CN106987536A - Complex micro organism fungicide for river regulation and its preparation method and application - Google Patents
Complex micro organism fungicide for river regulation and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of for complex micro organism fungicide of river regulation and its preparation method and application, in complex micro organism fungicide, microbial inoculum A is individually to cultivate formed five kinds of zymotic fluids by bacillus megaterium CCTCC No.M2012352, Bacillus subtillis CGMCC No.1.2162, bacillus licheniformis CGMCC No.1.6510, bacillus amyloliquefaciens CGMCC No.1.7463, Phanerochaete chrysosporium BKM F1767 to mix；Microbial inoculum B is individually to cultivate the zymotic fluid formed by the inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese.Solid suspended particle, somatic cells and the colloidal particle in ammonia nitrogen and nitrate, reduction phosphorus element concentration, and precipitation water body in the complex micro organism fungicide degradable water.
Description
Technical field
The invention belongs to technical field of environmental microorganism, be related to a kind of complex micro organism fungicide for river regulation and its
Preparation method and application.
Background technology
City river and lake be with human being's production and the closely coupled water environment of life, as population increases and urbanization
Process accelerates, and a large amount of pollutants have been directly discharged into river course and lake, cause organic matter in water body, ammonia nitrogen, total phosphorus and hydrogen sulfide
Continue to increase Deng pollutant, the natural purifying ability wretched insufficiency of water body, serious restriction economy and environment sustainable development, simultaneously
Jeopardize the healthy of the mankind.
Removing the traditional means of water pollutant mainly has physics and chemical method, although administered in river course and lake pollution
It is upper that there is certain effect, or but the investment of these methods and maintenance cost are high, or being difficult to reach permanent regulation effect, or even have
A little chemical methodes can cause secondary pollution of water.
Bioremediation technology is the new technology that the water pollutant that recent fast development is got up is repaired, and it passes through in water
High-effective microorganism bacterial strain is delivered in body, using these microorganisms are to the absorption of pollutant in water body, conversion or degrade, reach and slows down
Or finally eliminate water pollution, recover the biological control measure of water ecology function.The dominant mechanism of microorganism remediation river sewage has：
Microorganism is Self substances by assimilation meeting transform portion organic pollution, and another aspect microbes produce different lifes
Thing enzyme is used as catalyst degradation N-NH3With TP etc.；In addition, microorganism by way of nutrient competition, can suppress the life of algae
It is long；Also can be by suppressing the growth of some pathogens and spoilage organisms as dominant bacteria, so as to reduce the generation of ammonia and stink.It is micro-
Bioremediation technology is because having low, adaptable operating cost, the great efforts of reduction pollutant, will not cause pollutant transfer etc.
Feature, is expected to play an important role during polluted river water is administered.
Research of the China in terms of water pollution is repaired using microbial technique is started late, and microorganism remediation is controlled in pollution
Accounting in reason is smaller.Current Main is disposably to add complex micro organism fungicide in polluted-water, and these show
There is technology to there is problems at present：If 1) add species and quantity is very few, due to China's water pollution source category
Complexity, it is difficult to ensure that water pollution regulation effect；And add species and quantity is excessive, it is understood that there may be between strain mutually
Even there is the phenomenon of restriction or checking relation in five elements in competition, can also influence pollution control effect.In addition, the microorganism amount reproduction delivered, such as too late
When handle, can also cause secondary pollution to water body；2) deliver complex micro organism fungicide be based on degradable organic pollutant,
Because China's water pollution is in addition to Organic Pollution, various solid suspended particles, colloidal particle etc. are accompanied by, current this kind of dirt
The removal for contaminating thing is main with either physically or chemically, although also have a report of microbial flocculant, but filter out at present it is efficient
Bacterium for producing flocculant of microbe species is seldom, and the overwhelming majority rests on laboratory stage.Therefore, also lack comprehensive, high at present
The strain of effect and the recovery technique of maturation, causing microbiological comprehensive recovery technique, industrially application effect is not satisfactory.
The content of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art there is provided a kind of for the compound of river regulation
Microbial bacterial agent and preparation method thereof, the complex micro organism fungicide can not only decompose the amino in different organic matters, degradation water
The growth of phosphorus element concentration and suppression algae and harmful bacteria in nitrogen and nitrate, reduction water body, but also can precipitate in water body
Not degradable solid suspended particle, somatic cells and colloidal particle.Accordingly, the present invention also provides above-mentioned complex microorganism
Application of the microbial inoculum in river sewage processing.
In order to solve the above technical problems, the present invention proposes following technical scheme：
A kind of complex micro organism fungicide for river regulation, the complex micro organism fungicide includes complex micro organism fungicide
A and microbial bacterial agent B, the complex micro organism fungicide A are by bacillus megaterium CCTCC No.M2012352, Ko subtilis
Bacillus CGMCC No.1.2162, bacillus licheniformis CGMCC No.1.6510, bacillus amyloliquefaciens CGMCC
No.1.7463, Phanerochaete chrysosporium BKM-F1767, Klebsiella oxytoca CCTCC NO.M2011350 individually cultivate shape
Into six kinds of zymotic fluids mix；The microbial bacterial agent B is by the inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese and wine brewing
Two kinds of zymotic fluids that saccharomycete CGMCC No.3003 individually cultivate formation are mixed.
The above-mentioned complex micro organism fungicide for river regulation, it is preferred that in the complex micro organism fungicide A, each group
Point parts by volume be：10~20 parts of bacillus megaterium CCTCC No.M2012352 zymotic fluids, Bacillus subtillis
10~20 parts of CGMCCNo.1.2162 zymotic fluids, 10~20 parts of bacillus licheniformis CGMCC No.1.6510 zymotic fluids, solution starch
10~20 parts of bacillus CGMCC No.1.7463 zymotic fluids, 10~20 parts of Phanerochaete chrysosporium BKM-F1767 zymotic fluids,
10~20 parts of Klebsiella oxytoca CCTCC NO.M2011350 zymotic fluids.
The above-mentioned complex micro organism fungicide for river regulation, it is preferred that in the complex micro organism fungicide A, total bacterium
Fall number and be more than 10.0 × 1010cfu/mL。
The above-mentioned complex micro organism fungicide for river regulation, it is preferred that in the microbial bacterial agent B, flocculation activity
More than 70%.
As a total inventive concept, the present invention also provides a kind of above-mentioned composite microbial bacteria for river regulation
The preparation method of agent, comprises the following steps：
(1) complex micro organism fungicide A is prepared：
(1.1) bacillus megaterium CCTCC No.M2012352 are inoculated in solid slope culture medium I and activated,
Activation bacillus megaterium is made；Then activation bacillus megaterium is inoculated in fluid nutrient medium I and is enlarged culture；Connect again
Plant in fermentation medium I, fermented culture is made cell concentration and is not less than 3.0 × 109Cfu/mL bacillus megaterium
CCTCC No.M2012352 zymotic fluids；
(1.2) Bacillus subtillis CGMCC No.1.2162 are inoculated in solid slope culture medium II and activated, made
Bacillus subtillis must be activated；Then activation Bacillus subtillis is inoculated in fluid nutrient medium II and is enlarged culture；Connect again
Plant in fermentation medium II, fermented culture is made cell concentration and is not less than 10.0 × 107Cfu/mL Bacillus subtillis
CGMCC No.1.2162 zymotic fluids；
(1.3) bacillus licheniformis CGMCC No.1.6510 are inoculated in solid slope culture medium III and activated,
Activation bacillus licheniformis is made；Then activation bacillus licheniformis is inoculated in fluid nutrient medium III and is enlarged culture；Again
It is inoculated in fermentation medium III, fermented culture is made cell concentration and is not less than 8.0 × 109Cfu/mL lichens brood cell's bar
Bacterium CGMCC No.1.6510 zymotic fluids；
(1.4) bacillus amyloliquefaciens CGMCC No.1.7463 are inoculated in solid slope culture medium IV and activated,
Activation bacillus amyloliquefaciens are made；Then activation bacillus amyloliquefaciens are inoculated in fluid nutrient medium IV and are enlarged training
Support；It is inoculated in fermentation medium IV, fermented culture is made cell concentration and is not less than 6.0 × 109Cfu/mL solution starch
Bacillus CGMCC No.1.7463 zymotic fluids；
(1.5) Phanerochaete chrysosporium BKM-F1767 is inoculated in solid slope culture medium V and activated, be made and live
Change Phanerochaete chrysosporium；Then activation Phanerochaete chrysosporium is inoculated in fluid nutrient medium V and is enlarged culture；Inoculate
In fermentation medium V, fermented culture is made cell concentration and is not less than 6.0 × 108Cfu/mL Phanerochaete chrysosporium
BKM-F1767 zymotic fluids；
(1.6) Klebsiella oxytoca CCTCC NO.M2011350 are inoculated in solid slope culture medium VI and activated,
Activation Klebsiella oxytoca is made；Then activation Klebsiella oxytoca is inoculated in fluid nutrient medium VI and is enlarged culture；Again
It is inoculated in fermentation medium VI, fermented culture is made cell concentration and is not less than 5.0 × 108Cfu/mL Klebsiella oxytoca
CCTCCNO.M2011350 zymotic fluids；
(1.7) bacillus megaterium CCTCC No.M2012352 zymotic fluids, step (1.2) made from step (1.1) are made
Bacillus subtillis CGMCC No.1.2162 zymotic fluids, bacillus licheniformis made from step (1.3)
Bacillus amyloliquefaciens CGMCC No.1.7463 zymotic fluids, step made from CGMCCNo.1.6510 zymotic fluids, step (1.4)
(1.5) Klebsiella oxytoca prepared by Phanerochaete chrysosporium BKM-F1767 zymotic fluids, step (1.6) made from
After CCTCCNO.M2011350 zymotic fluids are proportionally mixed, complex micro organism fungicide A is obtained.
(2) microbial bacterial agent B is prepared：
The inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese are inoculated in solid slope culture medium VI and activated, are made
Activate the inferior Dbaly yeast bacterium of the Chinese；Then the inferior Dbaly yeast bacterium of the Chinese will be activated it is inoculated in fluid nutrient medium VI and is enlarged training
Support；It is inoculated in fermentation medium VI, the inferior Dbaly yeast bacterium of the Chinese that flocculation activity is more than 75% is made in fermented culture
CGMCC No.5770 zymotic fluids；
The preparation method of the above-mentioned complex micro organism fungicide for river regulation, it is preferable that in the step (2.1),
In the solid medium VI, containing 0.5~1g/100mL of beef extract, 0.5~1g/100mL of peptone, 0.5~1g/ of yeast extract
100mL, 5~6g/100mL of tomato juice, 0.5~1.0g/100mL of glucose, 0.5~1.0g/100mL of calcium bicarbonate, agar 1~
2 g/100mL, pH value 4~6.5；In the fluid nutrient medium VI, containing 1.0~2.0g/100mL of glucose, yeast extract 0.5~
1g/100mL, 1~1.5g/100mL of corn steep liquor, 0.01~0.02g/100mL of potassium dihydrogen phosphate, 0.5~1.0g/ of calcium bicarbonate
100mL；In the fermentation medium VI, containing 10~25g/100mL of bean dregs, 1.0~5.0g/100mL of yeast extract, maize pulp 5.0
~10.0/100mL, 0.01~0.1g/100mL of potassium dihydrogen phosphate, 0.5~2.0g/100mL of calcium bicarbonate, citric acid 0.2~
2.0g/100mL。
The preparation method of the above-mentioned complex micro organism fungicide for river regulation, it is preferable that the solid medium I
In, containing 0.5~1g/100mL of peptone, 1~1.5g/100mL of dusty yeast, 1~2g/100mL of glucose, dipotassium hydrogen phosphate 0.1
~0.5g/100mL, 1~2g/100mL of agar, pH value 6~8；In the solid medium II, containing 1~1.5g/ of peptone
100mL, 1~1.5g/100mL of glucose, 0.5~1g/100mL of sodium chloride, 1~2g/100mL of agar, pH value 6.5~7.5；Institute
State in solid medium III, containing 1~1.5g/100mL of tryptone, 0.5~1g/100mL of yeast extract, 1~1.5g/ of sodium chloride
100mL, 1.5~2g/100mL of agar, pH value 6~8；In the solid medium IV, containing 1~1.5g/100mL of tryptone,
0.5~1g/100mL of yeast extract, 1~1.5g/100mL of sodium chloride, 1.5~2g/100mL of agar, pH value 6~8；The solid training
Support in base V, containing 1~1.5g/100mL of peptone, 0.5~1g/100mL of yeast extract, 0.5~1g/100mL of sodium chloride, agar 1.5
~2g/100mL, pH value 6~8；In the solid medium VI, containing 0.5~1g/100mL of glucose, 0.2~0.5g/ of sodium acetate
100mL, 0.5~1g/100mL of yeast extract, 0.25~0.5g/100mL of ammonium chloride, 1~2g/100mL of agar, pH value 6.5~
7.5。
The preparation method of the above-mentioned complex micro organism fungicide for river regulation, it is preferable that the fluid nutrient medium I
In, containing 1~1.5g/100mL of glucose, 0.5~1g/100mL of yeast extract, 1~1.5g/100mL of peptone, potassium dihydrogen phosphate
0.05~0.1g/100mL, magnesium sulfate 0.04g/100mL；In the fluid nutrient medium II, containing 1~1.5g/100mL of glucose,
0.5~1g/100mL of yeast extract, 1~1.5g/100mL of peptone, 0.5~1.0g/100mL of sodium chloride；Fluid nutrient medium III
In, containing 2~2.5g/100mL of glucose, 0.2~0.5g/100mL of yeast extract, 0.02~0.0.5g/100mL of potassium dihydrogen phosphate；
In the fluid nutrient medium IV, containing 2~2.5g/100mL of glucose, yeast extract 0.2g/100mL, potassium dihydrogen phosphate 0.02g/
100mL；In the fluid nutrient medium V, 2.0g/100mL containing glycerine, 0.5~0.5g/100mL of yeast extract, 1.5~2g/ of corn steep liquor
100mL, 0.02~0.0.5g/100mL of potassium dihydrogen phosphate；In the fluid nutrient medium VI, containing 1~1.5g/100mL of glucose,
0.5~1g/100mL of sodium acetate, 0.5~1g/100mL of yeast extract, 0.5~1g/100mL of ammonium chloride, 0.2~0.5g/ of potassium nitrate
100mL, 0.02~0.05g/100mL of magnesium sulfate, 0.02~0.05g/100mL of dipotassium hydrogen phosphate.
The preparation method of the above-mentioned complex micro organism fungicide for river regulation, it is preferable that the fermentation medium I
In, containing 0.5~5g/100mL of glucose, 0.5~4g/100mL of yeast extract, 1.0~6g/100mL of analysis for soybean powder, potassium dihydrogen phosphate
0.01~0.1g/100mL, 0.01~0.1g/100mL of magnesium sulfate；In the fermentation medium II, containing 0.5~5g/ of glucose
100mL, yeast extract 0.3~3g/100mL, (NH4)2SO40.1~2.0g/100mL, 0.1~1g/100mL of sodium citrate, sulfuric acid
0.01~0.5g/100mL of magnesium；In the fermentation medium III, containing 0.5~5.0g/100mL of molasses, 0.3~3.0g/ of brown sugar
100mL, 0.2~2.0/100mL of yeast extract, 0.5~5.0/100mL of corn steep liquor, 0.01~0.05g/100mL of potassium dihydrogen phosphate,
0.005~0.05g/100mL of magnesium sulfate；In the fermentation medium IV, containing 0.5~5.0g/100mL of molasses, brown sugar 0.5~
5.0g/100mL, 0.5~3.0g/100mL of analysis for soybean powder, 1.0~5.0g/100mL of corn steep liquor, 0.01~0.1g/ of potassium dihydrogen phosphate
100mL；In the fermentation medium V, containing 1.0~5.0g/100mL of glycerine, 1.0~5.0/100mL of molasses, corn steep liquor 1.0~
5.0g/100mL, 0.5~3.0g/100mL of analysis for soybean powder, 0.01~0.1g/100mL of potassium dihydrogen phosphate, 0.01~0.1g/ of magnesium sulfate
100mL；In the fermentation medium VI, containing 1.0~5.0g/100mL of glucose, 0.5~2.0g/100mL of sodium acetate, yeast extract
1.0~5.0g/100mL, 0.5~2.0g/100mL of ammonium chloride, 0.05~0.5g/100mL of potassium nitrate, magnesium sulfate 0.01~
0.1g/100mL, 0.01~0.1g/100mL of dipotassium hydrogen phosphate, 0.1~1.0g/100mL of sodium potassium tartrate tetrahydrate.Preferably, the step
Suddenly in (1.1), the activation condition is：30~36 DEG C, 48~72h of activation culture；It is described expansion condition of culture be：Rotary
Under shaking table 200rpm rotating speeds, 30 DEG C of culture 48h；The fermentation culture conditions are：In rotary shaker 100~180rpm rotating speeds
Under, 30~36 DEG C of 48~96h of culture.
Preferably, in the step (1.2), the activation condition is：30~36 DEG C, 48~72h of activation culture；It is described to expand
Big condition of culture is：Under rotary shaker 200rpm rotating speeds, 30 DEG C of culture 48h；The fermentation culture conditions are：Rotary
Under shaking table 100~180rpm rotating speeds, 30~36 DEG C of 49~720h of culture.
Preferably, in the step (1.3), the activation condition is：32~36 DEG C, 48~72h of activation culture；It is described to expand
Big condition of culture is：Under rotary shaker 160rpm rotating speeds, 30 DEG C of culture 48h；The fermentation culture conditions are：Rotary
Under shaking table 100~180rpm rotating speeds, 30~36 DEG C of 48~72h of culture.
Preferably, in the step (1.4), the activation condition is：32~36 DEG C, 48~72h of activation culture；It is described to expand
Big condition of culture is：Under rotary shaker 150rpm rotating speeds, 37 DEG C of culture 48h；The fermentation culture conditions are：Rotary
Under shaking table 100~200rpm rotating speeds, 32~36 DEG C of 48~120h of culture.
Preferably, in the step (1.5), the activation condition is：32~36 DEG C, 48~72h of activation culture；It is described to expand
Big condition of culture is：Under rotary shaker 150rpm rotating speeds, 37 DEG C of culture 48h；The fermentation culture conditions are：Rotary
Under shaking table 100~200rpm rotating speeds, 30~36 DEG C of 48~120h of culture.
Preferably, in the step (1.6), the activation condition is：32~36 DEG C, 48~72h of activation culture；It is described to expand
Big condition of culture is：Under rotary shaker 150rpm rotating speeds, 32 DEG C of culture 72h；The fermentation culture conditions are：Rotary
Under shaking table 50~100rpm rotating speeds, 30~38 DEG C of 72~120h of culture.
Preferably, in the step (2), the activation condition is：32~36 DEG C, 48~72h of activation culture；It is described to expand
Condition of culture is：Under rotary shaker 150rpm rotating speeds, 35 DEG C of culture 72h；The fermentation culture conditions are：Rotatably shaking
Under bed 30~100rpm rotating speeds, 28~36 DEG C of 120~200h of culture.
As a total inventive concept, the present invention also provides a kind of above-mentioned complex micro organism fungicide or above-mentioned preparation
Application of the complex micro organism fungicide in river regulation prepared by method, comprises the following steps：First by complex micro organism fungicide A
Add, added once every 3~5 days by 1~the 1.5 ‰ of town and country river sewage volume, continuously added 3~5 weeks；Again by micro- life
Thing microbial inoculum B is added by the 1~1.5% of town and country river sewage volume
Compared with prior art, the advantage of the invention is that：
1st, complex micro organism fungicide of the invention, screened by bacillus megaterium CCTCC No.M2012352, withered grass
Bacillus CGMCC No.1.2162, bacillus licheniformis CGMCC No.1.6510, bacillus amyloliquefaciens CGMCC
No.1.7463, Phanerochaete chrysosporium BKM-F1767, Klebsiella oxytoca CCTCC NO.M2011350 individually cultivate shape
Into the complex micro organism fungicide A that mixes of six kinds of zymotic fluids there is strong adaptability the characteristics of, and unrestraint each other
Influence, can quickly form dominant microflora, and ammonia nitrogen and nitrate in different organic matters, degradation water, reduction can be decomposed simultaneously
The growth of phosphorus element concentration and suppression algae and harmful bacteria in water body.Screened by the inferior Dbaly yeast bacterium CGMCC of the Chinese
No.5770, which cultivates the two kinds of zymotic fluids (i.e. microbial bacterial agent B) to be formed, has the characteristics of microbial flocculant yield is high, Han Xunde
Bali saccharomycete CGMCC No.5770 are that applicant screens obtained efficient flocculant generation in disclosed microorganism
Bacterium, can make the aggegations such as solid suspended particle not degradable in water body, somatic cells and colloidal particle, precipitation.Utilize the present invention
Complex micro organism fungicide carry out town and country river sewage in-situ immobilization, with efficient, quick, thorough, non-secondary pollution, operation letter
Singly, the features such as repair rate is high, is conducive to large-scale promotion application.
2nd, complex micro organism fungicide of the invention is compound by first being added in river course in the application that river sewage is handled
Microbial bacterial agent A, decompose in different organic pollutions, the ammonia nitrogen in degradation water and nitrate, reduction water body phosphorus element concentration with
And suppress the growth of algae and harmful bacteria；Again by adding microbial bacterial agent B, the inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese
The characteristic high molecular polymer of generation, i.e. microbial flocculant, can make solid suspended particle not degradable in water body, compound micro-
Bacteria agent A adds the aggegations such as rear prolific somatic cells and colloidal particle, precipitation.Thus at the river sewage of the present invention
Reason method has the advantages that efficiently, thoroughly and non-secondary pollution.
Embodiment
Below in conjunction with specific preferred embodiment, the invention will be further described, but not thereby limiting the invention
Protection domain.
Embodiment 1：
The preparation of the complex micro organism fungicide of town and country river sewage in-situ immobilization
(1) complex micro organism fungicide A is prepared：
(1.1) bacillus megaterium CCTCC No.M2012352 seeds are taken, is inoculated in solid slope culture medium I and carries out
Activation culture, activation condition is：32 DEG C, activation culture 48h；Then by the bacillus megaterium CCTCC of activation
No.M2012352 is inoculated in fluid nutrient medium I and is enlarged culture, expands condition of culture and is：Turn in rotary shaker 200rpm
Under speed, 30 DEG C of culture 48h；The bacillus megaterium CCTCC No.M2012352 of culture be will be enlarged by by the 10% of fermentating liquid volume
It is inoculated in fermentation medium I, under rotary shaker 150rpm rotating speeds, after 32 DEG C of culture 96h, the bacillus megaterium of gained
Bacillus megaterium CCTCC No.M2012352 cell concentrations are 4.0 × 10 in CCTCC No.M2012352 zymotic fluids9cfu/
mL。
In the solid medium I, 1g/100mL containing peptone, dusty yeast 1.5g/100mL, glucose 2g/100mL, phosphorus
Sour hydrogen dipotassium 0.1g/100mL, agar 2g/100mL, pH value 6~8；
In the fluid nutrient medium I, 1.5g/100mL containing glucose, yeast extract 0.5g/100mL, peptone 1g/100mL,
Potassium dihydrogen phosphate 0.05g/100mL, magnesium sulfate 0.04g/100mL；
Described fermentation medium I components are as follows：Glucose 2g/100mL, yeast extract 1.5g/100mL, analysis for soybean powder 2/
100mL, potassium dihydrogen phosphate 0.02g/100mL, magnesium sulfate 0.01g/100mL.
(1.2) Bacillus subtillis CGMCC No.1.2162 seeds are taken, is inoculated in solid slope culture medium II and is lived
Change culture, activation condition is：32 DEG C, activation culture 48h；Then the Bacillus subtillis CGMCC No.1.2162 of activation are connect
Plant and be enlarged culture in fluid nutrient medium II, expanding condition of culture is：Under rotary shaker 200rpm rotating speeds, 30 DEG C of cultures
48h；The Bacillus subtillis CGMCC No.1.2162 that will be enlarged by culture are inoculated in fermentation medium by the 10% of fermentating liquid volume
In II, under rotary shaker 160rpm rotating speeds, after 32 DEG C of culture 96h, the Bacillus subtillis CGMCC No.1.2162 of gained
Bacillus subtillis CGMCC No.1.2162 cell concentrations are 12.0 × 10 in zymotic fluid7cfu/mL；
In the solid medium II, 1.5g/100mL containing peptone, glucose 1g/100mL, sodium chloride 0.5g/
100mL, agar 2g/100mL, pH value 7.0；
In the fluid nutrient medium II, 1.5g/100mL containing glucose, yeast extract 0.5g/100mL, peptone 1g/
100mL, sodium chloride 0.5g/100mL；
Described fermentation medium II components are as follows：Glucose 3g/100mL, yeast extract 1.5g/100mL, (NH4)2SO4
0.5g/100mL, sodium citrate 0.2g/100mL, magnesium sulfate 0.02g/100mL.
(1.3) bacillus licheniformis CGMCC No.1.6510 seeds are taken, is inoculated in solid slope culture medium III and carries out
Activation culture, activation condition is：32 DEG C, activation culture 48h；Then activation bacillus licheniformis is inoculated in fluid nutrient medium
III is enlarged culture, expands condition of culture and is：Under rotary shaker 160rpm rotating speeds, 30 DEG C of culture 48h；It will be enlarged by training
Foster bacillus licheniformis CGMCC No.1.6510 are inoculated in fermentation medium III by the 10% of fermentating liquid volume, in rotation
Under rotatable shaking table 160rpm rotating speeds, after 30 DEG C of culture 72h, in the bacillus licheniformis CGMCC No.1.6510 zymotic fluids of gained
Bacillus licheniformis CGMCC No.1.6510 cell concentrations are 9.0 × 109cfu/mL；
In the solid medium III, 1g/100mL containing tryptone, yeast extract 0.5g/100mL, sodium chloride 1g/
100mL, agar 2g/100mL, pH value 6.5；
In the fluid nutrient medium III, 2.5g/100mL containing glucose, yeast extract 0.2g/100mL, potassium dihydrogen phosphate
0.02g/100mL；
In the fermentation medium III, 1.5g/100mL containing molasses, brown sugar 1.0g/100mL, yeast extract 1.5/100mL,
Corn steep liquor 1.5/100mL, potassium dihydrogen phosphate 0.02g/100mL and magnesium sulfate 0.01g/100mL.
(1.4) bacillus amyloliquefaciens CGMCC No.1.7463 seeds are taken, is inoculated in solid slope culture medium IV and carries out
Activation culture, activation condition is：32 DEG C, activation culture 48h；Then the bacillus amyloliquefaciens of activation are inoculated in Liquid Culture
Base IV is enlarged culture, expands condition of culture and is：Under rotary shaker 150rpm rotating speeds, 37 DEG C of culture 48h；It will be enlarged by training
Foster bacillus amyloliquefaciens CGMCC No.1.7463 are inoculated in fermentation medium IV by the 5% of fermentating liquid volume, in rotation
Under formula shaking table 180rpm rotating speeds, after 37 DEG C of culture 72h, in the bacillus amyloliquefaciens CGMCC No.1.7463 zymotic fluids of gained
Bacillus amyloliquefaciens CGMCC No.1.7463 cell concentrations are 8.0 × 109cfu/mL；
In the solid medium IV, 1g/100mL containing tryptone, yeast extract 0.5g/100mL, sodium chloride 1g/
100mL, agar 2g/100mL, pH value 6.5；
In the fluid nutrient medium IV, 2.5g/100mL containing glucose, yeast extract 0.2g/100mL, potassium dihydrogen phosphate
0.02g/100mL；
Described fermentation medium IV components are as follows：Molasses 2.0g/100mL, brown sugar 1.5g/100mL, analysis for soybean powder 2.0g/
100mL, corn steep liquor 1.5g/100mL, potassium dihydrogen phosphate 0.02g/100mL.
(1.5) Phanerochaete chrysosporium BKM-F1767 seeds are taken, progress activation training in solid slope culture medium V is inoculated in
Support, activation condition is：32 DEG C, activation culture 48h；Then the Phanerochaete chrysosporium of activation is inoculated in into fluid nutrient medium V to enter
Row expands culture, expands condition of culture and is：Under rotary shaker 150rpm rotating speeds, 37 DEG C of culture 48h；It will be enlarged by the Huang of culture
The flat lead fungi BKM-F1767 of archespore hair is inoculated in fermentation medium V by the 10% of fermentating liquid volume, in rotary shaker
Under 160rpm rotating speeds, after 32 DEG C of culture 96h, yellow archespore Mao Pingge in the Phanerochaete chrysosporium BKM-F1767 zymotic fluids of gained
Bacterium BKM-F1767 cell concentrations are 1.0 × 109cfu/mL；
In the solid medium V, 1g/100mL containing peptone, yeast extract 0.5g/100mL, sodium chloride 1g/100mL, fine jade
Fat 2g/100mL, pH value 7；
In the fluid nutrient medium V, 2.0g/100mL containing glycerine, yeast extract 0.5g/100mL, corn steep liquor 1.5g/100mL,
Potassium dihydrogen phosphate 0.02g/100mL；
(1.6) Klebsiella oxytoca CCTCC NO.M2011350 seeds are taken, is inoculated in solid slope culture medium VI and carries out
Activation culture, activation condition is：32 DEG C, activation culture 48h；Then the Klebsiella oxytoca of activation is inoculated in fluid nutrient medium
VI is enlarged culture, expands condition of culture and is：Under rotary shaker 150rpm rotating speeds, 37 DEG C of culture 72h；It will be enlarged by culture
Klebsiella oxytoca CCTCC NO.M2011350 be inoculated in by the 10% of fermentating liquid volume in fermentation medium VI, rotation
Under rotatable shaking table 100rpm rotating speeds, after 32 DEG C of culture 96h, the Klebsiella oxytoca CCTCC NO.M2011350 zymotic fluids of gained
Middle Klebsiella oxytoca CCTCC NO.M2011350 cell concentrations are 9.0 × 108cfu/mL；
In the solid medium VI, 1g/100mL containing glucose, sodium acetate 0.2g/100mL, yeast extract 0.5g/
100mL, ammonium chloride 0.25g/100mL, agar 2g/100mL, pH value 7.0；
In the fluid nutrient medium VI, 1g/100mL containing glucose, sodium acetate 0.5g/100mL, yeast extract 0.5g/
100mL, ammonium chloride 0.5g/100mL, potassium nitrate 0.2g/100mL, magnesium sulfate 0.02g/100mL, dipotassium hydrogen phosphate 0.05g/
100mL；
Described fermentation medium VI components are as follows：Glucose 2.0g/100mL, sodium acetate 1.5g/100mL, yeast extract
1.0g/100mL, ammonium chloride 1.5g/100mL, potassium nitrate 0.2g/100mL, magnesium sulfate 0.03g/100mL, dipotassium hydrogen phosphate
0.02g/100mL, sodium potassium tartrate tetrahydrate 0.2g/100mL.
(1.7) bacillus megaterium CCTCC No.M2012352 zymotic fluids, step (1.2) made from step (1.1) are made
Bacillus subtillis CGMCC No.1.2162 zymotic fluids, bacillus licheniformis made from step (1.3)
Bacillus amyloliquefaciens CGMCC No.1.7463 zymotic fluids, step made from CGMCCNo.1.6510 zymotic fluids, step (1.4)
(1.5) Klebsiella oxytoca prepared by Phanerochaete chrysosporium BKM-F1767 zymotic fluids, step (1.6) made from
After CCTCCNO.M2011350 zymotic fluids are proportionally mixed, complex micro organism fungicide A is obtained.
(2) microbial bacterial agent B is prepared：
The inferior Dbaly yeast bacterium CGMCC No.5770 seeds of the Chinese are taken, is inoculated in solid slope culture medium VI and is activated
Cultivate, activation condition is：32 DEG C, activation culture 48h；Then the inferior Dbaly yeast bacterium of the Chinese will be activated and be inoculated in fluid nutrient medium VI
Culture is enlarged, expanding condition of culture is：Under rotary shaker 150rpm rotating speeds, 35 DEG C of culture 72h；It will be enlarged by culture
The inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese are inoculated in fermentation medium VI, under rotary shaker 50rpm rotating speeds,
After 32 DEG C of culture 150h, the inferior Dbaly yeast bacterium CGMCC No.5770 zymotic fluids of the Chinese that flocculation activity is 80% are made, that is, obtain
Microbial bacterial agent B；
In the solid medium VI, 0.5g/100mL containing beef extract, peptone 0.5g/100mL, yeast extract 0.5g/
100mL, tomato juice 5g/100mL, glucose 1.0g/100mL, calcium bicarbonate 1.0g/100mL, agar 2g/100mL, pH value
4.5；
In the fluid nutrient medium VI, 2.0g/100mL containing glucose, yeast extract 0.5g/100mL, corn steep liquor 1.5g/
100mL, potassium dihydrogen phosphate 0.02g/100mL, calcium bicarbonate 1.0g/100mL；
Described fermentation medium VI components are as follows：Glucose 2.5g/100mL, yeast extract 2.0g/100mL, corn steep liquor
3.0/100mL, potassium dihydrogen phosphate 0.02g/100mL, calcium bicarbonate 1.0g/100mL, citric acid 0.5g/100mL.
Embodiment 2
The application of the complex micro organism fungicide of town and country river sewage in-situ immobilization
, there are city domestic sewage, rural culture plantation sewage discharge in passage county of Hunan Huaihua suburb river course throughout the year, and
Mountain torrents water collects, and river is throughout the year muddy smelly, and COD, ammonia nitrogen and total phosphorus are exceeded.2L water samples are taken from the river course, 2mL embodiments are added
The liquid composite microbial microbial inoculum A of preparation, added microbial inoculum once, continuous dosing is after three weeks, and measurement result shows, water body every three days
COD drops to 14.0mg/L by delivering the 260mg/L before microorganism；Ammonia-nitrogen content is dropped to by preprosthetic 17.2mg/L
0.66mg/L；Total nitrogen drops to 0.91 by 32.4mg/L；Total phosphorus drops to 0.17 by 2.80mg/L；Add 20mL embodiments 1
The liquid microbe microbial inoculum B of preparation, adds water turbidity and colourity after 3 days and is obviously improved, transparency is brought up to by 10cm
22cm, transparency has brought up to 65cm after 15 days.Water body after reparation has reached People's Republic of China's surface water environment quality
Standard V class water standards.It is demonstrated by obvious water remediation effect (see the table below 1).
Repairing effect of the complex micro organism fungicide of table 1 to passage county river sewage
Embodiment 3
The application of the complex micro organism fungicide of town and country river sewage in-situ immobilization
Changde, hunan Shimen County river course, river is throughout the year muddy, and COD, ammonia nitrogen and total phosphorus are exceeded.2L water is taken from the river course
Sample, adds liquid composite microbial microbial inoculum A prepared by 2.5mL embodiments, added microbial inoculum once every four days, continuous dosing three weeks
Afterwards, measurement result shows, water body COD drops to 12.6mg/L by delivering the 245mg/L before microorganism；Ammonia-nitrogen content is before repairing
19.2mg/L drop to 0.62mg/L；Total nitrogen drops to 0.95 by 35.24mg/L；Total phosphorus drops to 0.14 by 3.03mg/L；
The liquid microbe microbial inoculum B of the preparation of 20mL embodiments 1 is added, water turbidity and colourity after 3 days is added and is obviously improved, transparency
27cm is brought up to by 15cm, transparency has brought up to 68cm after 15 days.Water body after reparation has reached the People's Republic of China (PRC)
Water environment quality standard V class water standards.It is demonstrated by obvious water remediation effect (see the table below 2).
Repairing effect of the complex micro organism fungicide of table 2 to Shimen County river sewage
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example.All technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It is noted that for the art
Those of ordinary skill for, improvements and modifications under the premise without departing from the principles of the invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of complex micro organism fungicide for river regulation, it is characterised in that the complex micro organism fungicide includes compound
Microbial bacterial agent A and microbial bacterial agent B, the complex micro organism fungicide A are by bacillus megaterium CCTCC
No.M2012352, Bacillus subtillis CGMCC No.1.2162, bacillus licheniformis CGMCC No.1.6510, solution starch bud
Spore bacillus CGMCC No.1.7463, Phanerochaete chrysosporium BKM-F1767, NO.M2011350 points of Klebsiella oxytoca CCTCC
Six kinds of zymotic fluids that not individually culture is not formed are mixed；The microbial bacterial agent B is by the inferior Dbaly yeast bacterium CGMCC of the Chinese
Two kinds of zymotic fluids that No.5770 and S. cervisiae CGMCC No.3003 individually cultivate formation are mixed.
2. the complex micro organism fungicide according to claim 1 for river regulation, it is characterised in that the composite microbial
In thing microbial inoculum A, the parts by volume of each component is：10~20 parts of bacillus megaterium CCTCC No.M2012352 zymotic fluids, withered grass bud
10~20 parts of born of the same parents bacillus CGMCC No.1.2162 zymotic fluids, bacillus licheniformis CGMCC No.1.6510 zymotic fluids 10~20
Part, 10~20 parts of bacillus amyloliquefaciens CGMCC No.1.7463 zymotic fluids, Phanerochaete chrysosporium BKM-F1767 zymotic fluids
10~20 parts, 10~20 parts of Klebsiella oxytoca CCTCC NO.M2011350 zymotic fluids.
3. the complex micro organism fungicide according to claim 1 or 2 for river regulation, it is characterised in that described compound
In microbial bacterial agent A, total clump count is more than 10.0 × 1010cfu/mL。
4. the complex micro organism fungicide according to claim 1 for river regulation, it is characterised in that the microbial bacteria
In agent B, flocculation activity is more than 70%.
5. a kind of preparation method of complex micro organism fungicide for river regulation as described in any one of Claims 1 to 4, bag
Include following steps：
(1) complex micro organism fungicide A is prepared：
(1.1) bacillus megaterium CCTCC No.M2012352 are inoculated in solid slope culture medium I and activated, is made
Activate bacillus megaterium；Then activation bacillus megaterium is inoculated in fluid nutrient medium I and is enlarged culture；It is inoculated in
In fermentation medium I, fermented culture is made cell concentration and is not less than 3.0 × 109Cfu/mL bacillus megaterium CCTCC
No.M2012352 zymotic fluids；
(1.2) Bacillus subtillis CGMCC No.1.2162 are inoculated in solid slope culture medium II and activated, be made and live
Change Bacillus subtillis；Then activation Bacillus subtillis is inoculated in fluid nutrient medium II and is enlarged culture；It is inoculated in
In fermentation medium II, fermented culture is made cell concentration and is not less than 10.0 × 107Cfu/mL Bacillus subtillis CGMCC
No.1.2162 zymotic fluids；
(1.3) bacillus licheniformis CGMCC No.1.6510 are inoculated in solid slope culture medium III and activated, is made
Activate bacillus licheniformis；Then activation bacillus licheniformis is inoculated in fluid nutrient medium III and is enlarged culture；Inoculate
In fermentation medium III, fermented culture is made cell concentration and is not less than 8.0 × 109Cfu/mL bacillus licheniformis
CGMCC No.1.6510 zymotic fluids；
(1.4) bacillus amyloliquefaciens CGMCC No.1.7463 are inoculated in solid slope culture medium IV and activated, is made
Activate bacillus amyloliquefaciens；Then activation bacillus amyloliquefaciens are inoculated in fluid nutrient medium IV and are enlarged culture；Again
It is inoculated in fermentation medium IV, fermented culture is made cell concentration and is not less than 6.0 × 109Cfu/mL solution starch gemma bar
Bacterium CGMCC No.1.7463 zymotic fluids；
(1.5) Phanerochaete chrysosporium BKM-F1767 is inoculated in solid slope culture medium V and activated, activation is made yellow
The flat lead fungi of archespore hair；Then activation Phanerochaete chrysosporium is inoculated in fluid nutrient medium V and is enlarged culture；It is inoculated in hair
In ferment culture medium V, fermented culture is made cell concentration and is not less than 6.0 × 108Cfu/mL Phanerochaete chrysosporium BKM-
F1767 zymotic fluids；
(1.6) Klebsiella oxytoca CCTCC NO.M2011350 are inoculated in solid slope culture medium VI and activated, is made
Activate Klebsiella oxytoca；Then activation Klebsiella oxytoca is inoculated in fluid nutrient medium VI and is enlarged culture；Inoculate
In fermentation medium VI, fermented culture is made cell concentration and is not less than 5.0 × 108Cfu/mL Klebsiella oxytoca
CCTCC NO.M2011350 zymotic fluids；
(1.7) it is bacillus megaterium CCTCC No.M2012352 zymotic fluids, step (1.2) made from step (1.1) is obtained
Bacillus licheniformis CGMCC No.1.6510 made from Bacillus subtillis CGMCC No.1.2162 zymotic fluids, step (1.3)
Bacillus amyloliquefaciens CGMCC No.1.7463 zymotic fluids made from zymotic fluid, step (1.4), yellow spore made from step (1.5)
Klebsiella oxytoca CCTCC NO.M2011350 zymotic fluids prepared by the flat lead fungi BKM-F1767 zymotic fluids of raw wool, step (1.6)
After proportionally mixing, complex micro organism fungicide A is obtained.
(2) microbial bacterial agent B is prepared：
The inferior Dbaly yeast bacterium CGMCC No.5770 of the Chinese are inoculated in solid slope culture medium VI and activated, activation is made
The inferior Dbaly yeast bacterium of the Chinese；Then the inferior Dbaly yeast bacterium of the Chinese will be activated it is inoculated in fluid nutrient medium VI and is enlarged culture；Again
It is inoculated in fermentation medium VI, the inferior Dbaly yeast bacterium CGMCC of the Chinese that flocculation activity is more than 75% is made in fermented culture
No.5770 zymotic fluids.
6. the preparation method of the complex micro organism fungicide according to claim 5 for river regulation, it is characterised in that institute
State in step (2.1), in the solid medium VI, containing 0.5~1g/100mL of beef extract, 0.5~1g/100mL of peptone, ferment
Female 0.5~1g/100mL of cream, 5~6g/100mL of tomato juice, 0.5~1.0g/100mL of glucose, 0.5~1.0g/ of calcium bicarbonate
100mL, 1~2g/100mL of agar, pH value 4~6.5；In the fluid nutrient medium VI, containing 1.0~2.0g/100mL of glucose,
0.5~1g/100mL of yeast extract, 1~1.5g/100mL of corn steep liquor, 0.01~0.02g/100mL of potassium dihydrogen phosphate, calcium bicarbonate
0.5~1.0g/100mL；In the fermentation medium VI, containing 10~25g/100mL of bean dregs, 1.0~5.0g/100mL of yeast extract,
5.0~10.0/100mL of maize pulp, 0.01~0.1g/100mL of potassium dihydrogen phosphate, 0.5~2.0g/100mL of calcium bicarbonate, lemon
0.2~2.0g/100mL of acid.
7. the preparation method of the complex micro organism fungicide for river regulation according to claim 5 or 6, its feature exists
In in the solid medium I, containing 0.5~1g/100mL of peptone, 1~1.5g/100mL of dusty yeast, 1~2g/ of glucose
100mL, 0.1~0.5g/100mL of dipotassium hydrogen phosphate, 1~2g/100mL of agar, pH value 6~8；In the solid medium II,
Containing 1~1.5g/100mL of peptone, 1~1.5g/100mL of glucose, 0.5~1g/100mL of sodium chloride, 1~2g/ of agar
100mL, pH value 6.5~7.5；In the solid medium III, containing 1~1.5g/100mL of tryptone, 0.5~1g/ of yeast extract
100mL, 1~1.5g/100mL of sodium chloride, 1.5~2g/100mL of agar, pH value 6~8；In the solid medium IV, containing pancreas
1~1.5g/100mL of peptone, 0.5~1g/100mL of yeast extract, 1~1.5g/100mL of sodium chloride, 1.5~2g/ of agar
100mL, pH value 6~8；In the solid medium V, containing 1~1.5g/100mL of peptone, 0.5~1g/100mL of yeast extract, chlorine
Change 0.5~1g/100mL of sodium, 1.5~2g/100mL of agar, pH value 6~8；In the solid medium VI, containing glucose 0.5~
1g/100mL, 0.2~0.5g/100mL of sodium acetate, 0.5~1g/100mL of yeast extract, 0.25~0.5g/100mL of ammonium chloride, fine jade
1~2g/100mL of fat, pH value 6.5~7.5.
8. the preparation method of the complex micro organism fungicide according to claim 7 for river regulation, it is characterised in that institute
State in fluid nutrient medium I, containing 1~1.5g/100mL of glucose, 0.5~1g/100mL of yeast extract, 1~1.5g/ of peptone
100mL, 0.05~0.1g/100mL of potassium dihydrogen phosphate, magnesium sulfate 0.04g/100mL；In the fluid nutrient medium II, containing grape
Sugar 1~1.5g/100mL, 0.5~1g/100mL of yeast extract, 1~1.5g/100mL of peptone, 0.5~1.0g/ of sodium chloride
100mL；In fluid nutrient medium III, containing 2~2.5g/100mL of glucose, 0.2~0.5g/100mL of yeast extract, potassium dihydrogen phosphate
0.02~0.0.5g/100mL；In the fluid nutrient medium IV, containing 2~2.5g/100mL of glucose, yeast extract 0.2g/100mL,
Potassium dihydrogen phosphate 0.02g/100mL；In the fluid nutrient medium V, 2.0g/100mL containing glycerine, 0.5~0.5g/ of yeast extract
100mL, 1.5~2g/100mL of corn steep liquor, 0.02~0.0.5g/100mL of potassium dihydrogen phosphate；In the fluid nutrient medium VI, contain
1~1.5g/100mL of glucose, 0.5~1g/100mL of sodium acetate, 0.5~1g/100mL of yeast extract, 0.5~1g/ of ammonium chloride
100mL, 0.2~0.5g/100mL of potassium nitrate, 0.02~0.05g/100mL of magnesium sulfate, 0.02~0.05g/ of dipotassium hydrogen phosphate
100mL。
9. the preparation method of the complex micro organism fungicide according to claim 8 for river regulation, it is characterised in that institute
State in fermentation medium I, containing 0.5~5g/100mL of glucose, 0.5~4g/100mL of yeast extract, 1.0~6g/ of analysis for soybean powder
100mL, 0.01~0.1g/100mL of potassium dihydrogen phosphate, 0.01~0.1g/100mL of magnesium sulfate；In the fermentation medium II, contain
0.5~5g/100mL of glucose, yeast extract 0.3~3g/100mL, (NH4)2SO40.1~2.0g/100mL, sodium citrate 0.1~
1g/100mL, 0.01~0.5g/100mL of magnesium sulfate；It is red containing 0.5~5.0g/100mL of molasses in the fermentation medium III
Sugar 0.3~3.0g/100mL, 0.2~2.0/100mL of yeast extract, 0.5~5.0/100mL of corn steep liquor, potassium dihydrogen phosphate 0.01~
0.05g/100mL, 0.005~0.05g/100mL of magnesium sulfate；In the fermentation medium IV, containing 0.5~5.0g/ of molasses
100mL, 0.5~5.0g/100mL of brown sugar, 0.5~3.0g/100mL of analysis for soybean powder, 1.0~5.0g/100mL of corn steep liquor, di(2-ethylhexyl)phosphate
0.01~0.1g/100mL of hydrogen potassium；In the fermentation medium V, containing 1.0~5.0g/100mL of glycerine, molasses 1.0~5.0/
100mL, 1.0~5.0g/100mL of corn steep liquor, 0.5~3.0g/100mL of analysis for soybean powder, 0.01~0.1g/100mL of potassium dihydrogen phosphate,
0.01~0.1g/100mL of magnesium sulfate；In the fermentation medium VI, containing 1.0~5.0g/100mL of glucose, sodium acetate 0.5~
2.0g/100mL, 1.0~5.0g/100mL of yeast extract, 0.5~2.0g/100mL of ammonium chloride, 0.05~0.5g/ of potassium nitrate
100mL, 0.01~0.1g/100mL of magnesium sulfate, 0.01~0.1g/100mL of dipotassium hydrogen phosphate, 0.1~1.0g/ of sodium potassium tartrate tetrahydrate
100mL。
10. as described in a kind of complex micro organism fungicide or any one of claim 5~9 as described in any one of Claims 1 to 4
Application of the complex micro organism fungicide in river regulation prepared by preparation method, comprises the following steps：First by complex microorganism
Microbial inoculum A is added by 1~the 1.5 ‰ of town and country river sewage volume, is added once every 3~5 days, is continuously added 3~5 weeks；Again will
Microbial bacterial agent B is added by the 1~1.5% of town and country river sewage volume.
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