CN104195070A - Strain and microbial agent capable of reducing content of cadmium as well as preparation and application methods of strain and microbial agent - Google Patents
Strain and microbial agent capable of reducing content of cadmium as well as preparation and application methods of strain and microbial agent Download PDFInfo
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- CN104195070A CN104195070A CN201410382461.XA CN201410382461A CN104195070A CN 104195070 A CN104195070 A CN 104195070A CN 201410382461 A CN201410382461 A CN 201410382461A CN 104195070 A CN104195070 A CN 104195070A
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Abstract
The invention discloses a strain and a microbial agent capable of reducing the content of cadmium as well as preparation and application methods of the strain and microbial agent. The strain is Paenibacillus polymyxa P1 of which the preservation number is CCTCC NO: M 2014319, when the microbial agent obtained by fermentation is applied to low-level and middle-level cadmium polluted paddy field, the content of cadmium in paddy rice can be effectively reduced and the microbial agent has huge application and popularization values.
Description
Technical field
The invention belongs to the technical field that heavy-metal contaminated soil is repaired, be specifically related to a kind of bacterial strain, microbial inoculum and preparation and application thereof that reduces cadmium content.
Background technology
Soil is the essential substance basis that the mankind depend on for existence.But because the mankind are increasing to the exploitation of heavy metal, smelting, processing and business manufacturing activities, cause many heavy metals as the heavy metals such as lead, cadmium, mercury enter in atmosphere, water and soil, cause serious environmental pollution.The whole world reaches 30,000 tons of left and right to the cadmium discharging in environment every year according to statistics, and wherein the cadmium of 82%-94% can enter into soil.Take in by the rice of cadmium severe contamination, vegetables, fruit for a long time, drink the water that cadmium content exceeds standard, all can occur the symptom of chronic poisoning, affect neural system, hemopoietic system, Digestive tract and the reproductive system of human body, be detrimental to health.Hunan Province is the main producing region of national grain, the good reputation that from ancient times just has " land of plenty " and " silo all over the world ", undertaking the weighty responsibility that ensures national food safety, but Hunan Province is also one of the most serious area of heavy metal contamination simultaneously, the quality of serious threat " Hunan rice ", has caused huge harm to local resident's health.
Remediation of heavy metal-contaminated soil mainly contains phytoremediation, Electroremediation method, leaching method, thermal desorption method, Vitrification and fixing-stable method etc. at present, in above heavy-metal contaminated soil improvement method, phytoremediation needs considerable time (decades are the centuries even) and have the processing problem of plant; Electrodynamics reparation is only applicable to small area and pollutes, and scene is not easy to operate; Leaching method need to be added tensio-active agent, has the danger of polluted underground water; The problems such as thermal desorption method is the method for utilizing heating recovery gas, and the method is not suitable for execute-in-place and expense is high, narrow application range; Vitrification has changed the character of soil, is only applicable to situation seriously polluted and that area is little; The stabilization method that adds lime has improved the pH value of soil, can cause soil compaction, and bad to alkaline soil effect.Therefore be all not suitable for for administering the heavy metal contamination of large-area rice field.
The in-situ passivation restorative procedure of microorganism can meet preferably from cost with the time administers the requirement that heavy metal in soil pollutes.Add the passivator that contains microorganism, regulate and the physicochemical property of change heavy metal in soil, make it produce the series reaction such as precipitation, absorption, ion-exchange, humify and oxidation-reduction, reduce its biological effectiveness and transportable property in edatope, can significantly reduce heavy metal and enter food chain, the sustainable development that can realize the coordinated development of environmental protection and production and guarantee society and economy.
This kind of recovery technique, do not occupy cultivated land, do not affect agriculture production and income, and cost is low,, not spoiled soil environment little to environmental influence, be applicable to being very much subject to for processing big area the agricultural land soil of mild or moderate cadmium pollution, if can successfully realize industrialization and large-area applications, to realize important breakthrough in heavy metal contamination original position reparation field, for the reparation of our province cadmium pollution soil, reduce heavy metal content in rice, realize agricultural produce, increasing peasant income etc. and all huge pushing effect will be played.
Summary of the invention
The object of the invention is, a kind of bacterial strain, microbial inoculum and their preparation and application that reduces cadmium content is provided, the present invention is easy and simple to handle, with low cost, security is good, practical, described microorganism strains or its microbial inoculum of preparing pass through effects such as the outer complexing of the born of the same parents of heavy metal, intracellular accumulation, precipitation and redox reactions, can effectively reduce the bioavailability of heavy metal in soil, and then reduce the content of cadmium in farm crop and agricultural-food.
The technical solution adopted for the present invention to solve the technical problems is:
It is a kind of that to reduce the bacterial strain of cadmium content in rice be that deposit number is bacillus polymyxa (Paenibacillus polymyxa) P1 of CCTCC NO:M 2014319.
Reducing a microbial inoculum for cadmium content in rice, is the microbial inoculum obtaining by after described bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation.The bacterial content of the bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is 1 * 10
6～1 * 10
8individual viable bacteria/milliliter.
Described microbial inoculum is the microbial inoculum obtaining after the bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is mixed with the sterilizing peat composed of rotten mosses.Bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is to wait mass ratio to mix with the sterilizing peat composed of rotten mosses.
The preparation method of the microbial inoculum of cadmium content in described reduction rice, comprises the following steps:
A, actication of culture
Bacillus polymyxa P1 claimed in claim 1 is inoculated on solid seed culture medium inclined-plane to activation culture;
B, seed liquor preparation
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, and concussion is cultivated, preparation seed liquor;
C, seeding tank fermentation
The seed liquor that step b is obtained is inoculated into seeding tank; Pass into sterile air and stir culture, obtain first class seed pot zymocyte liquid; By first class seed pot fermented liquid access secondary seed tank, pass into sterile air and stir culture, obtain secondary seed tank zymocyte liquid;
D, production fermentor cultivation
By secondary seed tank fermented liquid access fermentor tank, pass into sterile air and stir culture, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 * 10
6～1 * 10
8individual viable bacteria/milliliter;
E, the peat composed of rotten mosses shattering is carried out to sterilizing, the microbial fermentation solution then obtaining with steps d mixes, and 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 to activation culture 24～36h under 28～35 ℃ of conditions;
Solid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; Agar 2%; PH nature;
B, seed liquor preparation
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, and under 28～35 ℃ of conditions, 24～36h is cultivated in concussion, preparation seed liquor;
Liquid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; PH nature;
C, seeding tank fermentation
The seed liquor that step b is obtained is inoculated into seeding tank with the inoculum size of culture volume per-cent 5%-8% to be inoculated; Pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains first class seed pot zymocyte liquid; First class seed pot fermented liquid is pressed to culture volume per-cent 5%～8% inoculum size access secondary seed tank, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains secondary seed tank zymocyte liquid;
1L one-level or secondary seed tank culture medium prescription: extractum carnis 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl 0.1～0.6%, MgSO
47H
2o 0.01～0.06%, K
2hPO
40.01%～0.06%, MnSO
40.001%～0.008%, Yu Weishui, pH value sterilization is front 7.0～7.8, and sterilization is pH6.8～7.5 afterwards; The degree of described culture medium prescription composition is all weight percentage;
D, production fermentor cultivation
28 ℃～35 ℃, press the inoculum size of culture volume per-cent 5%～8% by secondary seed tank fermented liquid access fermentor tank, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 * 10
6～1 * 10
8individual viable bacteria/milliliter;
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH
2pO
40.08～0.22%, K
2hPO
40.10～2.2%, CaCO
30.1～2.20%, FeSO
47H
2o0.001～0.005%, Yu Weishui, pH value sterilization is front 8.5～11.0, sterilization afterwards 6.5～9.5; The degree of described culture medium prescription composition is all weight percentage.
E, the peat composed of rotten mosses shattering carry out sterilizing, then mix with microbial fermentation solution, are microbial inoculum product.It is standby that the peat composed of rotten mosses is crossed 3-5mm sieve after crushed.
The using method of the microbial inoculum of described reduction 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 evenly spread fertilizer over the fields by the full field of 75-100kg/ mu, plough, after ploughing the whole ground of rake, can plant.
Bacterial strain of the present invention mainly relies on hydroxyl (OH), carbonyl (CO-), amide group (CO-NH-), the phosphate (PO4 on cell walls
3-) etc. group carry out surface adsorption and by its meta-bolites, carry out the heavy metal cadmium in the complexings such as the outer complexing of born of the same parents, the outer precipitation of born of the same parents and fixing soil, thereby effectively reduce the effective cadmium content in soil.Bacillus polymyxa of the present invention derives from Xiangtan County river mouth town cadmium pollution paddy soil, Gram-positive, and amphimicrobian, shaft-like, produce gemma, through 16S rDNA Molecular Identification and Phylogenetic Analysis, be initially identified as bacillus polymyxa.And by adsorption test, show, this bacillus polymyxa can grow on the substratum of cadmium concentration 15mmol/L, in solution to the adsorption rate of cadmium up to 91.8%, freeze-dried vaccine powder to the adsorption rate of cadmium up to 98.5%.In soil, this bacterial strain colonization ability is strong, and the heavy metal in can efficient adsorption soil, reduces the absorption of plant.Described bacillus polymyxa (Paenibacillus polymyxa) P1 is preserved in Chinese Typical Representative 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 CCTCC NO:M 2014319.
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 matter (50wt%-80wt%) and soil ulmin (20wt%-40wt%), nitrogenous, phosphorus, potassium total content are more than 3wt%, (porosity is about 95%, and specific surface area is about 200m to have very large internal surface
2/ g) and good colloid surface character, as adsorptive power, cohesive force and high dispersion etc., contain a large amount of polar groups, comprise alcohols, aldehydes, carboxylic acid, hydroxyl etc., can complexing or chelating soil in heavy metal, meanwhile, can promote that soil is ventilative, water-permeable, improvement soil, improves utilization rate of fertilizer.
This product is administered repair process and is belonged to original position reparation, do not occupy cultivated land, do not affect agriculture production and income, and cost is low,, not spoiled soil environment little to environmental influence, is applicable to being very much subject to for processing big area the agricultural land soil of light moderate cadmium pollution.
Starting material wide material sources of the present invention, with low cost, preparation technology is simple, convenient and easy, has very wide application prospect.
Accompanying drawing explanation
Fig. 1 is microbiobacterial agent WSW of the present invention and contrast CK (not adding any passivator) and at paddy rice different growing stage, reduce the experimental result picture of each organ cadmium content of rice plant.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, and can not forms limitation of the present invention.Embodiment 1, the preparation of microorganism fermenting agent;
A, actication of culture
Bacillus polymyxa P1 is inoculated on solid seed culture medium inclined-plane to activation culture 24～36h under 28～35 ℃ of conditions;
Solid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; Agar 2%; PH nature;
B, seed liquor preparation
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, and under 28～35 ℃ of conditions, 24～36h is cultivated in concussion, preparation seed liquor;
Liquid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; PH nature;
C, seeding tank fermentation
The seed liquor that step b is obtained is inoculated into seeding tank with the inoculum size of culture volume per-cent 5%-8% to be inoculated; Pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains first class seed pot zymocyte liquid; By first class seed pot fermented liquid per-cent 5%～8% inoculum size access secondary seed tank by volume, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains secondary seed tank zymocyte liquid;
1L one-level or secondary seed tank culture medium prescription: extractum carnis 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl 0.1～0.6%, MgSO
47H
2o 0.01～0.06%, K
2hPO
40.01%～0.06%, MnSO
40.001%～0.008%, Yu Weishui, pH value sterilization is front 7.0～7.8, and sterilization is pH6.8～7.5 afterwards; The degree of described culture medium prescription composition is all weight percentage;
D, production fermentor cultivation
28 ℃～35 ℃, press the inoculum size of culture volume per-cent 5%～8% by secondary seed tank fermented liquid access fermentor tank, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 * 10
6～1 * 10
8individual viable bacteria/milliliter;
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH
2pO
40.08～0.22%, K
2hPO
40.10～2.2%, CaCO
30.1～2.20%, FeSO
47H
2o0.001～0.005%, Yu Weishui, pH value sterilization is front 8.5～11.0, sterilization afterwards 6.5～9.5; The degree of described culture medium prescription composition is all weight percentage.
The complete function yeast bacterium liquid of putting after tank that ferments, waits mass ratio to mix with the aseptic peat composed of rotten mosses, mixes, and is microbial inoculum product.
Embodiment 2
Microbiobacterial agent WSW of the present invention with contrast CK (not adding any passivator) and at paddy rice different growing stage, reduce the experimental result of each organ cadmium content of rice plant, see Fig. 1 and table 1.
Microbiobacterial agent use-pattern of the present invention: before whole ground, microbial inoculum is evenly spread fertilizer over the fields by the full field of 100kg/ mu paddy rice, plough, can plant after ploughing the whole ground of rake.
Table 1 microbiobacterial agent product of the present invention is applied on paddy rice falls cadmium effect
Claims (10)
1. a bacterial strain that reduces cadmium content, is characterized in that, described bacterial strain is that deposit number is bacillus polymyxa (Paenibacillus polymyxa) P1 of CCTCC NO:M 2014319.
2. a microbial inoculum that reduces cadmium content, is characterized in that, is the microbial inoculum obtaining by after bacillus polymyxa claimed in claim 1 (Paenibacillus polymyxa) P1 fermentation.
3. the microbial inoculum of reduction cadmium content according to claim 2, is characterized in that, the bacterial content of the bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is 1 * 10
6～1 * 10
8individual viable bacteria/milliliter.
4. the microbial inoculum of reduction cadmium content according to claim 3, is characterized in that, described microbial inoculum is the microbial inoculum obtaining after the bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is mixed with the sterilizing peat composed of rotten mosses.
5. the microbial inoculum of reduction cadmium content according to claim 4, is characterized in that, the bacterium liquid after bacillus polymyxa (Paenibacillus polymyxa) P1 fermentation is to wait mass ratio to mix with the sterilizing peat composed of rotten mosses.
6. the preparation method of the microbial inoculum of the reduction cadmium content described in claim 2-5 any one, is characterized in that, comprises the following steps:
A, actication of culture
Bacillus polymyxa P1 claimed in claim 1 is inoculated on solid seed culture medium inclined-plane to activation culture;
B, seed liquor preparation
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, and concussion is cultivated, preparation seed liquor;
C, seeding tank fermentation
The seed liquor that step b is obtained is inoculated into seeding tank; Pass into sterile air and stir culture, obtain first class seed pot zymocyte liquid; By first class seed pot fermented liquid access secondary seed tank, pass into sterile air and stir culture, obtain secondary seed tank zymocyte liquid;
D, production fermentor cultivation
By secondary seed tank fermented liquid access fermentor tank, pass into sterile air and stir culture, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 * 10
6～1 * 10
8individual viable bacteria/milliliter;
E, the peat composed of rotten mosses shattering is carried out to sterilizing, the microbial fermentation solution then obtaining with steps d mixes, and is microbial inoculum product.
7. the preparation method of the microbial inoculum of reduction cadmium content according to claim 6, is characterized in that,
A, actication of culture
Described bacillus polymyxa P1 is inoculated on solid seed culture medium inclined-plane to activation culture 24～36h under 28～35 ℃ of conditions;
Solid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; Agar 2%; PH nature;
B, seed liquor preparation
Bacillus polymyxa is inoculated in liquid seed culture medium from solid seed culture medium inclined-plane, and under 28～35 ℃ of conditions, 24～36h is cultivated in concussion, preparation seed liquor;
Liquid seed culture medium: Tryptones 10g/L; Yeast extract 5g/L; Sodium-chlor 10g/L; PH nature;
C, seeding tank fermentation
The seed liquor that step b is obtained is inoculated into seeding tank with the inoculum size of culture volume per-cent 5%-8% to be inoculated; Pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains first class seed pot zymocyte liquid; First class seed pot fermented liquid is pressed to culture volume per-cent 5%～8% inoculum size access secondary seed tank, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, obtains secondary seed tank zymocyte liquid;
1L one-level or secondary seed tank culture medium prescription: extractum carnis 0.3～0.8%, peptone 0.7～1.2%, glucose 0.1～0.6%, NaCl 0.1～0.6%, MgSO
47H
2o 0.01～0.06%, K
2hPO
40.01%～0.06%, MnSO
40.001%～0.008%, Yu Weishui, pH value sterilization is front 7.0～7.8, and sterilization is pH6.8～7.5 afterwards; The degree of described culture medium prescription composition is all weight percentage;
D, production fermentor cultivation
28 ℃～35 ℃, press the inoculum size of culture volume per-cent 5%～8% by secondary seed tank fermented liquid access fermentor tank, pass into sterile air and stirring, under 28～35 ℃ of conditions, cultivate 24～36h, air flow 1～2Vols/volmin, mixing speed 180-250rpm, bacillus polymyxa content in microbial fermentation solution after fermentation: 1 * 10
6～1 * 10
8individual viable bacteria/milliliter;
1L fermentor cultivation based formulas: starch 2%～3%, soybean cake powder 0.5～1.5%, yeast powder 0.5～1.0%, KH
2pO
40.08～0.22%, K
2hPO
40.10～2.2%, CaCO
30.1～2.20%, FeSO
47H
2o0.001～0.005%, Yu Weishui, pH value sterilization is front 8.5～11.0, sterilization afterwards 6.5～9.5; The degree of described culture medium prescription composition is all weight percentage.
E, the peat composed of rotten mosses shattering carry out sterilizing, then mix with microbial fermentation solution, are microbial inoculum product.
8. the preparation method of the microbial inoculum of reduction cadmium content according to claim 7, is characterized in that, it is standby that the peat composed of rotten mosses is crossed 3-5mm sieve after crushed.
9. the using method of the microbial inoculum of the reduction cadmium content described in claim 2-5 any one, is characterized 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, is characterized in that, for reducing the content of cadmium in rice terrace, before whole ground, described microbial inoculum is evenly spread fertilizer over the fields by the full field of 75-100kg/ mu, ploughs, and after ploughing the whole ground of rake, can plant.
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| 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 |
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| CN108795808A (en) * | 2018-06-15 | 2018-11-13 | 湖南省微生物研究院 | One plant of Batavia bacillus and its application |
| CN108821908A (en) * | 2018-06-15 | 2018-11-16 | 湖南省微生物研究院 | A kind of microbial inoculum comprising Batavia bacillus HJ-1 and its application |
| CN108795808B (en) * | 2018-06-15 | 2020-05-05 | 湖南省微生物研究院 | Bacillus badavirus and application thereof |
| 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 |
| CN109628345A (en) * | 2018-12-28 | 2019-04-16 | 天津开发区坤禾生物技术有限公司 | A kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application |
| 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 |
| CN109964765A (en) * | 2019-04-12 | 2019-07-05 | 徐州工程学院 | A kind of rice cadmium content reduction method based on Wei Si Salmonella |
| CN109964765B (en) * | 2019-04-12 | 2021-05-07 | 徐州工程学院 | Weissella-based rice cadmium content reduction method |
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