CN103966125B - Bacillus licheniformis, microbial agent and application of bacillus licheniformis or microbial agent in mine ecological remediation - Google Patents
Bacillus licheniformis, microbial agent and application of bacillus licheniformis or microbial agent in mine ecological remediation Download PDFInfo
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- CN103966125B CN103966125B CN201410171760.9A CN201410171760A CN103966125B CN 103966125 B CN103966125 B CN 103966125B CN 201410171760 A CN201410171760 A CN 201410171760A CN 103966125 B CN103966125 B CN 103966125B
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Abstract
The invention provides a bacillus licheniformis of which the preservation number is CCTCC NO: M2013497. The invention further provides a microbial agent. The microbial agent is prepared by the preparation method comprising the following steps: inoculating the bacillus licheniformis into a liquid culture medium for cultivation to obtain a seed liquid; (2) inoculating the seed liquid into a solid culture medium for fermentation to obtain fermented materials. The invention further provides application of the bacillus licheniformis or the microbial agent in mine ecological remediation. By the adoption of the technical scheme, the root growth of green plants can be remarkably promoted and reach to a higher flourishing degree, and the stability of side slopes is further increased.
Description
Technical field
A kind of the present invention relates to microbe application field, in particular it relates to Bacillus licheniformis (bacillus
Licheniformis), the microbial bacterial agent containing this Bacillus licheniformis and their purposes in ecological restoration of mine.
Background technology
Mine is repaired and Mining Wasteland pollution is repaired, and realizes land resource is reused.Mining
During can produce the non-soil that cannot use through improvement in a large number, also known as Mining Wasteland, exist discardedly and lead to because of production
Various pollutions.
The Mining Wasteland origin cause of formation is a lot, include stripping table mound amass form, rock fragment and poor value pile up formation,
Empty exploiting field and subsidence area are formed, mine tailing pile up formed, affected by mining and cannot exploitation soil etc..To examine from broad sense
Consider, also include metallurgical slag that the smelting of Ore formed aheap, and heavy metal and sulfur dioxide (SO2) emissions institute in smelting process
The degraded land leading to.
Mining Wasteland, how based on heavy metal pollution and acid mine drainage waste pollution, administers content with restoration of the ecosystem and dirt
Based on dye is administered.The impact to ecological environment for the mine development includes: the impact to land resource, the impact to water resource, to big
The impact of gas and bio-diversity loss.
Restoration of the ecosystem refers to stop to ecological interference and destruction, using the self-recovery ability of ecosystem, is aided with people
Work measure, makes the ecosystem being destroyed progressively recover or makes ecosystem to the process that develops of benign cycle direction.Mine
The measure of restoration of the ecosystem specifically includes that the control measures of side slope, the control measures of mine tailing, soil grass-root improvement and mine heavy metal
Phytoremediation of pollution etc..
Wherein, slope treatment groundwork seeks to stable side slope.The task of this process is to remove danger stone, descending grade to cut slope,
The steep cliff not forming step is constituted horizontal bench as far as possible, the gradient of side slope is dropped to below security standpoint, hidden to eliminate avalanche
Suffer from；Treated side slope will be carried out again green afterwards so as to keep stable further.
Side slope multiple green during, conventional plant includes Caulis Seu Folium Lespedezae Bicoloris (lespedeza bicolor turcz.)
And/or the shrub such as false indigo (amorpha fruticosa linn.), but find above-mentioned plant in actual use
Development degree in mine slope for the root system not high it is difficult to further keep mine slope stability.
Content of the invention
The present inventor has separated bacillus licheniformis, finds that it can significantly promote Caulis Seu Folium Lespedezae Bicoloris and purple fringe
The root system of Chinese scholartree grows in mine slope and reaches higher development degree, thus further increasing stablizing of mine slope
Property, resulting in the present invention.
The invention provides a kind of Bacillus licheniformis, this Bacillus licheniformis (bacillus licheniformis)
Deposit number is cctcc no:m2013497.
Present invention also offers a kind of microbial bacterial agent, this microbial bacterial agent is by the preparation method system comprising the steps
Standby obtain: Bacillus licheniformis as above for the present invention are inoculated in liquid culture medium, culture obtains seed liquor；(2) will
Described seed liquor is inoculated in solid medium and is fermented, the material after being fermented.
Present invention also offers Bacillus licheniformis as above or microbial bacterial agent as above are in Mine ecology
Purposes in reparation.
By technique scheme, the present invention can significantly promote the root system of green plants to grow simultaneously in mine slope
Reach higher development degree, thus further increasing the stability of mine slope.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Biomaterial preservation
The Bacillus licheniformis (bacillus licheniformis) of the present invention are that the present inventor is quiet from Tianjin
Detached pure culture in the soil sample of Hai Xian, its deposit number is cctcc no:m2013497, and preservation date is 2013
October 24, depositary institution is China typical culture collection center, and address is located at Wuhan, China Wuhan University, and Classification And Nomenclature is
bacillus licheniformis.
Specific embodiment
Hereinafter the specific embodiment of the present invention is described in detail.It should be appreciated that it is described herein concrete
Embodiment is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides a kind of Bacillus licheniformis, this Bacillus licheniformis (bacillus licheniformis)
Deposit number is cctcc no:m2013497.
Present invention also offers a kind of microbial bacterial agent, this microbial bacterial agent is by the preparation method system comprising the steps
Standby obtain: Bacillus licheniformis as above for the present invention are inoculated in liquid culture medium, culture obtains seed liquor；(2) will
Described seed liquor is inoculated in solid medium and is fermented, the material after being fermented.
Wherein, in step (1), the viable bacteria concentration in seed liquor has no particular limits, and can train for Bacillus licheniformis
During supporting, conventional concentration, for example, can be (2-20) × 109cfu/ml.Wherein, the condition of culture has no particular limits,
It is (2-20) × 10 that the condition of culture allows the viable bacteria concentration in seed liquor9Cfu/ml, for example, the temperature of culture can be
28-38 DEG C, the time of culture can be 10-40 hour.During culture, can be by conventional method, such as blood cell
Plate counting method or od value observational method obtain the concentration of viable bacteria.
Wherein, the condition of fermentation and fermentation after material in viable bacteria concentration particularly do not require, can be lichens bud
Conventional condition and concentration during spore bacillus fermentation, as a kind of currently preferred embodiment, in step (2), fermentation
Condition make ferment after material in viable bacteria concentration be (2-200) × 108Cfu/ml, the work in this preferred implementation
Under bacteria concentration, the material after the fermentation of acquisition uses as microbial bacterial agent, preferably can be colonized in side slope soil and build
Vertical microenvironment.The time of such as fermentation can be 20-200 hour, and temperature can be 28-36 DEG C.
Wherein, described liquid culture medium can be the conventional various liquid culture mediums that can cultivate Bacillus licheniformis,
For example include but is not limited at least one in beef-protein medium, broth bouillon and lb culture medium.
Wherein, described solid medium can expand the solid-state of Bacillus licheniformis number for conventional various can fermentation
Culture medium, for example described solid medium can containing in straw, wood flour, soybean cake powder, flour, starch, Testa oryzae and wheat bran extremely
Few one kind；And described solid medium can also the potassium dihydrogen phosphate containing 2-10g/kg and 1-10g/kg ferrous sulfate, with
Supplement required inorganic salt during Bacillus licheniformis fast-growth.
Wherein, as a kind of embodiment specifically preferred according to the invention, described solid medium contains 400-700g/kg's
Straw, the ferrous sulfate of the soybean cake powder of 100-300g/kg, the potassium dihydrogen phosphate of 2-10g/kg and 1-10g/kg.Wherein, this solid-state
Culture medium can also the water containing surplus and/or other composition.In the preferred embodiment, Bacillus licheniformis on the one hand can be allowed
Fast-growth during the fermentation, the material after the fermentation on the other hand obtaining uses as microbial bacterial agent, can be preferably
Side slope soil is colonized and sets up microenvironment.
Wherein, straw can pulverize the size for the routine that can be used for fermenting, and such as length can be 5-10mm, and width can
Think 3-8mm, thickness can be 3-8mm.Straw can be the straw in various sources, for example, include corn straw, sorghum stalks
At least one in stalk, rice straw, wheat stalk and soybean stalk.
Wherein, the material after fermentation can be directly as microbial bacterial agent using it is also possible to being dried by including and/or making
The microbial bacterial agent that the steps such as grain are further processed into the dosage form of more convenient storage uses.
Present invention also offers Bacillus licheniformis as above or microbial bacterial agent as above are in Mine ecology
Purposes in reparation.
Wherein, the present invention one kind preferred embodiment in, by Bacillus licheniformis as above or as above institute
The microbial bacterial agent stated is used for promoting side slope vegetation growth in ecological restoration of mine and builds side slope.
Wherein, in a kind of embodiment specifically preferred according to the invention, packed soil, described bag are laid on the basis of side slope
Dress soil includes flexible bag body and the soil being contained within this flexible bag body, and planting plants on described packed soil；
Wherein, Bacillus licheniformis as above or micro- as above are mixed being contained in the soil within described flexible bag body
Bacteria agent；Described plant includes Caulis Seu Folium Lespedezae Bicoloris (lespedeza bicolor turcz.) and/or false indigo (amorpha
fruticosa linn.).
The further description present invention by the following examples:
Embodiment 1
The present embodiment is used for the culture of Bacillus licheniformis of the present invention and the preparation of microbial bacterial agent are described.
The Bacillus licheniformis (bacillus licheniformis) for cctcc no:m2013497 for the deposit number are connect
Kind Dao lb culture medium (nacl of tryptone, the yeast extract of 5g/l and 10g/l containing 10g/l) in, 35 DEG C and 80 turns/
Divide the lower culture of vibration to be 1010cfu/ml to viable bacteria concentration, obtain seed bacterium solution.
Above-mentioned for 100ml seed bacterium solution is equably showered in 100kg fermentation medium (fermentation medium contains 600g/
The straw (corn straw, having pulverized is about 10mm length, 5mm width, the thick size of 5mm) of kg, the soybean cake powder of 300g/kg, 5g/kg
The water of potassium dihydrogen phosphate, the ferrous sulfate of 5g/kg and surplus), in 30 DEG C of bottom fermentations after turning uniformly, carry out during the fermentation
Sample and observed by ascites method, until the viable bacteria of the Bacillus licheniformis in every kilogram of solid medium
Number is 1010cfu, and the material after obtained fermentation is the microbial bacterial agent of the present embodiment.
Embodiment 2
The present embodiment is used for the culture of Bacillus licheniformis of the present invention and the preparation of microbial bacterial agent are described.
The Bacillus licheniformis (bacillus licheniformis) for cctcc no:m2013497 for the deposit number are connect
Kind Dao lb culture medium (nacl of tryptone, the yeast extract of 5g/l and 10g/l containing 10g/l) in, 35 DEG C and 80 turns/
The lower culture of vibration is divided to be 5 × 10 to viable bacteria concentration9Cfu/ml, obtains seed bacterium solution.
Above-mentioned for 100ml seed bacterium solution is equably showered in 100kg fermentation medium (fermentation medium contains 500g/
The straw (corn straw, having pulverized is about 10mm length, 5mm width, the thick size of 5mm) of kg, the soybean cake powder of 200g/kg, 5g/kg
The water of potassium dihydrogen phosphate, the ferrous sulfate of 5g/kg and surplus), in 30 DEG C of bottom fermentations after turning uniformly, carry out during the fermentation
Sample and observed by ascites method, until the viable bacteria of the Bacillus licheniformis in every kilogram of solid medium
Number is 109Cfu, the material after obtained fermentation is the microbial bacterial agent of the present embodiment.
Embodiment 3
The present embodiment is used for the culture of Bacillus licheniformis of the present invention and the preparation of microbial bacterial agent are described.
The Bacillus licheniformis (bacillus licheniformis) for cctcc no:m2013497 for the deposit number are connect
Plant Dao lb culture medium (nacl of tryptone, the yeast extract of 5g/l and 10g/l containing 10g/l) in, at 32 DEG C and 100
Rev/min lower culture of vibration is 2 × 10 to viable bacteria concentration9Cfu/ml, obtains seed bacterium solution.
Above-mentioned for 100ml seed bacterium solution is equably showered in 100kg fermentation medium (fermentation medium contains 450g/
The straw (corn straw, having pulverized is about 10mm length, 5mm width, the thick size of 5mm) of kg, the soybean cake powder of 150g/kg, 5g/kg
The water of potassium dihydrogen phosphate, the ferrous sulfate of 5g/kg and surplus), in 30 DEG C of bottom fermentations after turning uniformly, carry out during the fermentation
Sample and observed by ascites method, until the viable bacteria of the Bacillus licheniformis in every kilogram of solid medium
Number is 5 × 108Cfu, the material after obtained fermentation is the microbial bacterial agent of the present embodiment.
Comparative example 1
Article number purchased from atcc is14580tmBacillus licheniformis (bacillus
Licheniformis) it is inoculated into lb culture medium (nacl of tryptone, the yeast extract of 5g/l and 10g/l containing 10g/l)
In, cultivating to viable bacteria concentration under 35 DEG C and 80 revs/min vibrations is 1010cfu/ml, obtains seed bacterium solution.
Above-mentioned for 100ml seed bacterium solution is equably showered in 100kg fermentation medium (fermentation medium contains 600g/
The straw (corn straw, having pulverized is about 10mm length, 5mm width, the thick size of 5mm) of kg, the soybean cake powder of 300g/kg, 5g/kg
The water of potassium dihydrogen phosphate, the ferrous sulfate of 5g/kg and surplus), in 30 DEG C of bottom fermentations after turning uniformly, carry out during the fermentation
Sample and observed by ascites method, until the viable bacteria of the Bacillus licheniformis in every kilogram of solid medium
Number is 1010Cfu, the material after obtained fermentation is the microbial bacterial agent of this comparative example.
Testing example 1
The microbial bacterial agent that this testing example measures the present invention in outdoor potted plant experiment is promoting Caulis Seu Folium Lespedezae Bicoloris
Work in the root growth of (lespedeza bicolor turcz.) and false indigo (amorpha fruticosa linn.)
With.
The matrix soil of outdoor potted plant use is purchased from the flower cultivating soil of Beijing flowers market, soil organic matter content 10.24g/
Kg, full nitrogen 0.561g/kg, full phosphorus 1.14g/kg, full potassium 14.98g/kg, available nitrogen 39.19mg/kg, rapid available phosphorus 16.26mg/
Kg, available potassium 160.07mg/kg, ph6.5.And, in order to simulate the soil environment of mine slope, also it is mixed with flower cultivating soil
With the colliery powder (taking from the discarded colliery in Fangshan District of Beijing great An mountain area) of the weight such as flower cultivating soil, pot experiment basin height
50cm, upper diameter 30cm, lower diameter 23cm, every basin adds native 8.2kg, is separately added into embodiment 1-3 and the microbial bacteria of comparative example 1
Agent 100g is as experiment process, and (straw containing 600g/kg (pulverize by corn straw to add the fermentation medium of 100g
Be about 10mm length, 5mm width, the thick size of 5mm), the soybean cake powder of 300g/kg, the potassium dihydrogen phosphate of 5g/kg, 5g/kg sulphuric acid sub-
Ferrum and the water of surplus) as blank.
Caulis Seu Folium Lespedezae Bicoloris (lespedeza bicolor turcz.) and the false indigo of formed objects is implanted in different basins
(amorpha fruticosa linn.) seedling, is respectively used to, behind 30 days, 60 days and 90 days, measure Caulis Seu Folium Lespedezae Bicoloris
The Biomass of the under ground portion of (lespedeza bicolor turcz.) and false indigo (amorpha fruticosa linn.)
(i.e. the fresh weight of under ground portion), and with the Biomass of the under ground portion of experiment process divided by the under ground portion of blank biology
Amount, calculates weightening ratio, result is as shown in table 1.
Table 1
Data by table 1 is visible, and the bacillus licheniformis agent of the present invention can significantly promote Caulis Seu Folium Lespedezae Bicoloris and purple fringe
The root growth of Chinese scholartree simultaneously reaches higher development degree.
Testing example 2
The microbial bacterial agent that this testing example measures the present invention by side slope in-situ monitoring is promoting Caulis Seu Folium Lespedezae Bicoloris
The root growth of (lespedeza bicolor turcz.) and false indigo (amorpha fruticosa linn.) and steady
Effect in deckle slope.
Side slope is located at the discarded colliery slag bank in Fangshan District of Beijing great An mountain area, using slag bank as side slope basis.
Prepare the packed soil as experiment process and blank first, this packed soil is the plant biological bag of built-in soil
(107 × 74cm), often packed enter 250kg soil (being mixed by the soil moved in improve the original of 125kg and the colliery powder of 125kg).Exist respectively
The microbial bacterial agent 3kg of embodiment 1-3 and comparative example 1 is added as experiment process in every bag of 250kg soil, and to add
((corn straw, pulverized is about 10mm length to the straw containing 600g/kg to the fermentation medium of 3kg, 5mm width, thick big of 5mm
Little), the soybean cake powder of 300g/kg, the water of the potassium dihydrogen phosphate of 5g/kg, the ferrous sulfate of 5g/kg and surplus) as blank.
The zones of different laying packed soil of above-mentioned each group on the basis of side slope, every group of packed soil laying width is 15
Rice；The depth on side slope basis is 20 meters, and the gradient is 25 degree, the retaining wall of high 2 meters of setting at 5 meters from toe, leans on slope in retaining wall
Setting earth pressure gauge (the type vibration wire soil pressure being produced using Jiangsu marial rocks engineering material Instrument Ltd. at 1 meter of height of side
Power meter, model tyj-2021) to measure soil lateral pressure.In the packed grown on soil Caulis Seu Folium Lespedezae Bicoloris seedling laying and false indigo
Seedling, implantation time is March, and planting density is about every square metre 4 plants.
Behind 3 months after planting, measure during continuous no rainfall arid 20 days and each group side slope area of the 3rd day after rainfall
The soil lateral pressure in domain, and with the soil lateral pressure of experiment process divided by blank soil lateral pressure, to calculate Pressure reducing ratio,
Result is as shown in table 2.Additionally, in each group side slope region randomization (every arda sampling 10 times, each 1kg soil) packed soil
And (every arda sampling 10 times, each 1kg soil) measures the root system tissue weight in packed soil, and the root system with experiment process
Divided by the root system tissue weight of blank, to calculate root system weightening ratio, result is as shown in table 2 for tissue weight.
Table 2
| Microbial bacterial agent | Root system weightening ratio | Arid Pressure reducing ratio | Rainfall Pressure reducing ratio |
| Embodiment 1 | 1.257 | 0.729 | 0.541 |
| Embodiment 2 | 1.245 | 0.735 | 0.552 |
| Embodiment 3 | 1.238 | 0.742 | 0.559 |
| Comparative example 1 | 1.022 | 0.97 | 0.95 |
Data by table 2 is visible, and the bacillus licheniformis agent of the present invention can significantly promote Caulis Seu Folium Lespedezae Bicoloris and purple fringe
The root growth of Chinese scholartree simultaneously reaches higher development degree, thus further increasing the stability of side slope.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited in above-mentioned embodiment
Detail, in the range of the technology design of the present invention, multiple simple variant can be carried out to technical scheme, this
A little simple variant belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned specific embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to various can
The compound mode of energy no longer separately illustrates.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as it is without prejudice to this
The thought of invention, it equally should be considered as content disclosed in this invention.
Claims (9)
1. a kind of Bacillus licheniformis it is characterised in that: the guarantor of this Bacillus licheniformis (bacillus licheniformis)
Hiding numbering is cctcc no:m2013497.
2. a kind of microbial bacterial agent it is characterised in that: this microbial bacterial agent passes through the preparation method that comprises the steps and is prepared into
Arrive:
(1) Bacillus licheniformis described in claim 1 are inoculated in liquid culture medium, culture obtains seed liquor；
(2) described seed liquor is inoculated in solid medium and is fermented, the material after being fermented.
3. microbial bacterial agent according to claim 2 it is characterised in that: in step (1), the condition of culture makes seed liquor
In viable bacteria concentration be (2-20) × 109cfu/ml.
4. microbial bacterial agent according to claim 2 it is characterised in that: in step (2), after the condition of fermentation makes fermentation
Material in viable bacteria concentration be (2-200) × 108cfu/ml.
5. the microbial bacterial agent according to any one in claim 2-4 it is characterised in that: described liquid culture medium includes
At least one in beef-protein medium, broth bouillon and lb culture medium.
6. the microbial bacterial agent according to any one in claim 2-4 it is characterised in that: described solid medium contains
At least one in straw, wood flour, soybean cake powder, flour, starch, Testa oryzae and wheat bran；And described solid medium also contains 2-
The potassium dihydrogen phosphate of 10g/kg and the ferrous sulfate of 1-10g/kg.
7. microbial bacterial agent according to claim 6 it is characterised in that: described solid medium contains 400-700g/kg
Straw, the ferrous sulfate of the soybean cake powder of 100-300g/kg, the potassium dihydrogen phosphate of 2-10g/kg and 1-10g/kg.
8. in the Bacillus licheniformis described in claim 1 or claim 2-7 the microbial bacterial agent described in any one in ore deposit
Purposes in the restoration of the ecosystem of mountain.
9. purposes according to claim 8 it is characterised in that: by the Bacillus licheniformis described in claim 1 or right
The microbial bacterial agent described in any one in 2-7 is required to be used for promoting side slope vegetation growth in ecological restoration of mine and build side
Slope.
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| CN105149339A (en) * | 2015-03-23 | 2015-12-16 | 江南大学 | Method for preparing ionic rare earth mine tailing in-situ remediation plant nursing system |
| CN105133572B (en) * | 2015-08-30 | 2017-01-11 | 宁波市鄞州丰茂水利工程有限公司 | Method for reinforcing riverbank by waste oil, enzymolysis glutinous rice starch and rammed dredger fill |
| CN106929447B (en) * | 2017-03-21 | 2020-07-24 | 南京农业大学 | Bacillus licheniformis B L06 and application thereof |
| CN107836567A (en) * | 2017-11-22 | 2018-03-27 | 山东信得科技股份有限公司 | A kind of deoderizing feed addictive, preparation method, application method and evaluation method |
| CN111670778A (en) * | 2020-05-29 | 2020-09-18 | 北京林业大学 | Slag matrix and preparation method and application thereof |
| TW202205959A (en) * | 2020-08-06 | 2022-02-16 | 美商坎納爾地球科學有限公司 | Bioinsecticide |
| CN112080283A (en) * | 2020-08-27 | 2020-12-15 | 中地绿矿(北京)科技有限公司 | Soilless spray-seeding organic material, preparation method and application thereof |
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| CN102167639A (en) * | 2011-01-10 | 2011-08-31 | 艾应伟 | Microbial fertilizer special for slope artificial soil |
| CN102210332A (en) * | 2011-04-17 | 2011-10-12 | 赵颖 | Microbial agent for promoting rootage, preventing continuous cropping and performing ecological restoration on soil for planting bananas and preparation method thereof |
| CN102618295A (en) * | 2012-03-14 | 2012-08-01 | 山西金瓯土地矿产咨询服务有限公司 | Soil improving substrate suitable for ecological restoration of mine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102167639A (en) * | 2011-01-10 | 2011-08-31 | 艾应伟 | Microbial fertilizer special for slope artificial soil |
| CN102210332A (en) * | 2011-04-17 | 2011-10-12 | 赵颖 | Microbial agent for promoting rootage, preventing continuous cropping and performing ecological restoration on soil for planting bananas and preparation method thereof |
| CN102618295A (en) * | 2012-03-14 | 2012-08-01 | 山西金瓯土地矿产咨询服务有限公司 | Soil improving substrate suitable for ecological restoration of mine and preparation method thereof |
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