CN104195070B - A kind of reduce the bacterial strain of cadmium content, microbial inoculum and preparation and application thereof - Google Patents
A kind of reduce the bacterial strain of cadmium content, microbial inoculum and preparation and application thereof Download PDFInfo
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- CN104195070B CN104195070B CN201410382461.XA CN201410382461A CN104195070B CN 104195070 B CN104195070 B CN 104195070B CN 201410382461 A CN201410382461 A CN 201410382461A CN 104195070 B CN104195070 B CN 104195070B
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Abstract
The invention discloses and a kind of reduce the bacterial strain of cadmium content, microbial inoculum and preparation and application thereof.This bacterial strain is deposit number is bacillus polymyxa (Paenibacillus polymyxa) P1 of CCTCC NO:M 2014319；The microbial inoculum obtained afterwards by fermentation is applied to light moderate cadmium pollution rice field, it is possible to effectively reduces cadmium content in rice, has huge application value.
Description
Technical field
The invention belongs to the technical field that heavy-metal contaminated soil is repaired, be specifically related to a kind of reduce the bacterial strain of cadmium content, microbial inoculum and preparation and application thereof.
Background technology
Soil is the main matter basis that the mankind depend on for existence.But owing to the exploitation of mankind's heavy metal, smelting, processing and business manufacturing activities are increasing, cause many heavy metals to enter in air, water and soil earth such as heavy metals such as lead, cadmium, hydrargyrum, cause serious environmental pollution.The cadmium that the whole world discharges every year in environment according to statistics reaches about 30,000 tons, and wherein the cadmium of 82%-94% can enter in soil.Take in by the rice of cadmium severe contamination, veterinary antibiotics for a long time, drink the water that cadmium content exceeds standard, the symptom of chronic poisoning all can occur, affect the nervous system of human body, hemopoietic system, digestive system and reproductive system, be detrimental to health.Hunan Province is the main producing region of whole nation grain, just there are " land of plenty " and the good reputation of " all over the world silo " from ancient times, it is responsible for the weighty responsibility ensureing national food security, but simultaneously Hunan Province is also one of the most serious area of heavy metal pollution, the quality of serious threat " Hunan rice ", causes huge harm to the health of local resident.
Current remediation of heavy metal-contaminated soil mainly has phytoremediation, Electroremediation method, leaching method, thermal desorption method, Vitrification and fixing-stable method etc., in above heavy-metal contaminated soil administering method, phytoremediation needs considerable time (decades are the centuries even) and there is the process problem of plant；Electromotion repairing technique is only applicable to little area and pollutes, and scene not easily operates；Leaching method needs to add surfactant, there is the danger of polluted underground water；Thermal desorption method is the method utilizing heating recovery gas, and the method is not suitable for execute-in-place and costly, the problem such as narrow application range；Vitrification changes the character of soil, is only applicable to seriously polluted and that area is little situation；The stabilization method adding Calx improves the pH value of soil, can cause soil compaction, and basic soil effect is bad.Therefore it is unsuitable for for administering the heavy metal pollution of large-area rice field.
The in-situ passivation restorative procedure of microorganism is from cost and can meet the requirement administering heavy metal in soil pollution the time preferably.Add the passivator containing microorganism, regulate and change heavy metal physicochemical properties in soil, it is made to produce the series reaction such as precipitation, absorption, ion exchange, humification and oxidation-reduction, reduce its biological effectiveness in soil environment and transportable property, heavy metal can be substantially reduced and enter food chain, it is possible to achieve the coordinated development of environmental protection and production and guarantee the sustainable development of society and economy.
This kind of recovery technique, it is not take up ploughing, not affecting agricultural production and income, and cost is low, environmental effect is little, not spoiled soil environment, it is especially suitable for the agricultural land soil being subject to mild or moderate cadmium pollution for processing large area, if able to successfully realize industrialization and large-area applications, important breakthrough will be realized in heavy metal pollution in-situ immobilization field, and for the reparation of our province cadmium pollution soil, the content of beary metal reduced in rice, realize agricultural produce, increasing peasant income etc. and all will play huge impetus.
Summary of the invention
It is an object of the invention to, there is provided a kind of and reduce the bacterial strain of cadmium content, microbial inoculum and their preparation and application, the present invention is easy and simple to handle, with low cost, safety is good, practical, described microbial strains or its microbial inoculum prepared are by effects such as the outer complexation of the born of the same parents of heavy metal, intracellular accumulation, precipitation and redox reaction, can effectively reduce the bioavailability of heavy metal in soil, and then reduce the content of cadmium in crops and agricultural product.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of bacillus polymyxa (Paenibacilluspolymyxa) P1 reducing the bacterial strain of cadmium content in rice to be deposit number be CCTCCNO:M2014319.
A kind of reduce the microbial inoculum of cadmium content in rice, be by the microbial inoculum obtained after described bacillus polymyxa (Paenibacilluspolymyxa) P1 fermentation.The bacterial content of the bacterium solution after bacillus polymyxa (Paenibacilluspolymyxa) P1 fermentation is 1 × 106～1 × 108Individual viable bacteria/milliliter.
Described microbial inoculum is the microbial inoculum obtained after the bacterium solution after being fermented by bacillus polymyxa (Paenibacilluspolymyxa) P1 mixes with the sterilizing peat composed of rotten mosses.Bacterium solution after bacillus polymyxa (Paenibacilluspolymyxa) P1 fermentation is wait mass ratio to mix with the sterilizing peat composed of rotten mosses.
Described reduces the preparation method of the microbial inoculum of cadmium content in rice, comprises the following steps:
A, actication of culture
Bacillus polymyxa P1 described in claim 1 is inoculated on solid seed culture medium inclined-plane, activation culture；
Prepared by b, seed liquor
Being inoculated in liquid seed culture medium by bacillus polymyxa from solid seed culture medium inclined-plane, concussion is cultivated, and prepares seed liquor；
C, seed tank ferment
The seed liquor obtained by step b is inoculated into seed tank；Pass into filtrated air and stir culture, obtain first class seed pot zymocyte liquid；First class seed pot fermentation liquid is accessed secondary seed tank, passes into filtrated air and stir culture, obtain secondary seed tank zymocyte liquid；
D, production fermentor cultivation
Secondary seed tank fermentation liquid is accessed fermentation tank, passes into filtrated air and stir culture, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 × 106～1 × 108Individual viable bacteria/milliliter；
E, the peat composed of rotten mosses of smashing is carried out sterilizing, the microbial fermentation solution mix homogeneously then obtained with step d, it is microbial inoculum product.
The preparation method of the microbial inoculum of cadmium content in above-mentioned reduction rice, specific as follows:
A, actication of culture
Described bacillus polymyxa P1 is inoculated on solid seed culture medium inclined-plane, activation culture 24～36h under 28～35 DEG C of conditions；
Solid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar 2%；PH is natural；
Prepared by b, seed liquor
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, under 28～35 DEG C of conditions, shakes cultivation 24～36h, prepare seed liquor；
Liquid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；PH is natural；
C, seed tank ferment
The seed liquor obtained by step b is inoculated into seed tank with the inoculum concentration of culture volume percentage ratio 5%-8% to be seeded；Pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtain first class seed pot zymocyte liquid；First class seed pot fermentation liquid is pressed culture volume percentage ratio 5%～8% inoculum concentration and accesses secondary seed tank, pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtains secondary seed tank zymocyte liquid；
1L one-level or secondary seed tank culture medium prescription: Carnis Bovis seu Bubali cream 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl0.1～0.6%, MgSO4·7H2O0.01～0.06%, K2HPO40.01%～0.06%, MnSO40.001%～0.008%, Yu Weishui, pH value sterilization front 7.0～7.8, pH6.8～7.5 after sterilization；The degree of described culture medium prescription composition is all weight percentage；
D, production fermentor cultivation
28 DEG C～35 DEG C, by the inoculum concentration of culture volume percentage ratio 5%～8%, secondary seed tank fermentation liquid is accessed fermentation tank, pass into filtrated air and stirring, 24～36h is cultivated under 28～35 DEG C of conditions, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 × 106～1 × 108Individual viable bacteria/milliliter；
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH2PO40.08～0.22%, K2HPO40.10～2.2%, CaCO30.1～2.20%, FeSO4·7H2O0.001～0.005%, Yu Weishui, pH value sterilization front 8.5～11.0, after sterilization 6.5～9.5；The degree of described culture medium prescription composition is all weight percentage.
E, smashing the peat composed of rotten mosses carry out sterilizing, then with microbial fermentation solution mix homogeneously, be microbial inoculum product.It is standby that the peat composed of rotten mosses crosses 3-5mm sieve after crushed.
The using method of the described microbial inoculum reducing cadmium content, for reducing the content of Cadmium in Soil.
Specifically for reducing the content of cadmium in rice terrace, before whole ground, described microbial inoculum is uniformly spread fertilizer over the fields by the full field of 75-100kg/ mu, plough, can plant after ploughing and harrowing whole ground.
The bacterial strain of the present invention relies primarily on the hydroxyl (-OH) on cell wall, carbonyl (-CO-), amide groups (-CO-NH-), phosphate (-PO43-) etc. group carry out surface adsorption and carried out the heavy metal cadmium in the complexations such as precipitation outside born of the same parents outer complexation, born of the same parents and fixing soil by its metabolite, thus the effective cadmium content effectively reduced in soil.Bacillus polymyxa of the present invention derives from Xiangtan County river mouth town cadmium pollution paddy soil, Gram-positive, and amphimicrobian is shaft-like, produces spore, is initially identified as bacillus polymyxa through 16SrDNA Molecular Identification and Phylogenetic Analysis.And shown by adsorption test, this bacillus polymyxa can grow in the culture medium of cadmium concentration 15mmol/L, in the solution to the adsorption rate of cadmium up to 91.8%, lyophilizing mycopowder to the adsorption rate of cadmium up to 98.5%.In soil, this bacterial strain colonization ability is strong, it is possible to the heavy metal in efficient absorption soil, reduces the absorption of plant.Described bacillus polymyxa (Paenibacilluspolymyxa) P1 is preserved in China typical culture collection center on July 3rd, 2014 and (is called for short CCTCC, address: in the Wuhan University of Luo Jia Shan, Wuhan), deposit number is CCTCCNO:M2014319.
The present invention adopts the peat composed of rotten mosses and zymocyte liquid to be mixed to get microbial inoculum, the peat composed of rotten mosses is rich in organic (50wt%-80wt%) and humus (20wt%-40wt%), nitrogenous, phosphorus, potassium total content are at more than 3wt%, (porosity is about 95%, and specific surface area is about 200m to have very big inner surface2/ g) and good colloid surface character, such as absorption affinity, cohesion and high dispersion etc., containing substantial amounts of polar group, including alcohols, aldehydes, carboxylic acid, hydroxyl etc., can heavy metal in complexation or chelating soil, simultaneously, it is possible to promote Soil ventilation, water penetration, improvement soil, improves utilization rate of fertilizer.
This product is administered repair process and is belonged to in-situ immobilization, is not take up ploughing, not affecting agricultural production and income, and cost is low, environmental effect is little, not spoiled soil environment, is especially suitable for the agricultural land soil being subject to light moderate cadmium pollution for processing large area.
The raw material sources of the present invention is extensive, with low cost, and preparation technology is simple, convenient and easy, has very wide application prospect.
Accompanying drawing explanation
Fig. 1 is microbial bacterial agent WSW of the present invention and compare CK (without any passivator) and reduce at Oryza sativa L. different growing stage the experimental result picture of each organ cadmium content of rice plant.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, without forming limitation of the present invention.Embodiment 1, prepared by microbial fermentation inoculum；
A, actication of culture
Bacillus polymyxa P1 is inoculated on solid seed culture medium inclined-plane, activation culture 24～36h under 28～35 DEG C of conditions；
Solid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar 2%；PH is natural；
Prepared by b, seed liquor
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, under 28～35 DEG C of conditions, shakes cultivation 24～36h, prepare seed liquor；
Liquid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；PH is natural；
C, seed tank ferment
The seed liquor obtained by step b is inoculated into seed tank with the inoculum concentration of culture volume percentage ratio 5%-8% to be seeded；Pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtain first class seed pot zymocyte liquid；First class seed pot fermentation liquid by volume percentage ratio 5%～8% inoculum concentration is accessed secondary seed tank, pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtains secondary seed tank zymocyte liquid；
1L one-level or secondary seed tank culture medium prescription: Carnis Bovis seu Bubali cream 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl0.1～0.6%, MgSO4·7H2O0.01～0.06%, K2HPO40.01%～0.06%, MnSO40.001%～0.008%, Yu Weishui, pH value sterilization front 7.0～7.8, pH6.8～7.5 after sterilization；The degree of described culture medium prescription composition is all weight percentage；
D, production fermentor cultivation
28 DEG C～35 DEG C, by the inoculum concentration of culture volume percentage ratio 5%～8%, secondary seed tank fermentation liquid is accessed fermentation tank, pass into filtrated air and stirring, 24～36h is cultivated under 28～35 DEG C of conditions, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 × 106～1 × 108Individual viable bacteria/milliliter；
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH2PO40.08～0.22%, K2HPO40.10～2.2%, CaCO30.1～2.20%, FeSO4·7H2O0.001～0.005%, Yu Weishui, pH value sterilization front 8.5～11.0, after sterilization 6.5～9.5；The degree of described culture medium prescription composition is all weight percentage.
Ferment and complete put the function bacterium bacterium solution after tank, carry out with the aseptic peat composed of rotten mosses waiting mass ratio to mix, mix homogeneously, be microbial inoculum product.
Embodiment 2
Microbial bacterial agent WSW of the present invention reduces the experimental result of each organ cadmium content of rice plant with compareing CK (without any passivator) at Oryza sativa L. different growing stage, sees Fig. 1 and Biao 1.
Microbial bacterial agent occupation mode of the present invention: uniformly spread fertilizer over the fields by the full field of 100kg/ mu Oryza sativa L. by microbial inoculum before whole ground, plough, can plant after ploughing and harrowing whole ground.
What table 1 microbial bacterial agent product of the present invention was applied on Oryza sativa L. drops cadmium effect
Claims (10)
1. the bacterial strain reducing cadmium content, it is characterised in that described bacterial strain is deposit number is bacillus polymyxa (Paenibacilluspolymyxa) P1 of CCTCCNO:M2014319.
2. the microbial inoculum reducing cadmium content, it is characterised in that be the microbial inoculum obtained after bacillus polymyxa (Paenibacilluspolymyxa) the P1 fermentation described in claim 1.
3. the microbial inoculum of reduction cadmium content according to claim 2, it is characterised in that the bacterial content of the bacterium solution after bacillus polymyxa (Paenibacilluspolymyxa) P1 fermentation is 1 × 106～1 × 108Individual viable bacteria/milliliter.
4. the microbial inoculum of reduction cadmium content according to claim 3, it is characterised in that described microbial inoculum is the microbial inoculum obtained after the bacterium solution after being fermented by bacillus polymyxa (Paenibacilluspolymyxa) P1 mixes with the sterilizing peat composed of rotten mosses.
5. the microbial inoculum of reduction cadmium content according to claim 4, it is characterised in that the bacterium solution after bacillus polymyxa (Paenibacilluspolymyxa) P1 fermentation is wait mass ratio to mix with the sterilizing peat composed of rotten mosses.
6. the preparation method of the microbial inoculum reducing cadmium content described in any one of claim 2-5, it is characterised in that comprise the following steps:
A, actication of culture
Bacillus polymyxa P1 described in claim 1 is inoculated on solid seed culture medium inclined-plane, activation culture；
Prepared by b, seed liquor
Being inoculated in liquid seed culture medium by bacillus polymyxa from solid seed culture medium inclined-plane, concussion is cultivated, and prepares seed liquor；
C, seed tank ferment
The seed liquor obtained by step b is inoculated into seed tank；Pass into filtrated air and stir culture, obtain first class seed pot zymocyte liquid；First class seed pot fermentation liquid is accessed secondary seed tank, passes into filtrated air and stir culture, obtain secondary seed tank zymocyte liquid；
D, production fermentor cultivation
Secondary seed tank fermentation liquid is accessed fermentation tank, passes into filtrated air and stir culture, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 × 106～1 × 108Individual viable bacteria/milliliter；
E, the peat composed of rotten mosses of smashing is carried out sterilizing, the microbial fermentation solution mix homogeneously then obtained with step d, it is microbial inoculum product.
7. the preparation method of the microbial inoculum of reduction cadmium content according to claim 6, it is characterised in that
A, actication of culture
Described bacillus polymyxa P1 is inoculated on solid seed culture medium inclined-plane, activation culture 24～36h under 28～35 DEG C of conditions；
Solid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar 2%；PH is natural；
Prepared by b, seed liquor
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, under 28～35 DEG C of conditions, shakes cultivation 24～36h, prepare seed liquor；
Liquid seed culture medium: tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；PH is natural；
C, seed tank ferment
The seed liquor obtained by step b is inoculated into seed tank with the inoculum concentration of culture volume percentage ratio 5%-8% to be seeded；Pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtain first class seed pot zymocyte liquid；First class seed pot fermentation liquid is pressed culture volume percentage ratio 5%～8% inoculum concentration and accesses secondary seed tank, pass into filtrated air and stirring, under 28～35 DEG C of conditions, cultivate 24～36h, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, obtains secondary seed tank zymocyte liquid；
1L one-level or secondary seed tank culture medium prescription: Carnis Bovis seu Bubali cream 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl0.1～0.6%, MgSO4·7H2O0.01～0.06%, K2HPO40.01%～0.06%, MnSO40.001%～0.008%, Yu Weishui, pH value sterilization front 7.0～7.8, pH6.8～7.5 after sterilization；The degree of described culture medium prescription composition is all weight percentage；
D, production fermentor cultivation
28 DEG C～35 DEG C, by the inoculum concentration of culture volume percentage ratio 5%～8%, secondary seed tank fermentation liquid is accessed fermentation tank, pass into filtrated air and stirring, 24～36h is cultivated under 28～35 DEG C of conditions, ventilation 1～2Vols/vol min, speed of agitator 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 × 106～1 × 108Individual viable bacteria/milliliter；
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH2PO40.08～0.22%, K2HPO40.10～2.2%, CaCO30.1～2.20%, FeSO4·7H2O0.001～0.005%, Yu Weishui, pH value sterilization front 8.5～11.0, after sterilization 6.5～9.5；The degree of described culture medium prescription composition is all weight percentage；
E, smashing the peat composed of rotten mosses carry out sterilizing, then with microbial fermentation solution mix homogeneously, be microbial inoculum product.
8. the preparation method of the microbial inoculum of reduction cadmium content according to claim 7, it is characterised in that it is standby that the peat composed of rotten mosses crosses 3-5mm sieve after crushed.
9. the using method of the microbial inoculum reducing cadmium content described in any one of claim 2-5, it is characterised in that for reducing the content of Cadmium in Soil.
10. the using method of the microbial inoculum of reduction cadmium content according to claim 9, it is characterised in that for reducing the content of cadmium in rice terrace, uniformly spread fertilizer over the fields by the full field of 75-100kg/ mu by described microbial inoculum, plough before whole ground, can plant after ploughing and harrowing whole ground.
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| CN106007950B (en) * | 2016-05-23 | 2023-01-31 | 四川省兰月科技有限公司 | Application of bacillus LY152 in preparation of microbial fertilizer |
| CN108821908B (en) * | 2018-06-15 | 2021-03-02 | 湖南省微生物研究院 | Microbial inoculum containing Bacillus badensis HJ-1 and application thereof |
| CN108795808B (en) * | 2018-06-15 | 2020-05-05 | 湖南省微生物研究院 | Bacillus badavirus and application thereof |
| CN109628345B (en) * | 2018-12-28 | 2021-07-09 | 天津开发区坤禾生物技术有限公司 | Composite microbial inoculum for preventing and treating brown rot of succulent plants as well as preparation method and application of composite microbial inoculum |
| CN109964765B (en) * | 2019-04-12 | 2021-05-07 | 徐州工程学院 | Weissella-based rice cadmium content reduction method |
| CN111548230A (en) * | 2020-05-12 | 2020-08-18 | 四川嘉道博文生态科技有限公司 | Ligusticum wallichii cadmium-reducing special fertilizer and application method thereof |
| CN113699059B (en) * | 2021-07-27 | 2023-06-27 | 华南农业大学 | Cadmium-resistant growth-promoting paenibacillus strain and application thereof |
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