CN103013851A - Complex microbial inoculant and applications thereof - Google Patents
Complex microbial inoculant and applications thereof Download PDFInfo
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- CN103013851A CN103013851A CN2012103788027A CN201210378802A CN103013851A CN 103013851 A CN103013851 A CN 103013851A CN 2012103788027 A CN2012103788027 A CN 2012103788027A CN 201210378802 A CN201210378802 A CN 201210378802A CN 103013851 A CN103013851 A CN 103013851A
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- ssal
- composite fungus
- aquae
- fungus agent
- anabaena flos
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Abstract
The invention provides a complex microbial inoculant and applications thereof. The complex microbial inoculant is composed of bacillus SSAL-6 and acinetobacter SSAL-8 through compounding, wherein the biomass rate of the bacillus SSAL-6 to the acinetobacter SSAL-8 is (1-2):(2-1), preferably 1:1. The complex microbial inoculant provided by the invention can effectively degrade anabaena flos-aquae. When the biomass rate of the bacillus SSAL-6 to the acinetobacter SSAL-8 is 1:1, the degradation effect of the complex microbial inoculant on anabaena flos-aquae is increased with the increase of the concentration of the complex microbial inoculant; and when the concentration of the complex microbial inoculant is 15%, the inhibition rate to the chlorophyll a of anabaena flos-aquae can be as high as 96%.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of composite fungus agent and application thereof.
Technical background
Body eutrophication has become the ubiquity environmental problem of puzzlement countries in the world.The amount reproduction of planktonic algae causes the frequent outburst of wawter bloom, has had a strong impact on human life, production and healthy, has caused worldwide environmental disaster, and therefore, the effective way of exploring the generation of control wawter bloom is extremely urgent.At present, administering body eutrophication mainly is to adopt the physics and chemistry method, but these two kinds of methods not only can consume a large amount of financial resources and material resources, and can destroy to a certain extent ecotope.Molten phycomycete is biological as the control of wawter bloom and red tide, day by day is subject to home and abroad environment worker's extensive concern.The research of molten phycomycete is had the history of many decades abroad, since a kind of slime bacteria that colonizes on the bristle algae of Geitler report, the relevant report that molten phycomycete is arranged successively, research emphasis also are transitioned into bacterium from screening and the molten algae characteristic research of single molten phycomycete gradually---the aspects such as algae population ecology and molecular regulation mechanism.At present, domestic research to molten algae bacterium also is in the starting stage, therefore, seeks efficient molten phycomycete to the further investigation of molten phycomycete and uses significant.
Anabaena Flos-aquae is one of main algae kind that causes body eutrophication, it distributes wide, can produce the algae toxin, Direct and indirect damage is human, so up to now, the research that utilizes the microorganism mode to control Anabaena Flos-aquae very is rare, and the molten phycomycete agent that utilizes the molten phycomycete of different algicidal modes to be composited has the characteristics of efficient algicidal effect especially.
Summary of the invention
Purpose of the present invention, for a kind of composite fungus agent is provided, this composite fungus agent can affect growth effect and the photosynthetic pigments of Anabaena Flos-aquae, thereby plays algae-lysing exactly.
Respectively separation screening acquisition from the Anabaena Flos-aquae liquid of yellow of genus bacillus SSAL-6 among the present invention and acinetobacter calcoaceticus SSAL-8, the Classification And Nomenclature of genus bacillus SSAL-6 is genus bacillus, Latin name is Bacillussp.; (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) preservation, preserving number were CGMCC No.6195 at China Committee for Culture Collection of Microorganisms common micro-organisms center on June 6th, 2012.The Classification And Nomenclature of acinetobacter calcoaceticus SSAL-8 is acinetobacter calcoaceticus, Latin name is Acinetobactersp., (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) preservation, preserving number were CGMCC No.6196 at China Committee for Culture Collection of Microorganisms common micro-organisms center on June 6th, 2012.
In order to realize purpose of the present invention, the present invention has adopted following technical scheme:
A kind of composite fungus agent is composited by genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8, and the ratio of the biomass of genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1～2: 2～1.
Above-mentioned composite fungus agent, wherein, the ratio of the biomass of described genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1: 1.
The preparation method of above-mentioned composite fungus agent is, genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 are placed respectively 200mL LB liquid nutrient medium, at 250rpm, 37 ℃ of lower shaking culture 12h, to bacteria suspension OD value be 0.8～1.0, cell concentration is 10
8～10
9During cells/mL, that genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is compound.
The preparation method of above-mentioned composite fungus agent, wherein, the component of described LB liquid nutrient medium and proportioning are: yeast extract, 5g; Tryptones, 10g; NaCl, 10g; Distilled water 1000mL; The pH value of LB liquid nutrient medium is 7.0-7.2.
The application of above-mentioned composite fungus agent is used for the degraded Anabaena Flos-aquae.
The application of above-mentioned composite fungus agent, wherein, when the ratio of the biomass of genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1: 1, increase with composite fungus agent concentration increases composite fungus agent to the degradation effect of Anabaena Flos-aquae, when the concentration of composite fungus agent is 15%, the inhibiting rate of Anabaena Flos-aquae chlorophyll a is reached 96%.
Description of drawings
What Fig. 1 was composite fungus agent on Anabaena Flos-aquae affects transmission electron microscope(TEM) figure;
Fig. 2 is that composite fungus agent is on the synoptic diagram that affects of Anabaena Flos-aquae cell count;
Fig. 3 is that composite fungus agent is on the synoptic diagram that affects of Anabaena Flos-aquae dry weight;
Fig. 4 is that composite fungus agent is on the synoptic diagram that affects of Anabaena Flos-aquae absolution spectroscopy;
Fig. 5 is that composite fungus agent is on the synoptic diagram that affects of Anabaena Flos-aquae chlorophyll a.
Embodiment
Below illustrate the present invention, but the protection domain that is not intended to limit the present invention.
Genus bacillus (Bacillussp.) SSAL-6 and acinetobacter calcoaceticus (Acinetobactersp.) SSAL-8 are placed respectively 200mL LB liquid nutrient medium, 250rpm, 37 ℃ of lower shaking culture 12h are that 0.8～1.0 (cell concentration is 10 to bacteria suspension OD value
8～10
9Cells/mL) time, that genus bacillus (Bacillussp.) SSAL-6 and acinetobacter calcoaceticus (Acinetobactersp.) SSAL-8 is compound.
Anabaena Flos-aquae is cultivated the standard method (national environmental protection section) that suppresses experiment with reference to algal grown, adopts and cultivates without the nitrogen nutrient solution for aquatic No. 111, and pH is 7.5.30 ± 2 ℃ of culture temperature, continuously 24h illumination, intensity of illumination 3000 ± 2001x leaves standstill cultivation, regularly shakes every day 3 times.
Wherein, above-mentioned LB liquid nutrient medium component and proportioning are: yeast extract, 5g; Tryptones, 10g; NaCl, 10g; Distilled water 1000mL; The pH value of LB liquid nutrient medium is 7.0-7.2.Above-mentioned Anabaena Flos-aquae liquid nutrient medium component and proportioning are: dipotassium hydrogen phosphate (K
2HPO
4), 0.075g; Sal epsom (MgSO
4H
2O), 0.125g; Calcium carbonate (CaCO
3), 0.100g; Ironic citrate (1% aqueous solution), 0.5mL; Citric acid (1% aqueous solution), 0.5mL; Molybdic acid (1% aqueous solution), 5; Sodium hydroxide (1% aqueous solution), 1.5mL; Distilled water, 1000mL.
Be 5 * 10 in Anabaena Flos-aquae concentration
4～1 * 10
5Individual/during mL, (cell concentration is about 2 * 10 to add composite fungus agent in the 100mL algae liquid
9Individual/mL, wherein Bacillussp.SSAL-6 and Acinetobactersp.SSAL-8 cell concentration are 10
8-10
9Individual/mL), concentration for the treatment of (v/v) gradient is: 2.5%, 5%, 7.5%, 10%, 12.5% and 15%, and every group of sample established 3 repetitions.The nutrient solution (LB nutrient solution: aquatic No. 111 without the nitrogen nutrient solution) for preparing a series of different proportionings identical from experimental group is respectively applied to cultivate Anabaena Flos-aquae and forms control group.With determination of cell count frond cell quantity and measure the Anabaena Flos-aquae dry weight.Method for cell count: from the inoculation timing, every the 24h sampling, carry out cell counting with counting frame.Learnt from else's experience algae liquid 0.1mL that the ultrasonic disruption instrument smashed in counting frame with pipettor, and at the low power Microscopic observation, magnification 40 * 10 is got five visuals field at random, and the cell count that number goes out to see are got its mean value.N=10 * a * S
Meter/ S
Depending onA represents each visual field inner cell mean value; S
MeterRepresent the counting frame area; S
Depending onRepresent visual field area; N represents number of cells among every mL.Anabaena Flos-aquae dry weight measuring method: get quantitative algae liquid, centrifugal algae adds weighing disk, dries to constant weight at 80 ℃.
The composite fungus agent of different concns on the measurement result of the impact of the cell count of Anabaena Flos-aquae as shown in Figure 2, the result shows: the removal effect of Anabaena Flos-aquae cell and the concentration of composite fungus agent present certain dependency, namely along with the growth of composite fungus agent concentration, stronger to the removal effect of Anabaena Flos-aquae cell.When composite fungus agent concentration is 2.5%, 5%, 7.5%, 10%, 12.5% and 15%, cultivation 24h is respectively 5%, 13%, 24%, 33%, 37% and 46% to the clearance of Anabaena Flos-aquae cell, become respectively 10%, 35%, 54%, 58%, 63% and 70% after cultivating 168h, compared with the control, present significant difference (P＜0.05).
The composite fungus agent of different concns on the measurement result of the impact of the dry weight of Anabaena Flos-aquae as shown in Figure 3, the result shows: the input of pure LB liquid nutrient medium also can exert an influence to the Anabaena Flos-aquae dry weight, when LB liquid nutrient medium concentration is followed successively by 2.5%, 5%, 7.5%, 10%, in the time of 12.5% and 15%, the corresponding increase of Anabaena Flos-aquae dry weight behind the cultivation 168h, be respectively 15.89,16.40,16.77,17.74,18.30, and 19.38mg/mL, and after adding the composite fungus agent of different concns (2.5%～15%), be respectively 14.55,11.11,8.03,6.66,6.90 and 7.11mg/mL.By getting rid of LB liquid nutrient medium itself to Anabaena Flos-aquae affects on the growth factor, as seen, along with composite fungus agent concentration rises to 15% successively from 2.5%, composite fungus agent is followed successively by 8%, 32%, 52%, 63%, 62% and 63% to the inhibiting rate of Anabaena Flos-aquae dry weight.
Embodiment 2 composite fungus agents are on the impact of Anabaena Flos-aquae photosynthetic pigments
With absolution spectroscopy in Bhandari and Sharma method continuous sweep 400～750nm wavelength region, and the Anabaena Flos-aquae Chlorophyll-a Content measured.The extraction and determination of chlorophyll a: the magnificent anabena algae liquid 10mL that fetches water crosses the compound cellulose film of 0.45 μ m, with the film freeze overnight with frustule, in hot water bath, extract 2min with the 8mL hot ethanol rapidly after taking out, behind extraction liquid ultrasonication 5～20min, leave standstill 2～6h in the dark place, get supernatant liquor 3.5mL behind centrifugal (5000r/min, 4 ℃) 5min and place cuvette, survey light absorption value in 665nm and 750nm place, calculate the front optical density value (E of acidifying
665b=Abs
665b-Abs
750b), then drip the 1mol/L hcl acidifying of 200 μ L, survey again light absorption value in wavelength 665nm and 750nm place behind the 5min, calculate the optical density value (E after the acidifying
665a=Abs
665a-Abs
750a).The employing hot ethanol is extraction solvent, A=11.5, and K=2.43, cuvette light path are 1cm.
Wherein, v represents extracting liquid volume (mL), and V represents the volume (L) of sample.
The composite fungus agent of different concns is on the measurement result of the impact of Anabaena Flos-aquae absolution spectroscopy as shown in Figure 4 (it is 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15% that A, B, C, D, E, F, G represent respectively molten phycomycete concentration), the result shows when the composite fungus agent of different concns acts on Anabaena Flos-aquae that its pigment spectral absorption curve variation differs greatly.It is very not obvious that middle and high concentration (＞7.5%) is processed lower spectral absorption curve, and peak value is fuzzy very little everywhere, shows that composite fungus agent has suppressed kind and the content of frond pigment widely.Middle lower concentration (2.5～7.5%) is processed lower, increase along with composite fungus agent concentration, each pigment spectral absorption peak value all has reduction, and this is consistent on the impact of frond cell count and dry weight with composite fungus agent, and spectrogram demonstrates fully the concentration dependency of composite fungus agent.
Chlorophyll a is the captor of luminous energy, also is light conduction person in the chloroplast membranes, so what of Chlorophyll-a Content, fully reflects the power of frond light compositing ability.The composite fungus agent of different concns on the measurement result of the impact of Anabaena Flos-aquae chlorophyll a as shown in Figure 5, the result shows: composite fungus agent strengthens along with the increase of composite fungus agent concentration the restraining effect of chlorophyll a.Composite fungus agent concentration is 2.5%, 5%, 7.5%, 10%, 12.5% and 15%, 24h to the inhibiting rate of chlorophyll a be respectively 9%, 15%, 23%, 30%, 38% and 44%, 168h after rise to successively 76%, 81%, 91%, 95%, 96% and 96%.
The composite fungus agent of the present invention Anabaena Flos-aquae of can effectively degrading provides scientific basis to the control of body eutrophication, for the research of microbial treatment wawter bloom provides important foundation.Under certain condition, the increase with bacteria suspension concentration increases composite fungus agent to the degradation effect of Anabaena Flos-aquae, when the concentration of composite fungus agent is 15%, and can be up to 96% to the inhibiting rate of Anabaena Flos-aquae chlorophyll a.
Claims (8)
1. a bacillus, separation screening obtains from the Anabaena Flos-aquae liquid of yellow, and called after SSAL-6, preserving number are CGMCC No.6195.
2. a strain acinetobacter calcoaceticus, separation screening obtains from the Anabaena Flos-aquae liquid of yellow, and called after SSAL-8, preserving number are CGMCC No.6196.
3. a composite fungus agent is characterized in that, is composited by genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8, and the ratio of the biomass of genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1～2: 2～1.
4. composite fungus agent according to claim 3 is characterized in that, the ratio of the biomass of described genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1: 1.
5. according to claim 3 or the preparation method of 4 described composite fungus agents, it is characterized in that, genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 are placed respectively 200mL LB liquid nutrient medium, at 250rpm, 37 ℃ of lower shaking culture 12h, to bacteria suspension OD value be 0.8～1.0, cell concentration is 10
8～10
9During cells/mL, that genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is compound.
6. the preparation method of composite fungus agent according to claim 5 is characterized in that, the component of described LB liquid nutrient medium and proportioning are: yeast extract, 5g; Tryptones, 10g; NaCl, 10g; Distilled water 1000mL; The pH value of LB liquid nutrient medium is 7.0-7.2.
7. the application of composite fungus agent according to claim 3 is used for the degraded Anabaena Flos-aquae.
8. the application of composite fungus agent according to claim 7, it is characterized in that: when the ratio of the biomass of genus bacillus SSAL-6 and acinetobacter calcoaceticus SSAL-8 is 1: 1, increase with composite fungus agent concentration increases composite fungus agent to the degradation effect of Anabaena Flos-aquae, when the concentration of composite fungus agent is 15%, the inhibiting rate of Anabaena Flos-aquae chlorophyll a is reached 96%.
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| CN104030459A (en) * | 2014-06-24 | 2014-09-10 | 天津科技大学 | Microbial preparation for brackish water aquaculture, and preparation method and application thereof |
| CN104388341A (en) * | 2014-10-29 | 2015-03-04 | 青岛明月蓝海生物科技有限公司 | Bacillus mucilaginosus strain and application thereof in seaweed degradation |
| CN105969699A (en) * | 2016-07-12 | 2016-09-28 | 佛山杰致信息科技有限公司 | Agricultural ecological probiotic composite microbial inoculant |
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| CN102796685A (en) * | 2012-08-10 | 2012-11-28 | 上海交通大学 | Bacillus SSAL-6 and application thereof in degrading anabaena flos-aquae |
| CN102851236A (en) * | 2012-08-01 | 2013-01-02 | 上海交通大学 | Acinetobacter and construction method and application thereof |
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2012
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154162A (en) * | 2010-12-30 | 2011-08-17 | 北京大学 | Bacillus amyloliquefaciens and application thereof |
| CN102181384A (en) * | 2011-02-25 | 2011-09-14 | 中国科学院烟台海岸带研究所 | Acinetobacter calcoaceticus T32 capable of metabolizing furazolidone and application thereof |
| CN102250789A (en) * | 2011-05-31 | 2011-11-23 | 黑龙江大学 | Acinetobacter baumannii capable of efficiently degrading imazamox |
| CN102851236A (en) * | 2012-08-01 | 2013-01-02 | 上海交通大学 | Acinetobacter and construction method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104030459A (en) * | 2014-06-24 | 2014-09-10 | 天津科技大学 | Microbial preparation for brackish water aquaculture, and preparation method and application thereof |
| CN104388341A (en) * | 2014-10-29 | 2015-03-04 | 青岛明月蓝海生物科技有限公司 | Bacillus mucilaginosus strain and application thereof in seaweed degradation |
| CN104388341B (en) * | 2014-10-29 | 2017-06-13 | 青岛明月蓝海生物科技有限公司 | A kind of colloid bacillus cereus bacterial strain and its application in marine alga is degraded |
| CN105969699A (en) * | 2016-07-12 | 2016-09-28 | 佛山杰致信息科技有限公司 | Agricultural ecological probiotic composite microbial inoculant |
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| CN103013851B (en) | 2014-06-25 |
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