CN108751429A - A method of degradation microcystic aeruginosa - Google Patents
A method of degradation microcystic aeruginosa Download PDFInfo
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- CN108751429A CN108751429A CN201810611591.4A CN201810611591A CN108751429A CN 108751429 A CN108751429 A CN 108751429A CN 201810611591 A CN201810611591 A CN 201810611591A CN 108751429 A CN108751429 A CN 108751429A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
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Abstract
The invention discloses a kind of methods of degradation microcystic aeruginosa, including：Degradation bacterium solution is pressed into 0.05-10% additive amounts, it is inoculated into the culture solution of microcystic aeruginosa, co-incubation under conditions of temperature is 20-40 DEG C, pH 5-10, intensity of illumination are 1600-2300lux, wherein, the degradation bacterium solution is made of pseudomonas mendocina (Pseudomonas mendocina), the pseudomonas mendocina is preserved in Guangdong Province's Culture Collection on December 8th, 2017, and biological deposits number is：GDMCC 60297.The present invention is using degradation bacterium solution come microcystic aeruginosa of degrading, and input amount is few, processing time is short, and removal rate is high.
Description
Technical field
The present invention relates to water-treatment technology field more particularly to a kind of methods of degradation microcystic aeruginosa.
Background technology
In recent years, be continuously improved along with rapid development of economy and quality of the life, it is more and more it is full of nutrition, ingredient is multiple
Miscellaneous production and living waste water is discharged into river and lake.These waste water largely containing pollutants such as nitrogen, phosphorus cause to be discharged into water body richness battalion
Fosterization, it is serious to lead to blue algae bloom, the ecological environment of water body is destroyed, not only threatens the existence of aquatile, or even threaten
The health of the mankind is arrived.In recent years, the eutrophication of domestic poisons in freshwater is increasingly severe, some large-size lakes, reservoir
Algae raised growth, serious formation cyanobacterial bloom are found with breeding water body.Microcystic aeruginosa is that China causes cyanobacterial bloom
Main algae, algae toxin can be released in metabolic process, human being's production life is caused to seriously affect.Therefore, it administers blue
Algae wawter bloom is very urgent.
Currently, there are mainly three types of the methods of improvement cyanobacterial bloom：First, physical method, as enclosure fence, direct filtration process,
Artificial and machinery salvaging etc., advantage is the direct algae removed in water body and does not generate secondary pollution, but efficiency is low, expensive, big
Scale operations are difficult, and this method can not be suitable for larger landscape water body；Second is that chemical method, such as adds CuSO4Etc. killing
Algae agent and flocculant, advantage be it is easy to operate, it is rapid-action, but the chemical agent added is to the toxic effect of aquatile, Yi Zao
At secondary pollution；Third, biological method, Species Competition algal control, fish phagocytosis remove algae, microorganism except algae and integrated control are all
For the BIOLOGICAL CONTROL technology of lake eutrophication.Bioanalysis has cheap, efficient, safety, maintenance water ecology balance etc. excellent
Gesture has become the hot spot of body eutrophication Controlling research at present.For water body in large, physics, chemical method are equal
It is unfavorable for implementing, and biological rule is using effect between biology and biological natural propagation, can effectively realize water body in large removes algae
Control algae.
Bacterium and microalgae are most wide respectively, two kinds of biologies that quantity is most in water environment, in a natural environment, bacterium and micro-
Algae maintains the balance of nitrogen in aquatic ecosystem, phosphorus and other nutriments jointly.There are algae-lysing bacteriums in nature, play
The important function of algae bio amount balance is maintained, and balances and is broken when the nutriments such as nitrogen phosphorus are largely exceeded, algae is a large amount of
Breeding, naturally occurring algae-lysing bacterium are difficult to continue to algae bio amount balance.Therefore, isolated efficient alga-lysing bacterium,
Prevention and control cyanobacteria, which is administered, with bacterium has become the hot spot that current cyanobacterial bloom is administered.Currently, having reported multiple has molten algae work(
The bacterium of energy, wherein with bacillus, based on pseudomonad and Bacteroides etc..
Publication No. is that the patent of CN1055861395A discloses a kind of effective composite bacteria agent for inhibiting wawter bloom microcystic aeruginosa
DH-1 and its application.The composite bacteria agent DH-1 of the patent includes the bacterium of pseudomonas, the bacterium of protozoa category, hot unit cell
The bacterium of Pseudomonas, moral walsh this Pseudomonas bacterium and atypical uncommon Bordetella of going out bacterium.The patent passes through the aquatic micro- life of microorganism
State adjusts and the effect of " phycomycete interaction ", inhibits (removal) microcystic aeruginosa to reach.But the patent needs a variety of strains
Synergistic effect, and input amount is big, and processing time needs 8 days or more, and inhibiting rate only has 84.4%.
Invention content
Technical problem to be solved by the present invention lies in provide a kind of method of degradation microcystic aeruginosa, using degradation bacteria
Liquid is come microcystic aeruginosa of degrading, and input amount is few, processing time is short, and removal rate is high.
In order to solve the above technical problem, the present invention provides a kind of methods of degradation microcystic aeruginosa, including：It will degradation
Bacterium solution press 0.05-10% additive amounts, be inoculated into the culture solution of microcystic aeruginosa, temperature be 20-40 DEG C, pH 5-10, light
Co-incubation under conditions of being 1600-2300lux according to intensity, wherein the degradation bacterium solution is by pseudomonas mendocina
(Pseudomonas mendocina) is made, and the pseudomonas mendocina was preserved in the micro- life in Guangdong Province on December 8th, 2017
Object Culture Collection Center, biological deposits number are：GDMCC 60297.
As the improvement of said program, the degradation bacterium solution includes untreated bacterium solution, thalline re-suspension liquid and sterile supernatant.
As the improvement of said program, the additive amount of the degradation bacterium solution is 0.5-10%.
As the improvement of said program, the preparation method of the degradation bacterium solution includes：
(1) pseudomonas mendocina for taking Storage in refrigerator is crossed in nutrient broth solid medium tablets, is in temperature
10-16h is cultivated under conditions of 26-33 DEG C to growing single bacterium colony；
(2) inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth fluid nutrient medium, is 26-33 DEG C, revolution in temperature
It is to cultivate 10-16h under conditions of 150-250rpm to exponential phase, then is forwarded to fresh battalion by the additive amount of 0.5-2%
Meat soup fluid nutrient medium is supported, continues culture to exponential phase by above-mentioned condition, obtains untreated bacterium solution；
(3) untreated bacterium solution is centrifuged, collects the thalline after centrifugation, is resuspended with sterile water, obtains thalline re-suspension liquid；
(4) untreated bacterium solution is centrifuged, collects the supernatant after centrifugation, 0.5 μm of membrane filtration is less than with aperture
Supernatant obtains sterile supernatant.
As the improvement of said program, centrifuge RPMs 8000-15000rpm, temperature is 1-10 DEG C.
As the improvement of said program, the aperture of filter membrane is less than or equal to 0.22 μm.
As the improvement of said program, the preparation method of the culture solution of microcystic aeruginosa, including：Microcystic aeruginosa is pressed into 5-
20% additive amount is inoculated in BG11 culture mediums, is trained under conditions of temperature is 25-35 DEG C, intensity of illumination is 1600-2300lux
It supports to exponential phase, wherein the microcystic aeruginosa is preserved in country of Inst. of Hydrobiology, Chinese Academy of Sciences fresh water algae
Library, biological deposits number are：FACHB-930.
As the improvement of said program, the carbon source of 700-1500mg/L is added in the culture solution of microcystic aeruginosa.
As the improvement of said program, the carbon source be glucose, sodium acetate, trisodium citrate, one kind in starch or
It is several.
As the improvement of said program, the condition of culture for the culture solution that degradation bacterium solution is inoculated into microcystic aeruginosa is：PH is
6-9,25-35 DEG C of temperature.
Implement the present invention, has the advantages that：
A kind of method of degradation microcystic aeruginosa provided by the invention is thrown using degradation bacterium solution come microcystic aeruginosa of degrading
Enter that amount is few, processing time is short, and removal rate is high.Wherein, the present invention prepares drop by cultivating pseudomonas mendocina
Bacterium solution is solved, to improve the drop algae ability of strain.In addition, the present invention is by by pseudomonas mendocina culture to logarithmic growth
Phase so that the metabolite of molten algae reaches peak value, to improve the ability of strain degradation microcystic aeruginosa.
Description of the drawings
Fig. 1 is the algicidal effect curve graph of the embodiment of the present invention 1；
Fig. 2 a be the embodiment of the present invention 1 degrade under 400 power microscopes bacterium solution addition before microcystic aeruginosa microscope inspection
Figure；
Fig. 2 b be the embodiment of the present invention 1 degrade under 400 power microscopes bacterium solution addition after microcystic aeruginosa microscope inspection
Figure；
Fig. 3 is the algicidal effect curve graph of the embodiment of the present invention 2；
Fig. 4 is the algicidal effect block diagram of the embodiment of the present invention 3；
Fig. 5 is the algicidal effect block diagram of the embodiment of the present invention 4；
Fig. 6 is the algicidal effect block diagram of the embodiment of the present invention 5.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
The present invention provides a kind of methods of degradation microcystic aeruginosa, including：Degradation bacterium solution is added by 0.05-10%
Amount, be inoculated into the culture solution of microcystic aeruginosa, temperature be 20-40 DEG C, pH 5-10, intensity of illumination 1600-2300lux
Under conditions of co-incubation, wherein the degradation bacterium solution by pseudomonas mendocina (Pseudomonas mendocina) make
At the pseudomonas mendocina (Pseudomonas mendocina) was preserved in Guangdong Province microorganism on December 8th, 2017
Culture Collection Center, biological deposits number are：GDMCC 60297.It should be noted that the pseudomonas mendocina tool of the present invention
There is efficient algal control function.
It should be noted that temperature, pH value and care intensity have the solute effect and inhibition of microcystic aeruginosa
Important influence.It is prepared since the degradation bacterium solution of the application needs pseudomonas mendocina, and temperature and pH value can doors
Growth, breeding and the metabolism of more Sa pseudomonads.When pH value is less than 5 or is more than 10, pseudomonas mendocina can stop growing,
It is even dead.Preferably, the condition of culture of the degradation bacterium solution culture solution that is inoculated into microcystic aeruginosa is：PH is 6-9, temperature 25-
35℃。
Specifically, the degradation bacterium solution of the present invention includes untreated bacterium solution, thalline re-suspension liquid and sterile supernatant.
Preferably, degradation bacterium solution includes untreated bacterium solution and sterile supernatant, wherein the additive amount for bacterium solution of degrading is
0.05-10%.
Best, degradation bacterium solution includes untreated bacterium solution, wherein the additive amount for bacterium solution of degrading is 10%.
The secretion metabolite for containing a large amount of pseudomonas mendocina in untreated bacterium solution and sterile supernatant, has
Preferable algae-lysing.And thalline re-suspension liquid only has individual pseudomonas mendocina cell, because of life under no carbon source environment
Long unobvious, and algicidal effect is not notable；And under carbon source environment, pseudomonas mendocina growth secretion algicidal substances, and it is same
Sample has algal control and algicidal effect.Therefore, can be added in the culture solution of microcystic aeruginosa can provide CODcr 700-
The carbon source of 1500mg/L.Preferably, carbon source is one or more of glucose, sodium acetate, trisodium citrate, starch.
The preparation method of the untreated bacterium solution includes：
(1) pseudomonas mendocina for taking Storage in refrigerator is crossed in nutrient broth solid medium tablets, is in temperature
10-16h is cultivated under conditions of 26-33 DEG C to growing single bacterium colony；
(2) inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth fluid nutrient medium, is 26-33 DEG C, revolution in temperature
It is to cultivate 10-16h under conditions of 150-250rpm to exponential phase, then is forwarded to fresh battalion by the additive amount of 0.5-2%
Meat soup fluid nutrient medium is supported, continues culture to exponential phase by above-mentioned condition, obtains untreated bacterium solution；
(3) untreated bacterium solution is centrifuged, collects the thalline after centrifugation, is resuspended with sterile water, obtains thalline re-suspension liquid；
(4) supernatant after centrifugation is collected, 0.5 μm of membrane filtration supernatant is less than with aperture, obtains sterile supernatant.
Preferably, the pseudomonas mendocina for taking -90 DEG C to -60 DEG C Storage in refrigerator is flat in nutrient broth solid medium
Lining out, culture 12-14h is to growing single bacterium colony under conditions of temperature is 28-31 DEG C；It is inoculated with pseudomonas mendocina single bacterium
Nutrient broth fluid nutrient medium is dropped down onto, 12-14h is cultivated under conditions of temperature is 28-31 DEG C, revolution is 180-220rpm to right
Number growth period, then fresh nutrient broth fluid nutrient medium is forwarded to by the additive amount of 0.5-2%, continue to cultivate by above-mentioned condition
To exponential phase, untreated bacterium solution is obtained；The thalline after centrifugation is collected, is resuspended with sterile water, obtains thalline re-suspension liquid；It will be upper
The supernatant after centrifugation is stated, the membrane filtration degerming of 0.5um is less than with aperture, obtains sterile supernatant.
Preferably, the pseudomonas mendocina for taking -85 DEG C to -75 DEG C Storage in refrigerator is flat in nutrient broth solid medium
Lining out, culture 12-14h is to growing single bacterium colony under conditions of temperature is 28-31 DEG C；It is inoculated with pseudomonas mendocina single bacterium
Nutrient broth fluid nutrient medium is dropped down onto, 12-14h is cultivated under conditions of temperature is 28-31 DEG C, revolution is 180-220rpm to right
Number growth period, then fresh nutrient broth fluid nutrient medium is forwarded to by the additive amount of 0.5-2%, continue to cultivate by above-mentioned condition
To exponential phase, untreated bacterium solution is obtained；Above-mentioned untreated bacterium solution is centrifuged, centrifuge RPMs 8000-15000rpm,
Temperature is 1-10 DEG C, is resuspended with sterile water after collecting thalline, obtains thalline re-suspension liquid；By the supernatant after above-mentioned centrifugation, hole is used
Diameter is less than 0.3 μm of membrane filtration degerming, obtains sterile supernatant.
More preferably, the pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator, in nutrient broth solid medium tablets strokes
Line, culture 14h is to growing single bacterium colony under conditions of temperature is 30 DEG C；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth
Fluid nutrient medium cultivates 14h to exponential phase, then by 0.5-2%'s under conditions of temperature is 30 DEG C, revolution is 200rpm
Additive amount is forwarded to fresh nutrient broth fluid nutrient medium, continues culture to exponential phase by above-mentioned condition, is not located
Manage bacterium solution；Above-mentioned untreated bacterium solution is centrifuged, centrifuge RPMs 10000rpm, temperature is 4 DEG C, with sterile after collection thalline
Water is resuspended, and obtains thalline re-suspension liquid；By the supernatant after above-mentioned centrifugation, the membrane filtration degerming for being 0.22 μm with aperture obtains nothing
Bacterium supernatant.
Since pseudomonas mendocina mainly secretes the molten algae of metabolite by indirectly-acting, individually by Mendoza vacation unit cell
Bacterium be added BG11 culture mediums in, due to the culture medium lack carbon source thalline can not growth metabolism, can not Secretion answer metabolite
Carry out effectively molten algae.Therefore the present invention prepares degradation bacterium solution, to improve bacterium by cultivating pseudomonas mendocina
The drop algae ability of kind.In addition, the present invention is by by pseudomonas mendocina culture to exponential phase so that the metabolism of molten algae is produced
Object reaches peak value, to improve the ability of strain degradation microcystic aeruginosa.In addition, by the way that carbon source is added in the medium, can promote
Into the growth of pseudomonas mendocina, to improve the molten algae ability of strain.
Therefore, through the invention, no matter pseudomonas mendocina is in the form of which kind of bacterium solution, all can get preferable molten algae effect
Fruit.
The raised growth of phytoplankton is the principal phenomena of body eutrophication, and wherein chlorophyll a is all phytoplanktons
The chlorophyll type that class all contains.Wherein, chlorophyll a serves not only as the important indicator of water nutrition state division, Er Qieke
Standing crop for characterizing phytoplankton.Therefore, the present invention characterizes the amount of frustule in water body using Chlorophyll-a Content.Leaf
The detection method of green element a is as follows：
Glass fiber filter is placed on the nutsch filter for being connected with vacuum pump, it is fixed accurately to be measured according to water sample chlorophyll concentration
The water sample of amount volume is filtered.Filtered filter membrane is put into tool plug glass centrifuge tube, it is molten that 90% acetone of 10mL is added
Liquid covers tightly cock cap and acutely vibrates a moment, is placed in 4 DEG C of refrigerators and is protected from light and impregnate 2h, oscillation 3 times is needed in soaking process.Centrifuge tube is existed
Revolution is 3500rpm, temperature centrifuges 15min under conditions of being 4 DEG C.The supernatant after centrifugation is taken to pour into 1cm cuvettes, with
90% acetone does reference, measures light absorption value at 630nm, 647nm, 664nm and 750nm wavelength respectively.
Calculation formula is as follows：
ρ Chl-a=[11.85 (A664-A750) -1.54 (A647-A750) -0.08 (A630-A750)] V1/V2L；
Molten algae rate=(ρ 1- ρ 2)/ρ 1 × 100%；
Algal control rate=(ρ 3- ρ 2)/ρ 3 × 100%.
In formula：
The mass concentration of ρ Chl-a-chlorophyll a, μ g/L；
Absorbance value of the A630-extracting solution at wavelength 630；
Absorbance value of the A647-extracting solution at wavelength 647；
Absorbance value of the A664-extracting solution at wavelength 664；
Absorbance value of the A750-extracting solution at wavelength 750；
V1-extracting liquid volume, 10mL；
V2-volume of water sample, L；
L-cuvette light path, 1cm；
1-samples of ρ originate Chlorophyll-a Content, μ g/L；
Chlorophyll-a Content on the day of 2-samples of ρ, μ g/L；
ρ 3-control same day Chlorophyll-a Contents, μ g/L.
Specifically, the preparation method of the culture solution of microcystic aeruginosa, including：
Microcystic aeruginosa is pressed into 5-20% additive amounts, is inoculated in BG11 culture mediums, is 25-35 DEG C, intensity of illumination in temperature
To be cultivated 3-5 days under conditions of 1600-2300lux, wherein the microcystic aeruginosa is preserved in Chinese Academy of Sciences aquatile and grinds
Study carefully institute's national fresh water algae library, biological deposits number is：FACHB-930.
Preferably, microcystic aeruginosa is pressed into 5-20% additive amounts, is inoculated in BG11 culture mediums, be 28-32 DEG C, light in temperature
It is cultivated 3 days under conditions of being 1800-2100lux according to intensity.
More preferably, microcystic aeruginosa is pressed into 5-20% additive amounts, is inoculated in BG11 culture mediums, be 30 DEG C, illumination in temperature
Intensity is cultivated 4 days under conditions of being 2000lux.
Nutrient broth trains the preparation method that liquid supports base, including：Weigh peptone 10g, extracted beef powder 3g and sodium chloride
5g adjusts pH to 7.2 after 1000mL distilled water stirring and dissolvings are added, 121 DEG C of sterilizing 20min in high-pressure sterilizing pot.
Nutrient broth trains the preparation method that solid supports base, including：Weigh peptone 10g, extracted beef powder 3g, sodium chloride 5g
With agar powder 20g, pH to 7.2 is adjusted after 1000mL distilled water stirring and dissolvings are added, 121 DEG C of sterilizing 20min in high-pressure sterilizing pot.
The preparation method of BG11 culture mediums, including：Weigh NaNO3 1.5g、K2HPO4 0.04g、MgSO4·7H2O
0.075g、CaCl2·2H2O 0.036g, citric acid 0.006g, ferric citrate 0.006g, EDTA-Na2 0.001g、NaCO3
0.02g and trace element A5 solution 1mL adjust pH to 7.1 after 1000mL distilled water stirring and dissolvings are added, go out in high pressure in beaker
121 DEG C of sterilizing 20min in bacterium pot.
Wherein, the preparation method of micro- A5 solution, including：Weigh boric acid 2.86g, MnCl2·4H2O1.86g,
ZnSO4·7H2O 0.22g, Na2MoO4·2H2O 0.39g, CuSO4·5H2O 0.08g, Co (NO3)2·6H2O 0.05g, add
Enter 1000mL distilled water, stirring and dissolving.
The present invention is illustrated with specific embodiment below
Embodiment 1
1, degradation bacterium solution is prepared
The pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator is crossed in nutrient broth solid medium tablets, in temperature
12h is cultivated under conditions of being 30 DEG C to growing single bacterium colony；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth Liquid Culture
Base is cultivated 16h to exponential phase under the conditions of temperature is 30 DEG C, revolution is 200rpm, then is forwarded to by 1% additive amount fresh
Nutrient broth fluid nutrient medium, under conditions of temperature is 30 DEG C, revolution is 200rpm cultivate 12h to exponential phase, obtain
It is spare to untreated bacterium solution.
2, the culture solution of microcystic aeruginosa is prepared
Microcystic aeruginosa is inoculated in BG11 culture mediums by 10% additive amount, temperature is 30 DEG C, intensity of illumination is
It is cultivated 3 days under the conditions of 2000lux, it is 586.4 μ g/L to measure culture medium Determination of Chlorophyll a contents, is diluted to culture medium so that
The content of culture medium Determination of Chlorophyll a is 100 μ g/L, pH 7.
3, microcystic aeruginosa is dissolved using degradation bacterium solution
Above-mentioned untreated bacterium solution is added to 0.05%, 0.5% and 10% additive amount the culture solution of microcystic aeruginosa respectively
In, and be arranged and do not add the blank assay group of degradation bacterium solution as a contrast, temperature is 30 DEG C, intensity of illumination is 2000lux items
It is cultivated 5 days under part, Light To Dark Ratio 12:12, every the content of sampling sample Determination of Chlorophyll a for 24 hours, and do microscope inspection.As a result
As depicted in figs. 1 and 2.
Fig. 1 shows that when the additive amount for bacterium solution of degrading is 10% and 0.5%, pseudomonas mendocina has preferable molten algae effect
Fruit has dissolved 69.5% and 41.0% microcystic aeruginosa respectively after adding five days.When being added to 0.05%, algicidal effect is not
Significantly, microcystic aeruginosa remains to comparatively fast grow in shaking flask, but compares blank group, still has certain algal control effect.Compared to sky
It is white to compare, there is effect of algae restraint to press down after five days when wherein additive amount is 10%, 0.5% and 0.05% under the conditions of three kinds of additive amounts
Algae rate is respectively 98.5%, 97% and 35.1%.Fig. 2 and Fig. 2 b are microscope inspection figure, before wherein Fig. 2 a is addition degradation bacterium solutions,
Fig. 2 b are after adding degradation bacterium solution., it is evident that Microcystis aeruginosa Strains are transparent by green bleach after addition degradation bacterium solution, carefully
Born of the same parents are destructurized.
Embodiment 2
1, degradation bacterium solution is prepared
The pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator is crossed in nutrient broth solid medium tablets, in temperature
12h is cultivated under conditions of being 30 DEG C to growing single bacterium colony；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth Liquid Culture
Base is cultivated 16h to exponential phase under the conditions of temperature is 30 DEG C, revolution is 200rpm, then is forwarded to by 1% additive amount fresh
Nutrient broth fluid nutrient medium, under conditions of temperature is 30 DEG C, revolution is 200rpm cultivate 12h to exponential phase, obtain
It is spare to untreated bacterium solution；
Above-mentioned untreated bacterium solution is centrifuged, centrifuge RPMs 10000rpm, temperature is 4 DEG C, and nothing is used after collecting thalline
Bacterium water is resuspended, and obtains thalline re-suspension liquid；
By the supernatant after above-mentioned centrifugation, the membrane filtration degerming for being 0.22um with aperture obtains sterile supernatant.
2, the culture solution of microcystic aeruginosa is prepared
Microcystic aeruginosa is inoculated in BG11 culture mediums by 10% additive amount, temperature is 30 DEG C, intensity of illumination is
It is cultivated 5 days under the conditions of 2000lux, it is 1683.9 μ g/L to measure culture medium Determination of Chlorophyll a contents, is diluted, makes to culture medium
The content for obtaining culture medium Determination of Chlorophyll a is 400 μ g/L, pH 6.
3, microcystic aeruginosa is dissolved using degradation bacterium solution
Untreated bacterium solution, resuspension thalline and sterile supernatant are added to the training of microcystic aeruginosa respectively by 0.5% additive amount
In nutrient solution, and it is arranged and does not add the blank assay group of degradation bacterium solution as a contrast, temperature is 30 DEG C, intensity of illumination is
It is cultivated 5 days under the conditions of 2000lux, Light To Dark Ratio 12:12, every the content of sampling sample Determination of Chlorophyll a for 24 hours, as a result such as Fig. 3
It is shown.
Untreated bacterium solution and sterile supernatant all have preferable algae-lysing, and after adding 5 days, molten algae rate reaches respectively
54.7% and 45.2%, algal control rate is respectively 92.5% and 91.1%.And pseudomonas mendocina cell is individually added, molten algae effect
Fruit is not notable, and algal control rate is 51.5%.Thus it proves, pseudomonas mendocina relies primarily on secretion metabolite and reaches molten algae effect
Fruit.And individually pseudomonas mendocina is added in BG11 culture mediums, it can not grow generation since the culture medium lacks carbon source thalline
Thank, can not Secretion answer metabolite to carry out effectively molten algae, but it is suitable pseudomonas mendocina can be added in BG11 culture mediums
Carbon source solve, wherein carbon source may be selected：One or more of glucose, sodium acetate, trisodium citrate, starch.
Embodiment 3
1, degradation bacterium solution is prepared
The pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator is crossed in nutrient broth solid medium tablets, in temperature
12h is cultivated under conditions of being 30 DEG C to growing single bacterium colony；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth Liquid Culture
Base is cultivated 16h to exponential phase under the conditions of temperature is 30 DEG C, revolution is 200rpm, then is forwarded to by 1% inoculum concentration fresh
Nutrient broth fluid nutrient medium, under conditions of temperature is 30 DEG C, revolution is 200rpm cultivate 12h to exponential phase, obtain
It is spare to untreated bacterium solution.
2, the culture solution of microcystic aeruginosa is prepared
Microcystic aeruginosa is inoculated in BG11 culture mediums by 10% additive amount, temperature is 30 DEG C, intensity of illumination is
It is cultivated 4 days under the conditions of 2000lux, it is 1182.4 μ g/L to measure culture medium Chlorophyll-a Content, is diluted to culture medium so that
The content of culture medium Determination of Chlorophyll a is 200 μ g/L, pH 9.
3, microcystic aeruginosa is dissolved using degradation bacterium solution
Above-mentioned untreated bacterium solution is added in the culture solution of microcystic aeruginosa by 0.5% additive amount, setting does not add drop
Solve bacterium solution blank assay group as a contrast, intensity of illumination 2000lux, Light To Dark Ratio 12:12, respectively temperature be 25 DEG C,
It is cultivated 5 days under the conditions of 30 DEG C and 35 DEG C, every the content of sampling sample Determination of Chlorophyll a for 24 hours, the results are shown in Figure 4.
Under conditions of adding 0.5% degradation bacterium solution to microcystic aeruginosa culture solution, cultivation temperature is 25 DEG C, 30 DEG C and 35 DEG C
The efficient molten algae effect of algae restraint of Shi Douyou.When cultivation temperature is 25 DEG C, molten algae rate and algal control rate are respectively 46.2% He
94.6%；When cultivation temperature is 30 DEG C, molten algae rate and algal control rate are respectively 50.6% and 92.5%；When cultivation temperature is 35 DEG C
When, molten algae rate and algal control rate are respectively 41.3% and 91.2%.
Embodiment 4
1, degradation bacterium solution is prepared
The pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator is crossed in nutrient broth solid medium tablets, in temperature
12h is cultivated under conditions of being 30 DEG C to growing single bacterium colony；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth Liquid Culture
Base is cultivated 16h to exponential phase under the conditions of temperature is 30 DEG C, revolution is 200rpm, then is forwarded to by 1% inoculum concentration fresh
Nutrient broth fluid nutrient medium, under conditions of temperature is 30 DEG C, revolution is 200rpm cultivate 12h to exponential phase, obtain
It is spare to untreated bacterium solution.
2, the culture solution of microcystic aeruginosa is prepared
Microcystic aeruginosa is inoculated in BG11 culture mediums by 10% additive amount, temperature is 30 DEG C, intensity of illumination is
It is cultivated 4 days under the conditions of 2000lux, it is 1072.1 μ g/L to measure culture medium Chlorophyll-a Content, is diluted to culture medium so that
The content of culture medium Determination of Chlorophyll a is 300 μ g/L, respectively so that the pH of culture solution is 6,7,8 and 9.
3, microcystic aeruginosa is dissolved using degradation bacterium solution
Above-mentioned untreated bacterium solution is added in the culture solution of microcystic aeruginosa by 0.5% additive amount, setting does not add drop
Solve bacterium solution blank assay group as a contrast, intensity of illumination 2000lux, Light To Dark Ratio 12:12, under the conditions of temperature is 30 DEG C
Culture 5 days, every the content of sampling sample Determination of Chlorophyll a for 24 hours, the results are shown in Figure 5.
Under conditions of adding 0.5% degradation bacterium solution to microcystic aeruginosa culture solution, the culture solution pH of microcystic aeruginosa is 6,
7,8 and 9 when have efficient molten algae effect of algae restraint.When culture solution pH is 6, molten algae rate and algal control rate are respectively 42.5% He
92.8%；When culture solution pH is 7, molten algae rate and algal control rate are respectively 49.8% and 96.8%；It is molten when culture solution pH is 8
Algae rate and algal control rate are respectively 45.2% and 94.1%；When culture solution pH is 9, molten algae rate and algal control rate are respectively 42.9% He
93.4%.
Embodiment 5
1, degradation bacterium solution is prepared
The pseudomonas mendocina for taking -80 DEG C of Storage in refrigerator is crossed in nutrient broth solid medium tablets, in temperature
12h is cultivated under conditions of being 30 DEG C to growing single bacterium colony；Inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth Liquid Culture
Base is cultivated 16h to exponential phase under the conditions of temperature is 30 DEG C, revolution is 200rpm, then is forwarded to by 1% inoculum concentration fresh
Nutrient broth fluid nutrient medium, under conditions of temperature is 30 DEG C, revolution is 200rpm cultivate 12h to exponential phase, obtain
It is spare to untreated bacterium solution.
2, the culture solution of microcystic aeruginosa is prepared
Microcystic aeruginosa is inoculated in BG11 culture mediums by 10% additive amount, temperature is 30 DEG C, intensity of illumination is
It is cultivated 4 days under the conditions of 2000lux, it is 1072.1 μ g/L to measure culture medium Chlorophyll-a Content, is diluted to culture medium so that
The content of culture medium Determination of Chlorophyll a is respectively 100,200,300 and 400 μ g/L, pH 8.
3, microcystic aeruginosa is dissolved using degradation bacterium solution
Above-mentioned untreated bacterium solution is added in the culture solution of microcystic aeruginosa by 0.5% additive amount, setting does not add drop
Solve bacterium solution blank assay group as a contrast, intensity of illumination 2000lux, Light To Dark Ratio 12:12, under the conditions of temperature is 30 DEG C
Culture 5 days, every the content of sampling sample Determination of Chlorophyll a for 24 hours, the results are shown in Figure 6.
Under conditions of adding 0.5% degradation bacterium solution to microcystic aeruginosa culture solution, starting Chlorophyll-a Content be 100,200,
There is efficient molten algae effect of algae restraint when 300 and 400 μ g/L.When it is 100 μ g/L to originate Chlorophyll-a Content, molten algae rate and suppression
Algae rate is respectively 40.9% and 91.2%；When it is 200 μ g/L to originate Chlorophyll-a Content, molten algae rate and algal control rate are respectively
43.6% and 92.8%；When it is 300 μ g/L to originate Chlorophyll-a Content, molten algae rate and algal control rate are respectively 53.6% He
95.6%；When it is 400 μ g/L to originate Chlorophyll-a Content, molten algae rate and algal control rate are respectively 47.9% and 94.3%.
Algicidal effect obtained by Statistics Implementation example 1-5, when pseudomonas mendocina bacterium solution dosage >=0.5%, all implementations
Example be added degradation bacterium solution after 5 days frustule Chlorophyll-a Content reduce by 90% or more compared to the control group, compared to be added degradation bacterium solution before
Reduce by 40% or more.From the foregoing, it will be observed that the degradation bacterium solution of the present invention can be achieved under the conditions of different starting concentration of algae, temperature and pH
Efficient alga-lysing.
It is above disclosed to be only a preferred embodiment of the present invention, the power of the present invention cannot be limited with this certainly
Sharp range, therefore equivalent changes made in accordance with the claims of the present invention, are still within the scope of the present invention.
Claims (10)
1. a kind of method of degradation microcystic aeruginosa, which is characterized in that including：Degradation bacterium solution is pressed into 0.05-10% additive amounts, is connect
In kind to the culture solution of microcystic aeruginosa, in the item that temperature is 20-40 DEG C, pH 5-10, intensity of illumination are 1600-2300lux
Co-incubation under part, wherein the degradation bacterium solution is made of pseudomonas mendocina (Pseudomonas mendocina), institute
It states pseudomonas mendocina and is preserved in Guangdong Province's Culture Collection on December 8th, 2017, biological deposits number is：
GDMCC 60297。
2. the method for degradation microcystic aeruginosa as described in claim 1, which is characterized in that the degradation bacterium solution includes untreated
Bacterium solution, thalline re-suspension liquid and sterile supernatant.
3. as claimed in claim 2 degradation microcystic aeruginosa method, which is characterized in that it is described degradation bacterium solution additive amount be
0.5-10%.
4. the method for degradation microcystic aeruginosa as claimed in claim 2, which is characterized in that the preparation method of the degradation bacterium solution
Including：
(1) pseudomonas mendocina for taking Storage in refrigerator is crossed in nutrient broth solid medium tablets, is 26- in temperature
10-16h is cultivated under conditions of 33 DEG C to growing single bacterium colony；
(2) inoculation pseudomonas mendocina single bacterium drops down onto nutrient broth fluid nutrient medium, temperature is 26-33 DEG C, revolution is
10-16h is cultivated under conditions of 150-250rpm to exponential phase, then fresh nutrition is forwarded to by the additive amount of 0.5-2%
Meat soup fluid nutrient medium continues culture to exponential phase by above-mentioned condition, obtains untreated bacterium solution；
(3) untreated bacterium solution is centrifuged, collects the thalline after centrifugation, is resuspended with sterile water, obtains thalline re-suspension liquid；
(4) untreated bacterium solution is centrifuged, collects the supernatant after centrifugation, 0.5 μm of membrane filtration supernatant is less than with aperture
Liquid obtains sterile supernatant.
5. the method for degradation microcystic aeruginosa as claimed in claim 4, which is characterized in that centrifuge RPMs 8000-
15000rpm, temperature are 1-10 DEG C.
6. the method for degradation microcystic aeruginosa as claimed in claim 4, which is characterized in that the aperture of filter membrane is less than 0.3 μm.
7. the method for degradation microcystic aeruginosa as described in claim 1, which is characterized in that the system of the culture solution of microcystic aeruginosa
Preparation Method, including：Microcystic aeruginosa is pressed into 5-20% additive amounts, is inoculated in BG11 culture mediums, is 25-35 DEG C, illumination in temperature
Intensity is cultivated under conditions of being 1600-2300lux to exponential phase, wherein the microcystic aeruginosa is preserved in Chinese science
Country of aquatile research institute of institute fresh water algae library, biological deposits number are：FACHB-930.
8. the method for degradation microcystic aeruginosa as described in claim 1, which is characterized in that in the culture solution of microcystic aeruginosa
The carbon source of 700-1500mg/L is added.
9. the method for degradation microcystic aeruginosa as claimed in claim 8, which is characterized in that the carbon source is glucose, acetic acid
One or more of sodium, trisodium citrate, starch.
10. the method for degradation microcystic aeruginosa as described in claim 1, which is characterized in that it is micro- that degradation bacterium solution is inoculated into verdigris
The condition of culture of the culture solution of capsule algae is：PH is 6-9,25-35 DEG C of temperature.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109749969A (en) * | 2019-03-04 | 2019-05-14 | 河海大学 | A kind of preparation method and algal control application of pseudomonas microbial |
| CN113583906A (en) * | 2021-07-21 | 2021-11-02 | 首都师范大学 | Application of pseudomonas B5 in algae removal |
-
2018
- 2018-06-14 CN CN201810611591.4A patent/CN108751429A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 史顺玉: "溶藻细菌对藻类的生理生态效应及作用机理研究", 《中国博士学位论文全文数据库 工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109749969A (en) * | 2019-03-04 | 2019-05-14 | 河海大学 | A kind of preparation method and algal control application of pseudomonas microbial |
| CN113583906A (en) * | 2021-07-21 | 2021-11-02 | 首都师范大学 | Application of pseudomonas B5 in algae removal |
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