CN103289941A - A secondary salinization soil remediation bacterium and applications thereof - Google Patents
A secondary salinization soil remediation bacterium and applications thereof Download PDFInfo
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- CN103289941A CN103289941A CN2013102550815A CN201310255081A CN103289941A CN 103289941 A CN103289941 A CN 103289941A CN 2013102550815 A CN2013102550815 A CN 2013102550815A CN 201310255081 A CN201310255081 A CN 201310255081A CN 103289941 A CN103289941 A CN 103289941A
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Abstract
A strain for secondary salinization soil remediation and applications thereof belong to the technical field of secondary salinization soil remediation. The secondary salinization soil remediation strain is Klebsiella sp. (SNR-1), and the accession number thereof is CGMCC No. 7124. The secondary salinization soil remediation strain is used for degrading nitrates. The secondary salinization soil remediation strain of the invention has a good ability of degrading nitrates in soil, and very good adaptability to the environment, thus having good application prospects in salinization soil remediation for protected cultivation.
Description
Technical field
What the present invention relates to is the method in a kind of secondary salinization soil recovery technique field, specifically is that a kind of secondary salinization soil is repaired bacterium and application thereof.
Background technology
Industrialized agriculture obtains large-area applying in China in recent years, the mode of utilizing of this land consolidationization plays an important role in the anti-season of China vegetables and other cash crop and trans-regional plantation, also major contribution has been made in increasing peasant income and expanding economy.But because the excessive of chemical fertilizer used, irrational pattern of farming, the soil secondary salinization problem that irrational irrigation etc. cause is more and more serious, become the major obstacle factor that the restriction vegetables produce, the focus that salinification is polluted becomes current research of therefore how controlling effectively and improve the soil.
Because the soil under the cultivation conditions such as greenhouse, booth lacks rainwater drip washing, and temperature, humidity, aeration status and water and fertilizer management etc. all have than big difference with outdoor cropping, facility cultivation is in again under the production status of high intensive, high multiple crop index, high rate of fertilizer for a long time in addition, the ecotope that it is special and irrational water and fertilizer management measure have caused soil secondary salinization, show as mainly that nitrate content increases, crop is because nutritional trouble causes physiological and pathologic obstacle, causes the underproduction of crop and the decline of crop quality.
In order to reduce soil salt, the rehabilitating soil secondary salinization guarantees that farm crop well grow, and generally adopts washing salinity by irrigation, membrane covering, accurately prophylactico-therapeutic measuress such as drip irrigation, soil improving method in the past.But these measures not only will expend lot of manpower and material resources, and have also can bring disadvantageous effect to environment.Must be based upon as the washing salinity by irrigation measure on the basis of dynamical water exhaust system, to guarantee freely circulating of the soil solution, and pollutents such as the nitrogen phosphorus that produces run off be the land for growing field crops open country produce more than 3 times, can cause other environmental problem such as widespread pollution from the overuse of fertilizers and pesticides in rural area; The membrane covering cost is higher, reclaims and has any problem and may have a negative impact to the second stubble crop growth; The accurate drip irrigation technique professional operative technique that not only cost is higher but also needs are good, big area is promoted and is still had certain degree of difficulty; Soil improvement agent method cost might produce secondary pollution than higher.
Microorganism to each pollutant all have more by force, adaptability faster.Compare with traditional secondary salinization method of improving the soil, repairing method of microorganism has many outstanding advantages: do not produce secondary pollution, do not increase burden to surrounding environment, cost is low, and is easy to operate.Along with the expansion of China's facility cultivation area and becoming increasingly conspicuous of secondary salinization problem, utilize research and the application of microorganism rehabilitating soil secondary salinization that wide prospect is arranged.
Summary of the invention
The present invention is directed to the prior art above shortcomings, propose a kind of secondary salinization soil and repair bacterium and application thereof, nitrate in the soil there is very strong degradation capability, and environment there is extremely strong adaptability, therefore in the reparation of facility cultivation secondary salinization soil good prospects for application is arranged.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of secondary salinization soil and repair bacterium, be specially klebsiella (SNR-1), now be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science institute of microbiology.Date saved is on January 11st, 2013, and deposit number is CGMCC No.7124.
The present invention relates to above-mentioned secondary salinization soil and repair the separation purification method of bacterium, stablize bacterium colony by collected specimens from soil through using the inorganic salt liquid culture medium inoculated also to cultivate until acquisition after repeatedly taming.
Described soil is taken from the booth of industrial park, Nanhui, Shanghai City facility cultivation 12-15.
Described domestication refers to: pedotheque is inoculated in the enrichment medium, cultivated 7 days under 25 ℃, 120rpm condition.
Described repeatedly domestication refers to: each acclimation period is 7 days, and the domestication back is inserted in the enrichment medium again with 10% inoculum size, is preferably 4 times.
Described enrichment medium is the inorganic salt liquid substratum.
The component of described inorganic salt liquid substratum and content are: glucose 15g, KCl1.0g/L, MgSO
47H
2O0.5g/L, CaCl
21.0mg/L, FeSO
47H
2O0.1g/L, KH
2PO
40.5g/L pH6.8-7.2 can prepare by solid medium being added 15-18g/L agar.
Described inoculation refers to: the suspension of getting 100 μ L domesticated strains is coated in the inorganic salt solid medium, is inverted in 25 ℃ of fixed temperature and humidity incubators and cultivates 2d, treats that bacterium colony grows, and picking colony is rule on the inorganic salt solid medium, until obtaining single bacterium colony; Single bacterium colony that will obtain again, continuous passage is 6 times on the inorganic salt solid medium that with the nitric nitrogen is only nitrogen source, makes it to become the stable pure culture of colonial morphology.
The present invention relates to above-mentioned secondary salinization soil and repair preservation and the activation of bacterium, wherein:
Storage conditions is: adopts-80 ℃ of cryogenic freezing preserving processes, microorganism is in the lesser temps, thereby to reduce the purpose that its metabolism speed reaches the preservation bacterial classification;
Activation is: the bacterial classification inoculation of freezing state in the potato glucose liquid nutrient medium, is treated that bacterial classification adapts to culture environment gradually and breeds, to obtain the culture that quantity is enough, flush.
The component of described potato glucose liquid nutrient medium and content are: potato 200g, and glucose 20g/L, moisturizing is regulated pH to 7.0-7.2 to 1L.
The present invention relates to a kind of soil remediation method, adopt above-mentioned secondary salinization soil to repair bacterium and degrade.
Described degraded by bacterial strain SNR-1 microbial inoculum with the nitric nitrogen in the soil as nitrogenous source, stalk is as carbon source, transforms through microbiological deterioration and realizes.
Described microbial inoculum prepares in the following manner: with the SNR-1 inoculation in the potato glucose liquid nutrient medium of pH7.0-7.2, in 30 ℃, 150rpm shaking culture 24-48h, at last air-dry straw is crushed to its length less than 2mm after, bacterium liquid and stalk are mixed namely with 1:1 (mL/g).
Technique effect
Compared with prior art, the present invention has the following advantages: 1, klebsiella of the present invention reaches more than 90% the transformation efficiency of nitric nitrogen (300mg/L) in the nitrogenous substratum; 2, in simulation secondary salinization soil pot experiment, the microbial inoculum of klebsiella of the present invention reaches 60% to the degradation rate of nitric nitrogen in the secondary salinization soil (concentration is 200mg/kg soil); 3, klebsiella of the present invention has very strong degradation capability to nitrate in the soil, and environment is had extremely strong adaptability, therefore in the reparation of facility cultivation secondary salinization soil good prospects for application is arranged.
Description of drawings
Fig. 1 is colonial morphology (a) and thalli morphology (b) synoptic diagram of klebsiella (Klebsiella sp.) SNR-1.
Fig. 2 is nitric nitrogen transformation efficiency synoptic diagram in the substratum of klebsiella (Klebsiella sp.) SNR-1.
Fig. 3 is that klebsiella (Klebsiella sp.) SNR-1 microbial inoculum is to the changing effect synoptic diagram of nitrate in the secondary salinization soil.
Fig. 4 is that klebsiella (Klebsiella sp.) SNR-1 microbial inoculum is to the synoptic diagram that influences of little green vegetables growth.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Separation and the purifying of klebsiella (Klebsiella sp.) SNR-1
Pedotheque is taken from the booth of industrial park, Nanhui, Shanghai City facility cultivation 12-15, after the sampling sample is fully mixed.A bottle enrichment culture is shaken in employing, and soil sampling 10g is inoculated in the 100mL enrichment medium, cultivates under 25 ℃, 120rpm condition.Every 7d, insert again in the enrichment medium with 10% of inoculum size, so repeatedly 4 domestications.Enrichment medium adopts the inorganic salt liquid substratum, and the inorganic salt liquid medium component is glucose 15g, KCl1.0g/L, MgSO
47H
2O0.5g/L, CaCl
21.0mg/L, FeSO
47H
2O0.1g/L, KH
2PO
40.5g/L pH6.8-7.2, solid medium add 15-18g/L agar and get final product.
Get the suspension of 100 μ L domesticated strains and coat in the inorganic salt solid medium, be inverted in 25 ℃ of fixed temperature and humidity incubators and cultivate 2d, treat that bacterium colony grows, picking colony is rule on the inorganic salt solid medium, until obtaining single bacterium colony; Single bacterium colony that will obtain again, continuous passage is 6 times on the inorganic salt solid medium that with the nitric nitrogen is only nitrogen source, makes it to become the stable pure culture of colonial morphology.
SNR-1 colonial morphology and thalli morphology are as shown in Figure 1.
The evaluation of klebsiella (Klebsiella sp.) SNR-1
By the comparative analysis to 16SrDNA the preceding paragraph homology sequence, in conjunction with the utilize situation of this bacterial strain to the dull and stereotyped 95 kinds of different carbon sources of Biolog GN2, identify that the SNR-1 bacterial strain is klebsiella.
Naming this klebsiella is SNR-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on March 22nd, 2013, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science institute of microbiology, deposit number is CGMCC No.7124.
The nitric nitrogen conversion capability of klebsiella (Klebsiella sp.) SNR-1 detects
With 5mL SNR-1 bacterium liquid (OD
600=1.1) being inoculated in 95mL nitric nitrogen concentration is in the 300mg/L minimal medium.In 30 ℃, 120rpm shaking culture, and measure nitrate content and bacterium liquid OD in the nutrient solution every 24h
600Value.
The result shows (Fig. 2), along with the prolongation of incubation time, and bacterium liquid OD
600Value reaches maximum at 3d; Transformation efficiency to nitric nitrogen (300mg/L) during 3d reaches 92.5%, and is the highest to the transformation efficiency of nitric nitrogen when 5d, reaches 93.5%, afterwards kept stable.
Embodiment 4
The preparation of SNR-1 microbial inoculum
In the potato glucose liquid nutrient medium, potato glucose liquid culture based formulas is potato 200g with the SNR-1 inoculation, glucose 20g/L, and moisturizing is to 1L, and adjusting pH is 7.0-7.2.In 30 ℃, 150rpm shaking culture 24-48h, make the OD of bacterium liquid
600Value is about 2.With air-dry straw be crushed to its length less than 2mm after, bacterium liquid and stalk are mixed with 1:1 (mL/g), it is standby to spend the night.
Simulation SNR-1 microbial inoculum is to the secondary salinization soil repairing effect
Diameter was respectively the plastic tub of 18cm and 12cm about pot experiment was used, the about 1.5kg wind desiceted soil of every basin dress.Four groups of processing are respectively: (1) does not add KNO
3, blank CK1; (2) add KNO
3Solution makes NO in the soil
3-N concentration is 200mg/kg, CK2; (3) handle 2 soil+30g stalk (WS); (4) handle 2 soil+30g stalk+30mL bacteria suspension (WSBS).For blank CK1, KNO in interpolation and the processing 2
3The isopyknic tap water of solution.
All are handled basin and all are placed in the tray, and add the 150mL tap water to keep the soil water content every day in tray.The little green vegetables seed HgCl of plantation
2(1.0g/L) deionized water is cleaned behind the surface sterilization 10min, is placed on the filter paper after 28 ° of C incubators are cultivated 24h, gets 20 and evenly disseminates in all plastic tub.Respectively at the 0th, 3,6,9,12 and 15d gather soil sample, nitrate nitrogen content in the soil is measured in the air-dry back of room temperature; 6d adds up little green vegetables percentage of germination; 15d, it is long to measure little green vegetables root, indexs such as plant height and the number of blade.
Test-results shows that the SNR-1 microbial inoculum has good changing effect to nitrate in the soil, and in the soil after microbial inoculum is handled (WSBS) 15d, nitric nitrogen descends 59.4%, improves nearly 30% (Fig. 3,4) than stalk (WS) treatment group; Simultaneously the SNR-1 microbial inoculum also can improve the percentage of germination of little green vegetables in secondary salinization soil, in the time of can effectively reducing nitrate little green vegetables root is sprouted and the restraining effect during growth.
Claims (13)
1. a secondary salinization soil is repaired bacterium, it is characterized in that, be specially klebsiella (SNR-1), now be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science institute of microbiology.Date saved is on January 11st, 2013, and deposit number is CGMCC No.7124.
2. a secondary salinization soil according to claim 1 is repaired the separation purification method of bacterium, it is characterized in that, stablizes bacterium colony by collected specimens from soil through using the inorganic salt liquid culture medium inoculated also to cultivate until acquisition after repeatedly taming.
3. method according to claim 2 is characterized in that, described soil is taken from the booth of industrial park, Nanhui, Shanghai City facility cultivation 12-15.
4. method according to claim 2 is characterized in that, described domestication refers to: pedotheque is inoculated in the enrichment medium, cultivated 7 days under 25 ℃, 120rpm condition.
5. method according to claim 2 is characterized in that, described repeatedly domestication refers to: each acclimation period is 7 days, and the domestication back is inserted in the enrichment medium 4 times repeatedly again with 10% inoculum size.
6. method according to claim 4 is characterized in that, described enrichment medium is the inorganic salt liquid substratum.
7. according to claim 2 or 6 described methods, it is characterized in that the component of described inorganic salt liquid substratum and content are: glucose 15g, KCl1.0g/L, MgSO
47H
2O0.5g/L, CaCl
21.0mg/L, FeSO
47H
2O0.1g/L, KH
2PO
40.5g/L, pH6.8-7.2.
8. method according to claim 2, it is characterized in that, described inoculation refers to: the suspension of getting 100 μ L domesticated strains is coated in the inorganic salt solid medium, in 25 ℃ of fixed temperature and humidity incubators, be inverted and cultivate 2d, treat that bacterium colony grows, picking colony is rule on the inorganic salt solid medium, until obtaining single bacterium colony; Single bacterium colony that will obtain again, continuous passage is 6 times on the inorganic salt solid medium that with the nitric nitrogen is only nitrogen source, makes it to become the stable pure culture of colonial morphology.
9. preservation and activation according to an arbitrary described secondary salinization soil reparation bacterium among the claim 1-8 is characterized in that, wherein:
Storage conditions is: adopts-80 ℃ of cryogenic freezing preserving processes, microorganism is in the lesser temps, thereby to reduce the purpose that its metabolism speed reaches the preservation bacterial classification;
Activation is: the bacterial classification inoculation of freezing state in the potato glucose liquid nutrient medium, is treated that bacterial classification adapts to culture environment gradually and breeds, to obtain the culture that quantity is enough, flush.
10. method according to claim 9 is characterized in that, the component of described potato glucose liquid nutrient medium and content are: potato 200g, and glucose 20g/L, moisturizing is regulated pH to 7.0-7.2 to 1L.
11. a soil remediation method is characterized in that, arbitrary described secondary salinization soil reparation bacterium is degraded among the employing claim 1-10.
12. method according to claim 11 is characterized in that, described degraded by bacterial strain SNR-1 microbial inoculum with the nitric nitrogen in the soil as nitrogenous source, stalk is as carbon source, transforms through microbiological deterioration and realizes.
13. method according to claim 11, it is characterized in that, described microbial inoculum prepares in the following manner: with the SNR-1 inoculation in the potato glucose liquid nutrient medium of pH7.0-7.2, in 30 ℃, 150rpm shaking culture 24-48h, at last air-dry straw is crushed to its length less than 2mm after, bacterium liquid and stalk are mixed namely with 1:1 (mL/g).
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| CN103980906A (en) * | 2014-05-14 | 2014-08-13 | 扬州大学 | Biological-reinforced carbon conditioning agent and method of reducing soluble salt content in secondary salinized soil using same |
| CN104190703A (en) * | 2014-08-20 | 2014-12-10 | 上海华土生态环境科技有限公司 | Co-treatment method for soil salinization |
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| CN102703344A (en) * | 2012-05-10 | 2012-10-03 | 上海交通大学 | Straw degradation actinomycete and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980906A (en) * | 2014-05-14 | 2014-08-13 | 扬州大学 | Biological-reinforced carbon conditioning agent and method of reducing soluble salt content in secondary salinized soil using same |
| CN104190703A (en) * | 2014-08-20 | 2014-12-10 | 上海华土生态环境科技有限公司 | Co-treatment method for soil salinization |
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Application publication date: 20130911 |