significantly greater than control group not The volume for the mineral precipitation that SRB3 is formed is added;Mineralising can occur for SRB3 itself simultaneously.To the ball that b white box is chosen in Fig. 7 Shape and dumbbell shape mineral carry out energy spectrum analysis, the results showed that the mineral mainly contain C, O and Ca these three elements (e in Fig. 7), say The mineral of bright formation are CaCO3, in addition mineral also contain a small amount of Na elements and Si elements (e in Fig. 7), thus it is speculated that sodium element may Sodium chloride in culture solution, Si elements are probably derived from objective table, because the objective table main component of scanning electron microscope is just It is monocrystalline silicon.

Fig. 8 is scanning electron microscope (SEM) photograph and the energy spectrum analysis for the mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium second.It sweeps Retouch Electronic Speculum the result shows that, huge change has occurred in the surface topography for the mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium second Change is in peanut shape and spherical (a in Fig. 8), and the surface of peanut shape mineral grows several laminar mineral (b in Fig. 8), No longer it is nano-scale particle shape mineral;Peanut shape mineral length is about 30 μm, and large amount of thin sheets shape mineral growth flocks together A small amount of random mineral (c in Fig. 8) are formed, part thalline (in Fig. 8 shown in the white arrow of d) has been inlayed in mineral precipitation, But do not find that itself mineralising occurs for thalline.Energy spectrum analysis is carried out to the mineral that b white box is chosen in Fig. 8, the results showed that the mine Main these three elements (e in Fig. 8) containing C, O and Ca of object illustrate that the mineral to be formed are CaCO3, in addition mineral also contain a small amount of Si elements (e in Fig. 8), Si elements are probably derived from objective table, because the objective table main component of scanning electron microscope is exactly monocrystalline silicon.

The mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium third is formed with experimental group in calcium-magnesium-containing culture medium second Mineral precipitation scanning electron microscope (SEM) photograph and energy spectrum analysis it is essentially identical.

Fig. 9 is scanning electron microscope (SEM) photograph and the energy spectrum analysis for the mineral that experimental group is formed in calcium-magnesium-containing culture medium fourth.Scanning electricity Mirror the result shows that, the surface topography of the mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium fourth is in addition to ellipse and random Except mineral, also there are a large amount of long column shape mineral (a and b in Fig. 9), there is the crisp thalline of a large amount of appearances to be gathered in mineral table On face (a and b in Fig. 9), when the long axis for the thalline being attached on mineral is just parallel to the long axis of long column shape mineral, mineral are just It can then be embedded in the position of recess in the recess for thering is the position generation that thalline adheres to be parallel to long axis, thalline, it is seen that the presence of thalline Induction produces long column shape mineral crystal, while thalline would also adhere to the surface of crystal.A black box in Fig. 9 is chosen Mineral carry out energy spectrum analysis, the results showed that main these three elements (c in Fig. 9) containing C, O and Ca of the mineral illustrate the mineral to be formed For CaCO3, for mineral also containing a small amount of Si elements and Mg elements (c in Fig. 9), Mg elements may be due to highly concentrated in solution in addition The Mg of degree2+It is attached to and is formed on mineral, Si elements are probably derived from objective table, because of the objective table main component of scanning electron microscope It is exactly monocrystalline silicon.The long column shape mineral chosen to b black box in Fig. 9 carry out energy spectrum analysis, the mineral mainly contain C, O and Mg this Three kinds of elements illustrate that the mineral to be formed are MgCO3, mineral are also containing a small amount of Ca and Si elements (d in Fig. 9), Ca elements in addition May be the Ca due to solution middle and high concentration2+It is attached to caused by mineral surfaces, Si elements are probably derived from objective table, because sweeping The objective table main component for retouching Electronic Speculum is exactly monocrystalline silicon.As it can be seen that the mineral that experimental group is formed in calcium-magnesium-containing culture medium fourth are CaCO3And MgCO3

The mineral precipitation that experimental group is formed in calcium-magnesium-containing culture medium penta is formed with experimental group in calcium-magnesium-containing culture medium fourth Mineral precipitation scanning electron microscope (SEM) photograph and energy spectrum analysis it is essentially identical.

Three, high resolution TEM (the High Resolution Transmission Electron of mineral precipitation Microscopy, HRTEM) and nano-area electronic diffraction Conjoint Analysis

Each culture medium bottom precipitation that 12 days are cultivated in 4 step 5 of Example is placed in 1.5mL centrifuge tubes, stands 5min, Supernatant is abandoned, the distilled water that 1mL is then respectively added in each centrifuge tube is washed, and stands 5 minutes, abandons supernatant, is washed three times, To remove various salt ions, then precipitation is fully ground with agate mortar, absolute ethyl alcohol is added and suspends, carried out HRTEM and receive Rice regional Electronic diffraction Conjoint Analysis.

Experimental result is as follows:

The interplanar crystal spacing d values for the mineral that experimental group is formed in calcium-magnesium-containing culture medium firstFor 1.1711,1.3502, 1.2938 and 1.4258 (a in Figure 10), the interplanar distance of this experimental result and calcite in PDF#24-0027 card datas 1.1778,1.3553,1.2950 and 1.4168 are very close, thus the corresponding indices of crystallographic plane be respectively (0114), (217), (128) and (0012), show that the mineral are calcite.The mineral that experimental group is formed in calcium-magnesium-containing culture medium first also have other one Kind, the interplanar crystal spacing d values of the mineralFor 3.5036,4.1162 and 5.8041 (b in Figure 10), this experimental result and PDF# Vaterite interplanar distance 3.4966,4.1730 and 5.8147 are very close in 72-1616 card datas, therefore corresponding crystal face Index is respectively (104), (103) and (101), shows that second of mineral is vaterite.As it can be seen that experimental group is in calcium-magnesium-containing culture medium The mineral formed in first are calcite and vaterite.

The interplanar crystal spacing d values for the mineral that experimental group is formed in calcium-magnesium-containing culture medium secondFor 2.4859,2.4220, 2.6457 and 2.9145 (c in Figure 10), the interplanar of this experimental result and monohydrocalcite in PDF#83-1923 card datas Be sufficiently close to away from 2.4905,2.4247,2.6384 and 2.9091, thus the corresponding indices of crystallographic plane be respectively (221), (103), (220) and (022), show that the mineral are monohydrocalcites.

The interplanar crystal spacing d values for the mineral that experimental group is formed in calcium-magnesium-containing culture medium thirdFor 2.3728,2.4232, 2.5526 and 2.9249 (d in Figure 10), the interplanar of this experimental result and monohydrocalcite in PDF#83-1923 card datas Away from 2.3701,2.4247,2.5476 and 2.9091, the very close therefore corresponding indices of crystallographic plane are respectively (032), (103) (212) and (022), show that the mineral are monohydrocalcites.

The interplanar crystal spacing d values for the mineral that experimental group is formed in calcium-magnesium-containing culture medium fourthFor 1.7836,2.0819, 2.1836 and 2.3737 (e in Figure 10), the interplanar of this experimental result and nesquehonite in PDF#70-1433 card datas Be sufficiently close to away from 1.7833,2.0726,2.1840 and 2.3726, thus the corresponding indices of crystallographic plane be respectively (206), (- 222), (- 214) and (204) show that the mineral are nesquehonite.The mineral that experimental group is formed in calcium-magnesium-containing culture medium fourth Also another, the interplanar crystal spacing d values of the mineralFor 3.4899,3.9162 and 5.8203 (f in Figure 10), this reality It tests result and monohydrocalcite interplanar distance in PDF#83-1923 card datas 3.4869,3.9087 and 5.8183 is very close, Therefore the corresponding indices of crystallographic plane are respectively (012), (201) and (101), show that second of mineral is monohydrocalcite.As it can be seen that The mineral that experimental group is formed in calcium and magnesium culture medium fourth are nesquehonite and monohydrocalcite.

The interplanar crystal spacing d values for the mineral that experimental group is formed in calcium-magnesium-containing culture medium pentaFor 2.0312,2.0754, 2.2003 and 2.2677 (g in Figure 10), the interplanar of this experimental result and nesquehonite in PDF#70-1433 card datas Be sufficiently close to away from 2.0202,2.0726,2.2020 and 2.2726, thus the corresponding indices of crystallographic plane be respectively (006), (- 222), (220) and (311) show that the mineral are nesquehonite.In the mineral that experimental group is formed in calcium-magnesium-containing culture medium penta As well as another, the interplanar crystal spacing d values of the mineralFor 2.8136,3.4478 and 5.2679 (h in Figure 10), this The interplanar distance 2.8249,3.4544 and 5.2768 ten of one experimental result and monohydrocalcite in PDF#83-1923 card datas Split-phase is close, therefore the corresponding indices of crystallographic plane are respectively (031), (120) and (110), shows that second of mineral is that single water side solves Stone.As it can be seen that the mineral that experimental group is formed in calcium and magnesium culture medium penta are nesquehonite and monohydrocalcite.

In conclusion experimental group is formed in calcium-magnesium-containing culture medium first, calcium-magnesium-containing culture medium second and calcium-magnesium-containing culture medium third Mineral include calcite, vaterite and monohydrocalcite, be CaCO3, i.e., the main component of mineral be Ca, C and O, This conclusion is just coincide with EDX analysis results in step 1;Experimental group is in calcium-magnesium-containing culture medium fourth and calcium-magnesium-containing culture medium penta The mineral of middle formation include nesquehonite and monohydrocalcite, i.e. the main component of mineral is Mg, Ca, C and O, this Conclusion also matches with the analysis result of EDX in step 1.

Claims (9)

  1. Bacillus licheniformis 1. (Bacillus licheniformis) SRB3, in Chinese microorganism strain preservation conservator The deposit number of meeting common micro-organisms center is CGMCC No.10971.
  2. 2. a kind of microbial inoculum, it is characterised in that:The active constituent of the microbial inoculum is bacillus licheniformis described in claim 1 (Bacillus licheniformis)SRB3 CGMCC No.10971。
  3. 3. (Bacillus licheniformis) the SRB3 CGMCC No.10971 of bacillus licheniformis described in claim 1 or Application of the microbial inoculum described in claim 2 in removing calcium ion and/or magnesium ion.
  4. 4. (Bacillus licheniformis) the SRB3 CGMCC No.10971 of bacillus licheniformis described in claim 1 or Application of the microbial inoculum described in claim 2 in removing water in calcium ion and/or magnesium ion.
  5. 5. (Bacillus licheniformis) the SRB3 CGMCC No.10971 of bacillus licheniformis described in claim 1 or Application of the microbial inoculum described in claim 2 in sedimentation calcium ion and/or magnesium ion.
  6. 6. (Bacillus licheniformis) the SRB3 CGMCC No.10971 of bacillus licheniformis described in claim 1 or Application of the microbial inoculum described in claim 2 in settling water in calcium ion and/or magnesium ion.
  7. 7. a kind of method obtaining calcite and/or vaterite, includes the following steps:To containing 0.005~0.015mol/L Ca2 +Liquid-phase system in be added claim 1 described in bacillus licheniformis (Bacillus licheniformis) SRB3 CGMCC No.10971;It is described to contain 0.005~0.015mol/L Ca2+Liquid-phase system in contain carbanion and/or bicarbonate radical Ion.
  8. 8. a kind of method obtaining monohydrocalcite, includes the following steps:To containing 0.005~0.015mol/LCa2+With 0.05 ~0.20mol/L Mg2+Liquid-phase system in be added claim 1 described in bacillus licheniformis (Bacillus licheniformis)SRB3 CGMCC No.10971;It is described to contain 0.005~0.015mol/LCa2+With 0.05~ 0.20mol/L Mg2+Liquid-phase system in contain carbanion and/or bicarbonate ion.
  9. 9. a kind of method obtaining nesquehonite, includes the following steps:To containing 0.10~0.20mol/L Mg2+Liquid phase body Bacillus licheniformis (Bacillus licheniformis) SRB3 CGMCC No.10971 described in claim 1 are added in system; It is described to contain 0.10~0.20mol/L Mg2+Liquid-phase system in contain carbanion and/or bicarbonate ion.
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Publication number Priority date Publication date Assignee Title
CN102628066A (en) * 2012-04-17 2012-08-08 山西大学 Preparation method and application of microbial flocculant

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Struvite Precipitation Induced by a Novel Sulfate-Reducing Bacterium Acinetobacter calcoaceticus SRB4 Isolated from River Sediment;ZUOZHEN HAN等;《Geomicrobiology Journal》;20150513;第32卷;868-877页 *
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