CN101525582A - Multi-functional bacterial strain and application thereof - Google Patents
Multi-functional bacterial strain and application thereof Download PDFInfo
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- CN101525582A CN101525582A CN200810101495A CN200810101495A CN101525582A CN 101525582 A CN101525582 A CN 101525582A CN 200810101495 A CN200810101495 A CN 200810101495A CN 200810101495 A CN200810101495 A CN 200810101495A CN 101525582 A CN101525582 A CN 101525582A
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Abstract
The invention provides a Bacillus edaphicus CGMCC No.2315 which is high in salt tolerance, heat resistance and adversity-resistance, can secrete various phytohormones such as indopropionic acid, zeatin, gibberellin, and the like, and can control nematodes and various fungal diseases. The invention also provides a method for producing the spore powder of the Bacillus edaphicus, which effectively reduces the death rate of the core bacterial strains, enables the survival rate of the core bacterial strains to reach 93 percent above and prolongs the guarantee period of the spore powder to more than 12 months. The multi-functional bacterial strain and the production method of the spore powder thereof are suitable for producing biological organic fertilizer, ecological formula fertilizer, biological pesticide, soil improvement agent and other biological preparations.
Description
Technical field
The present invention relates to the biotechnological formulation field, relate to a strain multifunction soil genus bacillus and an application thereof particularly.
Background technology
In China, the long-term excessive chemical fertilizer of using has caused a series of problems such as soil compaction, fertility degeneration.Simultaneously, people are to agricultural chemicals, the chemical fertilizer residue problem growing interest of agricultural-food, and green food, Organic food are subjected to the heat in market and hold in both hands.Under this overall situation, China's bio-feritlizer industry is developed rapidly, the annual production of bio-feritlizer reaches 4,000,000 tons, and the product category of registering in the Ministry of Agriculture reaches 12, comprises root nodule bacterium fertilizer, vinelandii fertilizer, phosphorus decomposing mushroom fertilizer, potassium decomposing mushroom fertilizer etc.
But the bio-feritlizer ubiquity of Xiao Shouing problems such as function singleness, quality instability, soil suitability difference in the market, thereby has limited its large-scale promotion.The key that overcomes the problems referred to above is to filter out the multi-functional bacterial strain of strong stress resistance.The present invention just is being based on this thinking, goes out to possess the soil genus bacillus of multiple function through experiment screening for many years, and has improved existing spore powder production technique, has realized the breakthrough that bio-bacterial manure is produced.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a strain multifunction soil genus bacillus, another object of the present invention provides the method for utilizing this bacterial strain production spore powder.
(2) technical scheme
Soil genus bacillus of the present invention (Bacillus edaphicus) YZH-002, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 25th, 2008, be called for short CGMCC, deposit number is CGMCC No.2315.
Soil genus bacillus of the present invention (Bacillus edaphicus) CGMCC No.2315 possesses multiple functions such as the soil property of improvement, secretion plant growth hormones, prevention and elimination of disease and pests.
The invention provides a kind of method of producing aforementioned soil subtilis spore powder, comprise the steps:
(1) seed activation: the bacterium liquid of picking freezing, on the solid medium flat board, rule, cultivate 14-16h in 30 ℃; Picking list colony inoculation is in liquid nutrient medium then, in 30 ℃, 250rpm shaking culture to OD
600nm=1-1.5;
(2) gemma generates: get step (1) gained bacterium liquid 0.2mL, be inoculated in the Kolle flask that solid medium is housed, smoothen, in 30 ℃ of cultivations 3-4 days, form tangible lawn until media surface, obtain the gemma seed;
(3) inoculation seeding tank: use the aseptic water washing lawn, be inoculated in the seeding tank of the bacterium of going out, inoculation temp 70-80 ℃, fermentation parameter is set to: temperature 30-35 ℃, and tank pressure 0.03-0.05MPa, air flow 1: 0.2-0.3 (m
3/ t/h), pH 6.8-7.2 stirs revolution 200-400rpm, incubation time 20-24h;
(4) fermentation culture: the seed liquor input fermentor tank that step (3) is obtained, fermentation parameter same step (3), incubation time 28-34h be set;
(5) dust: the bacterium liquid input hold tank that step (4) fermentation is obtained, by bacterium liquid: the ratio adding lime carbonate of lime carbonate=1: 3-4, stirring and evenly mixing; Enter spray tower then, inlet temperature is 180-220 ℃, and temperature out is 50-60 ℃, obtains spore powder.
The composition of used substratum is specific as follows in the aforesaid method:
(1) the used substratum composition of activated spawn is (be used for seed activation and gemma and generate step) by mass percentage:
Peptone 5～10 ‰
Glucose 3～8 ‰
Yeast powder 0.5～2 ‰
NaCl 3～6‰
KCl 0.5～1.5‰
MgSO
4·7H
2O 0.2～0.4‰
pH 7.0；
(2) fermention medium is (being used for inoculation jar and fermentation culture step) by mass percentage::
Corn steep liquor 1～10%
Soybean cake powder 0.5～5%
Starch 0.5～5%
NH
2SO
4 0.01～0.3％
K
2HPO
4 0.01～0.1％
MgSO
4 0.1％
FeSO
4 0.01％
pH 7.0～7.2。
Soil genus bacillus of the present invention can be applicable to prepare biotechnological formulations such as biological organic fertilizer, ecological bulk-blending fertilizer, biological pesticide and soil improvement agent.Correspondingly, spore powder production method of the present invention also can be applicable to the production of above-mentioned preparation.
(3) beneficial effect
Soil genus bacillus salt tolerant of the present invention, heat-resisting, strong stress resistance, can secrete various plants hormones such as indolepopionic acid, zeatin, Plant hormones regulators,gibberellins, and can prevent and treat nematode and multiple fungal disease, be applicable to biotechnological formulations such as producing biological organic fertilizer, ecological bulk-blending fertilizer, biological pesticide and soil improvement agent.Simultaneously, spore powder production method provided by the invention has effectively reduced the mortality ratio of core bacterial strain, makes survival rate reach more than 93%, and the quality guaranteed period of spore powder was brought up to more than 12 months.
Description of drawings:
Fig. 1, chitin decompose the experimental result circle.
Soil genus bacillus of the present invention (Bacillus edaphicus) YZH-002, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 25th, 2008, be called for short CGMCC, deposit number is CGMCC No.2315.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Character and the functional experiment of embodiment 1 soil genus bacillus of the present invention
Soil genus bacillus of the present invention is that the contriver separates from the paddy rice rhizosphere soil and obtains, and its character and function are as follows:.
One, form and physio-biochemical characteristics: G
+, shaft-like, size is (0.4-0.6) * (1.5-3) μ m; Gemma adnation, majority are oval; Vp
-, nutrient solution pH 7.0～7.2; 5.5～9.8 times growths of pH
+On anaerobic culture medium, do not grow, in the NaCl more than 7.5%, do not grow; The starch hydrolysis
-, gelatine liquefication
+, decompose casein
+, catalase
+, oxydase
-, the Citrate trianion utilization
-, the glucose aerogenesis
-, glucose produces acid
+, L-arabinose produces acid
-, wood sugar produces acid
-, N.F,USP MANNITOL produces acid
+, nitrate reduction
+, methyl red test
+
More than used detection method be classical way in the Experiment on Microbiology textbook,
+The expression experimental result positive,
-Expression experimental result feminine gender.
Two, functional experiment:
1. the secretion of plant growth hormones:
Table 1
Used measuring method is classical liquid phase chromatography.
2, chitin decomposes:
Interpolation concentration is 2% chitin in activation medium (seeing the record of present techniques scheme), the described soil genus bacillus of dull and stereotyped cultivation.Behind the 36h, observe periphery of bacterial colonies and transparent circle occurs, as shown in Figure 1, show that this bacterium possesses very strong decomposition chitin ability.Chitin is the main component of line eggs outer wall and pathomycete cell walls, and this illustrates that this bacterium possesses the function of control nematode and fungal disease, and field experiment proves that also its effect is remarkable.
3. resistance experiment:
3.1 salt tolerance:
Get 35% (NH
4)
2SO
4Solution 10mL adds 1 milliliter of soil genus bacillus bacterium liquid (zymocyte liquid, hundred million of thalline quantity 40-50/mL), soaked the soil genus bacillus 0～15 day.(NH with 10mL distilled water replacement 35%
4)
2SO
4Contrast is done in immersion.Then, soak solution is added in the triangular flask that the 90mL sterilized water is housed (in sterile glass beads is arranged) 50～100rpm shaking culture 30min.Get the adding of 1mL diluent and be equipped with in the test tube of 9mL sterilized water, shake up, repeat aforesaid operations, until being diluted to 10
-8Doubly.Get 10
-6, 10
-7, 10
-8Each 0.1mL of diluent doubly places flat board, smoothens, and cultivates 1～2 day counting for 30 ℃.Survival results is as shown in table 2, can find out that from table 2 this bacterial strain has stronger salt tolerance.
The experiment of table 2 salt tolerance
| Test duration | Viable count | Surviving rate |
| 0 day | 4,400,000,000/milliliter | 100.00％ |
| 4 days | 4,400,000,000/milliliter | 100.00％ |
| 8 days | 4,400,000,000/milliliter | 100.00％ |
| 15 days | 43.6 hundred million/milliliter | 99.09％ |
3.2 thermotolerance:
With 80 ℃ and 90 ℃ be Temperature Treatment, 1,3 hour to be to handle the time, be contrast with natural storage temperature, described soil genus bacillus is carried out heat-resisting processing.(zymocyte liquid, hundred million of thalline quantity 40-50/mL) add in the triangular flask that the 99ml sterilized water is housed (in sterile glass beads is arranged), 50～100rpm shaking culture 30min to get 1 milliliter of bacterium liquid then.Get the adding of 1mL diluent and be equipped with in the test tube of 9mL sterilized water, shake up, repeat aforesaid operations, until being diluted to 10
-8Doubly.Get 10
-6, 10
-7, 10
-8Each 0.1mL of diluent doubly places flat board, smoothens, cultivated 1～2 day for 30 ℃, and counting, the result is as shown in table 3.According to the data of table 3, we as can be seen 80 ℃ handle that the gemma survival rate is 93.25% in 3 hours the microbial inoculum, show very high thermotolerance.
The experiment of table 3 thermotolerance
| Sample | Handle | Viable count | Average survival |
| Former bacterium number (4,400,000,000/milliliter) | 80℃180min | 41.03 hundred million/milliliter | 93.25％ |
| Former bacterium number (4,400,000,000/milliliter) | 90℃60min | 35.14 hundred million/milliliter | 79.86％ |
The preparation of embodiment 2 spore powder
(1) seed activation: the bacterium liquid of picking freezing, on the solid medium flat board, rule, cultivate 14h in 30 ℃; Picking list colony inoculation is in liquid nutrient medium then, in 30 ℃, 250rpm shaking culture to OD
600nm=1.5;
(2) gemma generates: get step (1) gained bacterium liquid 0.2mL, be inoculated in the Kolle flask that solid medium is housed, smoothen, in 30 ℃ of cultivations 3 days, form tangible lawn until media surface, obtain the gemma seed;
(3) inoculation seeding tank: use the aseptic water washing lawn, be inoculated in the seeding tank of the bacterium of going out, 70 ℃ of inoculation temps, fermentation parameter is set to: 35 ℃ of temperature, tank pressure 0.03MPa, 1: 0.2 (m of air flow
3/ t/h), pH 6.8, stir revolution 250rpm, incubation time 20h;
(4) fermentation culture: the seed liquor input fermentor tank that step (3) is obtained, fermentation parameter same step (3), incubation time 34h be set;
(5) dust: the bacterium liquid input hold tank that step (4) fermentation is obtained, by bacterium liquid: the ratio adding lime carbonate of lime carbonate=1: 3, stirring and evenly mixing; Enter spray tower then, inlet temperature is 220 ℃, and temperature out is 60 ℃, obtains spore powder.
More than used substratum see technical scheme.
After testing, the spore survival rate reaches 95%, 18 months quality guaranteed perioves of spore powder.
The preparation of embodiment 3 spore powder
(1) seed activation: the bacterium liquid of picking freezing, on the solid medium flat board, rule, cultivate 16h in 30 ℃; Picking list colony inoculation is in liquid nutrient medium then, in 30 ℃, 250rpm shaking culture to OD
600nm=1;
(2) gemma generates: get step (1) gained bacterium liquid 0.2mL, be inoculated in the Kolle flask that solid medium is housed, smoothen, in 30 ℃ of cultivations 4 days, form tangible lawn until media surface, obtain the gemma seed;
(3) inoculation seeding tank: use the aseptic water washing lawn, be inoculated in the seeding tank of the bacterium of going out, 80 ℃ of inoculation temps, fermentation parameter is set to: 30 ℃ of temperature, tank pressure 0.05MPa, 1: 0.3 (m of air flow
3/ t/h), pH 7.2, stir revolution 400rpm, incubation time 24h;
(4) fermentation culture: the seed liquor input fermentor tank that step (3) is obtained, fermentation parameter same step (3), incubation time 28h be set;
(5) dust: the bacterium liquid input hold tank that step (4) fermentation is obtained, by bacterium liquid: the ratio adding lime carbonate of lime carbonate=1: 4, stirring and evenly mixing; Enter spray tower then, inlet temperature is 180 ℃, and temperature out is 50 ℃, obtains spore powder.
More than used substratum see technical scheme.
After testing, the spore survival rate reaches 96%, 14 months quality guaranteed perioves of spore powder.
The application of embodiment 4 in the preparation bio-bacterial manure
Embodiment 2 gained spore powder are mixed stirring with the good organic matter that becomes thoroughly decomposed,, produce water ratio and be no more than 8% organic-biological particle through 70 ℃ of-180 ℃ of hyperthermia dryings.Be cooled to normal temperature, screening obtains different big or small particles, chooses standard compliant particle packaging and warehousing, makes biological organic fungi-manure.
The application of embodiment 5 in the preparation ecological bulk-blending fertilizer
According to the N in the soil, P, K content and different crops N, P, K demand, N, the P, the K inorganic nutrients that in the biological organic fungi-manure that embodiment 4 makes, add different content, mix, make the ecological bulk-blending fertilizer that organic matter, inorganic nutrients and microorganism three organically combine.
The preparation of embodiment 6 nematode inhibitor
Get embodiment 3 gained spore powder, shrimp shell meal or crab shell powder to the starch that wherein adds 5%-20%, 3%-5% make the nematode inhibitor.Above ratio all is weight percentage.
Claims (5)
1, strain soil genus bacillus (Bacillus edaphicus) CGMCC No.2315 is characterized in that this bacterial strain possesses multiple functions such as the soil property of improvement, secretion plant growth hormones, prevention and elimination of disease and pests.
2, a kind of method of producing soil subtilis spore powder as claimed in claim 1 comprises the steps:
(1) seed activation: the bacterium liquid of picking freezing, on the solid medium flat board, rule, cultivate 14-16h in 30 ℃; Picking list colony inoculation is in liquid nutrient medium then, in 30 ℃, 250rpm shaking culture to OD
600nm=1-1.5;
(2) gemma generates: get step (1) gained bacterium liquid 0.2mL, be inoculated in the Kolle flask that solid medium is housed, smoothen, in 3 ℃ of cultivations 3-4 days, form tangible lawn until media surface, obtain the gemma seed;
(3) inoculation seeding tank: use the aseptic water washing lawn, be inoculated in the seeding tank of the bacterium of going out, inoculation temp 70-80 ℃, fermentation parameter is set to: temperature 30-35 ℃, and tank pressure 0.03-0.05MPa, air flow 1: 0.2-0.3, pH 6.8-7.2 stirs revolution 200-400rpm, incubation time 20-24h;
(4) fermentation culture: the seed liquor input fermentor tank that step (3) is obtained, fermentation parameter same step (3), incubation time 28-34h be set;
(5) dust: the bacterium liquid input hold tank that step (4) fermentation is obtained, by bacterium liquid: the ratio adding lime carbonate of lime carbonate=1: 3-4, stirring and evenly mixing; Enter spray tower then, inlet temperature is 180-220 ℃, and temperature out is 50-6 ℃, obtains spore powder.
3, method according to claim 2, wherein used substratum are formed:
(1) the used substratum of activated spawn is formed:
Peptone 5～10 ‰
Glucose 3～8 ‰
Yeast powder 0.5～2 ‰
NaCl 3～6‰
KCl 0.5～1.5‰
MgSO
4·7H
2O 0.2～0.4‰
pH 7.0；
(2) fermention medium:
Corn steep liquor 1～10%
Soybean cake powder 0.5～5%
Starch 0.5～5%
NH
2SO
4 0.01～0.3％
K
2HPO
4 0.01～0.1％
MgSO
4 0.1％
FeSO
4 0.01％
pH 7.0～7.2。
4, the application of bacterial classification according to claim 1 in biotechnological formulations such as preparation biological organic fertilizer, ecological bulk-blending fertilizer, biological pesticide and soil improvement agent.
5, the application of method according to claim 2 in biotechnological formulations such as preparation biological organic fertilizer, ecological bulk-blending fertilizer, biological pesticide and soil improvement agent.
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|---|---|---|---|
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ID=41093652
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571766A (en) * | 2012-07-20 | 2014-02-12 | 上海绿乐生物科技有限公司 | Liquid microbial leaf fertilizer, and production method and applications thereof |
| CN109518703A (en) * | 2019-01-15 | 2019-03-26 | 内蒙古工业大学 | A kind of administering method of the cementing treatment fluid of microbial mineralization and chiltern side slope |
-
2008
- 2008-03-07 CN CN200810101495A patent/CN101525582A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571766A (en) * | 2012-07-20 | 2014-02-12 | 上海绿乐生物科技有限公司 | Liquid microbial leaf fertilizer, and production method and applications thereof |
| CN103571766B (en) * | 2012-07-20 | 2015-04-22 | 上海绿乐生物科技有限公司 | Liquid microbial leaf fertilizer, and production method and applications thereof |
| CN109518703A (en) * | 2019-01-15 | 2019-03-26 | 内蒙古工业大学 | A kind of administering method of the cementing treatment fluid of microbial mineralization and chiltern side slope |
| CN109518703B (en) * | 2019-01-15 | 2021-07-02 | 内蒙古工业大学 | Microbial mineralization and cementation treatment fluid and treatment method of sandy slope |
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Open date: 20090909 |