CN103525739A - Nitrate assimilating bacterium and application of bacterium to restoring of facility secondary salinized soil - Google Patents
Nitrate assimilating bacterium and application of bacterium to restoring of facility secondary salinized soil Download PDFInfo
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Abstract
The invention relates to a nitrate assimilating bacterium and application of the bacterium to restoring of facility secondary salinized soil in the technical field of soil remediation. According to the invention, nitrate nitrogen, serving as a nitrogen source, in soil is assimilated into inorganic nitrogen or cellular components of other forms through the nitrate assimilating bacterium NCT-2, so that the degradation transformation of the nitrate nitrogen is realized, the nitrate nitrogen content in the soil is reduced and the soil is restored. The nitrate assimilating bacterium NCT-2 is a bacillus megaterium and is preserved in CGMCC (China General Microbiological Culture Collection Center) with the preservation number CGMCC NO. 4698. The nitrate assimilating bacterium disclosed by the invention has a very strong assimilation transformation capacity on nitrate nitrogen in environments, is high in adaptability to the environment and also has a phosphorus dissolving capacity, so that the bacterium has broad application prospects on restoring the facility secondary salinized soil and preparing microbial fertilizers.
Description
Technical field
What the present invention relates to is a kind of method of soil remediation technical field, specifically a kind of efficient nitrate assimilation bacterium and the application in restoration facilities secondary salinization soil thereof.
Background technology
Along with the development of industrialized agriculture, cultivated area constantly expands, and chemical nitrogen fertilizer usage quantity significantly rises, and substantially exceeds the demand of plant.Because nitrate in soil fails to be absorbed in time conversion by plant, cause a large amount of salinities to accumulate in plant materials and soil, thereby cause facility cultivation soil secondary salinization.
If miscarriage, there is salinity obstacle in glasshouse 2-3 soil; There is salinification obstacle in various degree in plastic greenhouse 3-5.Too high salt branch suppresses growing of crop, causes vegetable growth short and small, hypoevolutism, withered death when serious; Root hair Growth and Differentiation ability, plant resistance against diseases declines, and causes physiological disease, causes crop yield reduction, quality to decline; In addition, also can cause fertilizer waste, unnecessary chemical fertilizer causes widespread pollution from the overuse of fertilizers and pesticides in rural area by agricultural surface runoff and farmland seepage.
Research shows, removes HCO in facility cultivation secondary salinization soil
3 -outward, Na
+, K
+, Ca
2+, Mg
2+, Cl
-, SO
4 2 -, NO
3 -all there is accumulation in various degree.Research shows, in the cation composition of secondary salinization soil, and Na
+be not leading ion, positively charged ion is with Ca
2+be main, its content accounts for the more than 60% of positively charged ion total amount, Mg
2+content is between 15%-20%; Negatively charged ion is with NO
3 -and SO
4 2-be main, NO
3 -content is about the 56%-76% of negatively charged ion total amount.NO
3 -can in plant materials, accumulate, finally by food chain, enter human body, excessive NO
3 -easily be reduced into as NO in vivo
2 -, NO
2 -can make cell tissue anoxic, when serious, make people's death by suffocation.Therefore in treatment facility cultivation, excessive nitric nitrogen has become important and instant work of industrialized agriculture.For China's facility cultivation booth salinification, taked some control measures at present, as washing salinity by irrigation, the methods such as soil improvement agent method and half decomposed manure method.Although these methods have many advantages, also there is very large deficiency: washing salinity by irrigation meeting to underground, not only causes the loss of soil nitrogen nitric nitrogen drip washing, but also polluted underground water; Soil improvement agent method cost compare is high, easily produces secondary pollution; Half decomposed manure method exists fertilizer efficiency slow, uses the shortcomings such as inconvenience.
Administer secondary salinization method with tradition and compare, biological process especially microbial method has many outstanding advantages, and such as breeding is fast, adaptive faculty is strong; Do not produce secondary pollution, to surrounding environment, do not increase burden; Cost is low, and instant effect is easy to operate, manageability etc.The key that microbial method is processed nitric nitrogen in secondary salinization soil is to filter out the bacterial strain with efficient nitric nitrogen assimilative capacity.Along with the development of China's facility cultivation and becoming increasingly conspicuous of soil secondary salinization problem, utilize the nitric nitrogen tool accumulating in microbiological treatment soil to have broad application prospects.
Through the retrieval of prior art is found, Chinese patent literature CN103203356, open day 2013-07-17, a kind of " secondary salinization soil microbiobacterial agent and preparation thereof, purposes " disclosed, this technology relates to described secondary salinization soil microbiobacterial agent and is comprised of bacillus megaterium (Bacillus megaterium) NCT-2CGMCC NO.4698 and carrier, and described carrier is rice wheat straw and glucose.During preparation, in every liter of bacillus megaterium bacterium liquid, add 0.5～0.6kg rice wheat straw, the glucose of 0.3～0.4kg, mixes, and obtains described secondary salinization soil microbiobacterial agent; The bacteria containing amount of every milliliter of described bacillus megaterium liquid is 2.0 * 10
8～2.32 * 10
8individual.The present invention's advantage is compared with prior art: thereby the present invention has measured NCT-2, the content of total soil nitrogen before and after the reparation of facility secondary salinization soil has been confirmed to NCT-2 is a strain nitric nitrogen assimilation bacterium; The present invention also confirms that NCT-2 transforms the fast degradation of nitric nitrogen in fermented liquid and the good repairing effect to secondary salinization booth; In addition, the present invention also confirms that NCT-2 has molten phosphorus ability.
Summary of the invention
The present invention is directed to prior art above shortcomings, a kind of nitrate assimilation bacterium and the application in restoration facilities secondary salinization soil thereof are provided, nitric nitrogen in environment is had to very strong assimilation conversion capability, strong to environmental compatibility; In addition, also there is molten phosphorus ability.Therefore this bacterium has broad application prospects with preparing on microbial fertilizer at restoration facilities cultivation secondary salinization soil.
The present invention is achieved by the following technical solutions:
The mass production method that the present invention relates to a kind of nitrate assimilation bacterium NCT-2, comprising: culture presevation, actication of culture and strain expanded culture, wherein:
Culture presevation: adopt-80 ℃ of cryogenic freezing preservations, by nitrate assimilation bacterium NCT-2 in freezing state, to reduce its metabolism to reach preservation object.
Actication of culture: the nitrate assimilation bacterium NCT-2 under freezing is put into potato culture shaking culture, until obtain the culture (OD that quantity is enough, flush
600for 1.5-2.0).
Strain expanded culture: the culture after activation is inoculated in potato culture, cultivates two days under 28 ℃, 150rpm shaking table environment.
Described nitrate assimilation bacterium NCT-2 is bacillus megaterium (Bacillus megaterium), now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science institute of microbiology.Date saved is on March 21st, 2011, and deposit number is CGMCC No.4698.
Acquisition is inoculated and cultivated to stable single bacterium colony of described nitrate assimilation bacterium NCT-2, by collected specimens from soil, after repeatedly taming, then at solid medium.
Described soil is taken from the booth of Chongming Island of Shanghai City facility cultivation 12-15.
Described repeatedly refers to: each acclimation period is 7 days, after domestication, with 10% inoculum size, again accesses in liquid nutrient medium, repeatedly tames 4 times.
Described domestication refers to: pedotheque is inoculated in liquid nutrient medium, under 25 ℃, 120rpm condition, cultivates 7 days.
Described substratum is inorganic salt liquid substratum.
Component and the content of described inorganic salt liquid substratum are: glucose 15g/L, KCl1.0g/L, MgSO
47H
2o0.5g/L, CaCl
20.001g/L, FeSO
47H
2o0.1g/L, KH
2pO
40.5g/L, surplus is ultrapure water, pH is 6.8-7.2.
Described inoculation is: the bacteria suspension of getting 100 μ L domestications is coated in inorganic salt solid medium, in 25 ℃ of fixed temperature and humidity incubators, is inverted and cultivates 2d, treats that bacterium colony grows, and picking colony is rule on inorganic salt solid medium, until obtain single bacterium colony; Again by the single bacterium colony obtaining, take on the inorganic salt solid medium that nitric nitrogen is only nitrogen source continuous passage 6 times, until obtain the pure culture of form stable.
Described solid medium by adding 15-18g/L agar to prepare in inorganic salt liquid substratum.
Described potato culture prepares in the following manner: take peeling potato 200g, filter to such an extent that soak juice after boiling 20min, add sucrose 20g, mending ultrapure water to volume is 1L; Then regulating pH is 7.0-7.2, is finally heated to sterilizing 20min under 121 ℃ of environment.
Technique effect
Compared with prior art, advantage of the present invention comprises:
1, bacterium of the present invention assimilates nitric nitrogen, is converted into self cellular component, can not cause nitrogen loss, can be used for processing Nitrate Accumulation in soil, has further improved the utilization ratio of nitrogen, can not cause any harm to nature nitrogen cycle and environment;
2, bacterium of the present invention is in nitric nitrogen 5d in fermented liquid, degradation efficiency Gao Keda is more than 75%, and transformation efficiency is high;
3, bacterium of the present invention is to NO
3-n content is 100mgkg
-1nitrified nitrogen in soil transformation efficiency can reach more than 98%, and cost is low, easy to operate, is easy to industrializationization and implements;
4, bacterium of the present invention is high to nitric nitrogen tolerance concentration, and can bear and process nitrate is 1600mgkg
-1soil;
5, bacterium of the present invention has phosphorus decomposing ability, can be used for preparing microbial-bacterial fertilizer.
Accompanying drawing explanation
The colonial morphology (a) of bacillus megaterium in Fig. 1 the present invention (Bacillus megaterium) and thalli morphology (b) figure;
Bacillus megaterium in Fig. 2 the present invention (Bacillus megaterium) 16SrDNA sequence electrophorogram;
Bacillus megaterium in Fig. 3 the present invention (Bacillus megaterium) the most suitable growth curve;
Bacillus megaterium in Fig. 4 the present invention (Bacillus megaterium) is to nitric nitrogen transformation efficiency figure in fermented liquid;
The transformation efficiency figure of bacillus megaterium in Fig. 5 the present invention (Bacillus megaterium) microbial inoculum to nitrate in simulation secondary salinization soil;
Bacillus megaterium in Fig. 6 the present invention (Bacillus megaterium) microbial inoculum is on total nitrogen content impact figure in simulation secondary salinization soil;
The transformation efficiency figure of bacillus megaterium in Fig. 7 the present invention (Bacillus megaterium) microbial inoculum to nitrate in slight secondary salinization soil;
Bacillus megaterium in Fig. 8 the present invention (Bacillus megaterium) effect of solubilizing phosphate figure.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Separation and the purifying of bacillus megaterium (Bacillus megaterium) NCT-2
Pedotheque is taken from the booth of Chongming Island of Shanghai City facility cultivation 12-15, after sampling, sample is fully mixed.Adopt shaking flask enrichment culture, soil sampling 10g is inoculated in 100mL enrichment medium, under 25 ℃, 150rpm condition, cultivates.Every 7d, with 10% of inoculum size, again access in enrichment medium, repeatedly tame 4 times.
Get 100 μ L domestication bacteria suspensions and coat in 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 inorganic salt solid medium, until obtain single bacterium colony; Again by the single bacterium colony obtaining, take on the inorganic salt solid medium that nitric nitrogen is only nitrogen source continuous passage 6 times, until obtain the pure culture of form stable.NCT-2 colonial morphology and thalli morphology are as shown in Figure 1.
Described enrichment medium adopts inorganic salt liquid substratum, and inorganic salt liquid medium component is glucose 15g, KCl1.0g, MgSO
47H
2o0.5g, CaCl
20.001g, FeSO
47H
2o0.1g, KH
2pO
40.5g, ultrapure water 1000mL, adjusting pH is 6.8-7.2.
Described solid medium is in inorganic salt liquid substratum, add 15-18g/L agar again and get final product.
The evaluation of bacillus megaterium (Bacillus megaterium) NCT-2
By the contrast to bacillus megaterium (Bacillus megaterium) 16SrDNAV3 region sequence, the situation of utilizing in conjunction with this bacterial strain to the dull and stereotyped 95 kinds of different carbon sources of Biolog GP2, identifies that this bacterial strain is bacillus megaterium (Bacillus megaterium).
Name bacillus megaterium (Bacillus megaterium) is NCT-2, on March 21st, 2011, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science institute of microbiology, deposit number is CGMCC No.4698.
Determining of bacillus megaterium (Bacillus megaterium) NCT-2 the most suitable growth curve
After measured, determine that this bacterium optimum medium is potato glucose liquid nutrient medium, regulating pH is 7.0, to activate microbionation to LB liquid nutrient medium, inoculum size is 1.0% of culture volume, on 35 ℃, 150rpm shaking table, cultivating, is 1,2,4,6,8,10,12,14,18,22,26,30,34,38 hour at incubation time, measures respectively bacterium liquid OD
600value, curve plotting as shown in Figure 3.
Bacillus megaterium (Bacillus megaterium) NCT-2 measures the conversion capability of nitric nitrogen in fermented liquid.
Inoculum size with 10% is by NCT-2 bacterium access inorganic salt liquid substratum, and adjusting nitrate is 200mg/kg.In 25 ℃, 120rpm shaking culture, and respectively at the 0th, 1,3,5 day, measure in substratum and remain nitric nitrogen amount.Result shows, along with the prolongation of incubation time, in substratum, nitrate constantly reduces, the fastest the 1st day lowering speed, slow gradually by the 3rd day, by the 5th day, substantially reaches steady.This bacterium can reach 78% to nitric nitrogen transformation efficiency in 5 days.
Embodiment 5
Simulation bacillus megaterium (Bacillus megaterium) NCT-2 microbial inoculum is to facility secondary salinization soil repairing effect
To grind 2mm sieve after air-dry for examination soil natural.Test nitric nitrogen establish A, B, C3 concentration processing (0,100mgkg
-1and 200mgkg
-1), every basin dress 0.5kg soil, each processes straw powder (gkg
-1) and NCT-2 bacterium liquid (mlkg
-1) addition is respectively 0+0 (CK), 15+0 (MF), three groups of processing of 15+20 (MF+JY), totally 9 processing, 3 repetitions are established in each processing.Nitrate, straw powder and NCT-2 bacterium liquid is disposable applying when dress basin all, and room temperature is placed and kept soil moisture in 20% left and right.0th, sampling in 3,6,9,12,15 days is standby.
Described straw powder is standby with collection after crossing 2mm sieve after micromill pulverizing.
Described NCT-2 bacterium liquid be by the inoculation of activation in potato liquid nutrient medium, 28 ℃, 150rpm shaking table are cultivated two days to bacterium liquid OD
600after being about 2.0, stop fermentation.
Soil extraction is suitably changed with AA3 flow injection instruments mensuration Content of Nitrate-Nitrogen in Soil after dilution.As Fig. 5, compared with the control, MF, MF+JY nitrified nitrogen in soil significantly reduce.Result shows that when adding nitric nitrogen be 100mgkg
-1time, in 15d MF+JY group nitric nitrogen transformation efficiency up to 98.4%, than MF, organize high by 15.7%, higher more than 95% than blank; When adding nitric nitrogen, be 200mgkg
-1time, MF+JY group nitric nitrogen transformation efficiency is 45.5% in 15d, than MF, organize high by 15.3%, higher more than 40% than blank.Result shows, MF+JY group can reduce the conversion that soil nitrate-N and nitric nitrogen transformed bacteria NCT-2 can promote nitrified nitrogen in soil better.
The mensuration of full nitrogen is soil extraction after perchloric acid disappears and boils, and suitably dilution is measured again.At different nitric nitrogens, add under concentration level CK
0, CK, MF and MF+JY total nitrogen content of soil no significant difference (as Fig. 6).Generally speaking, the conversion of nitrified nitrogen in soil has following two kinds of approach: 1) solidification by microorganism is converted into self biological organic nitrogen by soil nitrate-N, then is converted into ammonia by ammonification, is finally converted into ammonium nitrogen and absorbs for plant; 2) by denitrification, by nitrate-nitrogen reduction, be NO, N
2o or N
2discharge into the atmosphere.Soil total N content does not have considerable change before and after experiment, illustrates that nitrified nitrogen in soil may be converted into microbial components and denitrification does not occur.
Bacillus megaterium (Bacillus megaterium) NCT-2 microbial inoculum is to slight secondary salinization field soil repairing effect
Choose at random 3 booths (each 0.5 mu) in slight saliferous neat new garden spot, each booth is established 3 ridges and is repeated, and four sampling spots are established on every ridge.Planting plant is Hua Qiangqing stalk dish, and No. 1 canopy is used NCT-2 solid fungicide, and No. 2 canopy is used commercially available biological organic fertilizer, and No. 3 booth is not used any fertilizer as blank.
After getting original soil sample and little green vegetables results, soil sample is measured nitrate nitrogen content.Result as shown in Figure 7, process rear Content of Nitrate-Nitrogen in Soil and reduce by 54.87% by NCT-2 microbial inoculum, and biological organic fertilizer is processed rear nitrified nitrogen in soil and reduced by 31.68%, and control group nitrate content and original soil sample difference are little.Than control group and biological organic fertilizer group, NCT-2 bacterial manure can be at the content that reduces to a greater extent nitrate.
Described NCT-2 solid fungicide is by bacterium liquid is mixed with the ratio of 1:1 (V:W) with straw powder, and 28 ℃ of standing for fermentation 1d obtain, and guarantee viable count>=2.0 * 10
9cfu/g.
Embodiment 7
The molten phosphorus aptitude tests of bacillus megaterium (Bacillus megaterium) NCT-2
NCT-2 bacterium is inoculated on this calcium phosphate substratum, observes flat board and whether produce transparent molten phosphorus circle after 4d, observed result records as Fig. 8.After measured, hydrolytic circle (D) is 5.4 with colony diameter (d) mean ratio D/d.Generally speaking, molten phosphorus capacity of water is directly proportional to the value of D/d.
Described calcium phosphate nutrient media components and content are: glucose 10g/L, (NH
4)
2sO
40.5g/L, NaCl0.2g/L, KCl0.2g/L, MgSO
47H
2o0.002g/L, MnSO
4h
2o0.002g/L, FeSO
47H
2o0.5g/L, yeast extract 5g/L, Ca
3(PO
4)
22g/L, agar 18g/L, all the other are ultrapure water, pH is 7.0.
Result shows, NCT-2 bacterium can be dissolved the inorganic salt of indissoluble.This bacterium can be converted into the soluble phosphoric manure being easily absorbed by plants by the Inorganic phosphate of indissoluble in soil.Can reduce the input of chemical fertilizer like this, not only reduce cost, and avoid a large amount of chemical fertilizer to drop into soil compaction and the secondary salinization of bringing, there is huge environmental benefit.
Claims (10)
1. a mass production method of nitrate assimilation bacterium NCT-2, is characterized in that, comprising: preservation, activation and enlarged culturing, wherein:
Culture presevation: adopt-80 ℃ of cryogenic freezing preservations, by nitrate assimilation bacterium NCT-2 in freezing state, to reduce its metabolism to reach preservation object;
Actication of culture: the nitrate assimilation bacterium NCT-2 under freezing is put into potato culture shaking culture, until obtain the culture (OD that quantity is enough, flush
600for 1.5-2.0);
Strain expanded culture: the culture after activation is inoculated in potato culture, cultivates two days under 28 ℃, 150rpm shaking table environment;
Described nitrate assimilation bacterium NCT-2 is bacillus megaterium (Bacillus megaterium), this nitrate assimilation bacterium NCT-2 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is CGMCC No.4698.
2. method according to claim 1, is characterized in that, acquisition is inoculated and cultivated to stable single bacterium colony of described nitrate assimilation bacterium NCT-2, by collected specimens from soil, after repeatedly taming, then at solid medium.
3. method according to claim 2, is characterized in that, described soil is taken from the booth of Chongming Island of Shanghai City facility cultivation 12-15.
4. method according to claim 2, is characterized in that, described repeatedly refers to: each acclimation period is 7 days, after domestication, with 10% inoculum size, again accesses in liquid nutrient medium, repeatedly tames 4 times.
5. method according to claim 2, is characterized in that, described domestication refers to: pedotheque is inoculated in liquid nutrient medium, under 25 ℃, 120rpm condition, cultivates 7 days.
6. according to the method described in claim 2 or 4 or 5, it is characterized in that, described substratum is inorganic salt liquid substratum.
7. method according to claim 6, is characterized in that, component and the content of described inorganic salt liquid substratum are:
Glucose 15g/L, KCl1.0g/L, MgSO
47H
2o0.5g/L, CaCl
20.001g/L, FeSO
47H
2o0.1g/L, KH
2pO
40.5g/L, surplus is ultrapure water, pH is 6.8-7.2.
8. method according to claim 2, it is characterized in that, described inoculation is: the bacteria suspension of getting 100 μ L domestications is coated in 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 inorganic salt solid medium, until obtain single bacterium colony; Again by the single bacterium colony obtaining, take on the inorganic salt solid medium that nitric nitrogen is only nitrogen source continuous passage 6 times, until obtain the pure culture of form stable.
9. according to the method described in claim 2 or 8, it is characterized in that, it is characterized in that, described solid medium is in inorganic salt liquid substratum, add 15-18g/L agar again and get final product.
10. method according to claim 1, is characterized in that, described potato culture prepares in the following manner: take peeling potato 200g, filter to such an extent that soak juice after boiling 20min, add sucrose 20g, mending ultrapure water to volume is 1L; Then regulating pH is 7.0-7.2, is finally heated to sterilizing 20min under 121 ℃ of environment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154357A (en) * | 2015-08-18 | 2015-12-16 | 上海交通大学 | Preparation method and application of secondary-salinization soil remediation microbial inoculum |
| CN105238808A (en) * | 2015-09-29 | 2016-01-13 | 上海交通大学 | Expression and application method of green fluorescent protein used for soil secondary salinization |
| CN110132871A (en) * | 2019-06-06 | 2019-08-16 | 扬州大学 | A kind of determination method that Chinese cabbage is endangered by soil secondary salinization |
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| CN102220267A (en) * | 2011-05-16 | 2011-10-19 | 上海交通大学 | Assimilated nitrate nitrogen strain and application thereof |
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2013
- 2013-10-25 CN CN201310512371.3A patent/CN103525739A/en active Pending
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| CN102220267A (en) * | 2011-05-16 | 2011-10-19 | 上海交通大学 | Assimilated nitrate nitrogen strain and application thereof |
Non-Patent Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154357A (en) * | 2015-08-18 | 2015-12-16 | 上海交通大学 | Preparation method and application of secondary-salinization soil remediation microbial inoculum |
| CN105154357B (en) * | 2015-08-18 | 2018-06-08 | 上海交通大学 | The preparation method and application of secondary salinization soil remediation microbial inoculum |
| CN105238808A (en) * | 2015-09-29 | 2016-01-13 | 上海交通大学 | Expression and application method of green fluorescent protein used for soil secondary salinization |
| CN110132871A (en) * | 2019-06-06 | 2019-08-16 | 扬州大学 | A kind of determination method that Chinese cabbage is endangered by soil secondary salinization |
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Application publication date: 20140122 |