CN102888354B - Lysinibacillusfusiformis and method for degrading microcystis aeruginosa by using lysinibacillusfusiformis - Google Patents
Lysinibacillusfusiformis and method for degrading microcystis aeruginosa by using lysinibacillusfusiformis Download PDFInfo
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Abstract
The invention discloses lysinibacillusfusiformis and a method for degrading microcystis aeruginosa by using lysinibacillusfusiformis, belongs to the technical field of environment friendliness and water treatment, provides algicidal bacteria TL which is suggested to be classified and named ysinibacillusfusiformis with the collection number CGMCC NO.6108, and also provides a method for degrading microcystis aeruginosa by using lysinibacillusfusiformis. The characteristics of the separated algae-lysing bacteria lysinibacillusfusiformis are exerted by a mode of indirectly dissolving algae by secreting extracellular non-protease algae-killing substances and a mode of directly dissolving algae by contacting algae cells, and the lysinibacillusfusiformis has high degradation rate of microcystis aeruginosa, and has wide application prospect in the aspect of preventing cyanobacterial bloom in which microcystis aeruginosa is used as preponderant algae by a microbiological method.
Description
Technical field
The invention belongs to environment-protective water processing technology field, be specifically related to a kind of 1 strain Methionin genus bacillus filtering out and utilize its efficient method of removing microcystic aeruginosa from yellow microcystic aeruginosa liquid.
Background technology
Progress along with modern science and technology, industry and agricultural automation are fast-developing, resident living level improves constantly, river or lake near the production of a large amount of rich Nitrogen-and Phosphorus-containings, sanitary wastewater enter continuously, increased the loads of nutrition of water body, cause Eutrophic Extent to increasingly sharpen, blue-green alga bloom frequently breaks out.Water surface cannot cause reoxygenation rate to reduce with extraneous exchange of air because algae covers, dissolved oxygen is rare, water quality stench, water transparency declines, the hydrobiont such as planktonic organism and fish and shrimp is dead because living environment worsens, biomass and kind reduce, and water body species diversity structure is changed.And after rotting, the algae that quivers in Cyanophyta, nostoc, joint ball algae, microcystis kutz etc. constantly to water body, discharge the larger algae toxin of toxicity, Microcystin (MCs) toxicity wherein producing with Microcystis aeruginosa is maximum, with solvability state, exist for a long time, be difficult for degraded, can be by food chain enrichment in water body organism, serious threat human life is healthy.Lake eutrophication and blue-green alga bloom are administered has become a global problem, a large amount of human and material resources are all dropped in the lakes such as the Dian Chi of China, Taihu Lake, Chaohu every year, financial resources are controlled or administer blue-green alga bloom, but produce little effect, Lake Eutrophication is not obviously changed.
In recent years, large quantity research shows, the unexpected extinction of wawter bloom may be that the infection due to molten algae bacterium causes, therefore separation screening has caused domestic and international field of Environment Protection experts and scholars' extensive concern to the inhibited molten algae bacterium of water body blue algae water bloom, at present existing increasing researchist is studied under laboratory condition aspect the screening of molten phycomycete, molten algae characteristic, Physiology and biochemistry, yet still has certain limitation for molten algae bacterium is applied to the research breaking out in blue-green alga bloom water body.The method of the molten algae bacterium of screening mainly contains liquid infection method and solid infection method at present, as the patent of invention of publication number CN101955904A, molten algae bacterium separation method in a kind of Natural Water environment is disclosed, adopt the solid method that infects take to be placed in the microbial film of eutrophication water inert support to screen molten algae bacterium as separation source, the bacterium of the method separation can effectively be controlled the growth of Anabaena Flos-aquae.Because Taihu Lake basin be take Microcystis aeruginosa as main, and the molten algae bacterium of aforesaid method screening only has high efficiency for anabena.< < Fudan Journal (natural science edition) > > the 49th volume the 1st phase 94-98 page in 2010 has reported that Wang Xiangrong separation from yellow algae liquid obtains the molten algae bacterium of 1 strain, through be accredited as bacillus brevis (
bacillus brevis), this bacterium only shows higher removal speed at the 1st d to microcystic aeruginosa, and bacterium algae is than under 1:1 condition, clearance 71.6%, after 2 d, remove speed and be almost 0, for the clearance reaching more than 90% can only strengthen throwing bacterium amount, when practical engineering application, improved cost.
The inventor is carrying out the research of microcystic aeruginosa artificial culture, the BG11 substratum that adopts improvement is during as microcystic aeruginosa substratum, once chance on and wherein have 2 bottles of microcystic aeruginosa nutrient solutions to produce gradually etiolation, the basic dead sinking of frond after 7 d, in nutrient solution, there is no other additives, got rid of the interference of exogenous material to the yellow of algae liquid, the yellow of deduction algae liquid may be infected relevant with bacterium, excite thus us to it, to do further further investigation, explore and whether have molten algae bacterium.
Being take in the strain separating source that the microcystic aeruginosa liquid of this yellow is molten algae bacterium in the present invention, by 4-5 liquid, infects enrichment, targetedly the bacterial strain of separation screening degraded microcystic aeruginosa therefrom.Methionin genus bacillus provided by the invention is by two kinds of molten algaes of the mode of action, secrete the indirect molten algae of non-protease Algicidal substances and contact the direct molten algae of frustule with thalline, microcystic aeruginosa is shown to high efficiency, therefore, make to take blue-green alga bloom that microcystic aeruginosa is advantage algae kind to be effectively controlled and become possibility.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency of the prior art, a kind of Methionin genus bacillus with efficient molten algae performance filtering out from yellow microcystic aeruginosa liquid that the microcystic aeruginosa of take provides as control object, and utilize its degraded microcystic aeruginosa method.
the technical solution adopted in the present invention is as follows:
The invention provides molten algae bacterium TL, the Classification And Nomenclature of suggestion be fusiform Methionin genus bacillus (
lysinibacillus fusiformis).On May 11st, 2012, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, deposit number is CGMCC NO.6108.
Utilize the method for above-mentioned fusiform Methionin genus bacillus degraded microcystic aeruginosa, according to following step, carry out:
The single colony inoculation of fusiform Methionin genus bacillus is cultivated to 28 h to logarithmic phase in LB liquid nutrient medium under 30 ℃ of temperature, shaking speed 130 r/min conditions, according to the inoculum size of volume fraction 5%, be forwarded in fresh LB liquid nutrient medium again, under the same terms, cultivate 28 h to logarithmic phase, standby;
Above-mentioned logarithmic phase Methionin genus bacillus is inoculated in fresh microcystic aeruginosa liquid than the volume ratio of 1:2-10 according to bacterium algae, in 28 ℃ of temperature, light intensity 2500 lux illumination box bacterium algae, cultivate altogether the microcystic aeruginosa of can degrading efficiently, the photoperiod arranges respectively light circulate 12 h:12 h, complete dark, full exposure.
Wherein said Methionin genus bacillus is untreated bacterium liquid, aseptic supernatant liquor, and thalline and high-temperature inactivation are processed bacterium liquid.
Wherein said fresh microcystic aeruginosa liquid Determination of Chlorophyll a starting point concentration is 478.85-1081.13mg/m
3, pH value is 7.0-7.5.
The method of above-mentioned degraded microcystic aeruginosa is preferred: in circulate 12 h:12 h illumination box bacterium algae of 28 ℃ of temperature, light intensity 2500 lux, light, cultivate altogether.
major advantage of the present invention:
1, to take the microcystic aeruginosa liquid of yellow be strain separating source in the present invention, adopts liquid to infect the molten algae bacterium of method enrichment mixed bacterial, by the molten algae bacterium of LB substratum line separation and purification, has the advantages such as cost is low, easy, safe, with strong points, efficient.
2, the molten algae bacterium Methionin genus bacillus of separation of the present invention by the outer indirect molten algae of non-proteolytic enzyme Algicidal substances of secretion born of the same parents with contact two kinds of modes of action of the direct molten algae of frustule and bring into play molten algae characteristic, microcystic aeruginosa is had to higher degradation rate, for microbial method control, take the blue-green alga bloom that microcystic aeruginosa is advantage algae kind and there is more wide application prospect.
Embodiment
the separation screening of bacterial strain and evaluation:
1, sample collecting
Under aseptic condition, collect the supernatant liquor of yellow microcystic aeruginosa liquid
2, substratum preparation
LB substratum: take Tryptones 10 g/L, yeast extract 5 g/L and NaCl 10g/L are in beaker, add 1000 mL distilled water, heated and stirred, make it quick dissolving, regulate pH to 7.0-7.2, then get respectively 100 mL and cultivate based in Erlenmeyer flask, with gauze and kraft paper, Erlenmeyer flask is sealed, put into 121 ℃ of sterilizing 20 min of pressure kettle.Solid medium adds agar 2.4%.
Microcystic aeruginosa substratum: take NaNO
31.5 g, K
2hPO
40.04 g, MgSO
47H
2o 0.075 g, CaCl
22H
2o 0.036 g, citric acid 0.006 g, ferric ammonium citrate 0.006 g, EDTA-Na
20.001 g, NaCO
30.02 g and micro solution 1 mL, in beaker, adjust pH to 7.1,121 ℃ of sterilizing 20 min in pressure kettle after adding 1000 mL distilled water stirring and dissolving.
Micro solution: take boric acid 2.86 g, MnCl
24H
2o 1.86 g, ZnSO
47H
2o 0.22 g, Na
2moO
42H
2o 0.39 g, CuSO
45H
2o 0.08 g, Co (NO
3)
26H
2o 0.05 g, adds 1000 mL distilled water, stirring and dissolving.
3, separating screening method
(1) acclimating
By the yellow microcystic aeruginosa supernatant liquor of collecting, via hole diameter 0.8 μ m filtering with microporous membrane, filtrate is the membrane filtration of via hole diameter 0.22 μ m again, hold back the bacterium in yellow algae liquid, after the filter membrane of holding back bacterium being shredded under aseptic condition, be added in the microcystic aeruginosa liquid that 50 mL are fresh, be placed in the circulate standing cultivation of illumination box of 12 h:12 h of 28 ℃, light intensity 2500 lux, light, after yellow, in the ratio of 1:5, be forwarded to and in fresh algae liquid, again carry out enrichment culture, repeat 4-5 time, with enrichment, obtain having the bacteria flora of solubilized stable algae ability.
(2) screening, plate isolation
By the bacterium algae co-culture media through 4-5 acclimating, by gradient dilution (10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6), be inoculated in respectively on LB solid medium, cultivate after 48 h for 30 ℃, the different single bacterium colony of picking, adopts method of scoring on LB solid medium, again to carry out plate isolation respectively, the constant incubator that is placed in 30 ℃ is cultivated 48 h, repeat 3-4 time, obtain the bacterium of purifying, by respectively its molten algae performance being carried out to decision analysis, obtain 1 plant height and imitate molten algae bacterium, its single bacterium colony is saved backup in slant medium.
4, strain morphology and physiological biochemical property
By bacterial strain is carried out to morphologic observation, staining reaction and Physiology and biochemistry, measure, and according to the < < of Science Press common bacteria system identification handbook > > (2001), it is identified.Result is as follows:
Colony morphology characteristic: bacterium colony is circular, larger, is faint yellow, and translucent, edge-smoothing is neat, has an even surface more moistening, toughness.
Bacterium colony physiological and biochemical property: Gram-negative, catalase is positive, sugar or alcohols fermentation and acid, glucose oxidase fermentation test fermentation and acid, the test of acetylmethyl alcohol (V.P), methyl red test (M.R), hydrogen sulfide production test, Starch Hydrolysis and nitrate reduction are all negative.
5, the 16S rDNA sequence homology analysis of bacterial strain
By the known array in the GenBank of the 16S rDNA sequence of bacterial strain and the U.S. state-run biotechnology information center (NCBI) is compared, the fusiform Methionin genus bacillus LQ88(that this bacterial strain and sequence accession number are EF472269
lysinibacillus fusiformis) similarity is up to 99.7%, and two bacterial strains are in same branch in the phylogenetic tree building based on distance method, the value of bootstrapping is 99, wherein repeat number is set to 1000, with a high credibility.
According to 16S rDNA sequence homology analysis, in conjunction with morphological feature and the physio-biochemical characteristics of bacterial strain, the molten algae bacterium that preliminary evaluation the present invention filters out be fusiform Methionin genus bacillus (
lysinibacillus fusiformis).The sequence accession number that this bacterium is applied in the GenBank of NCBI is JQ991004, the invention provides molten algae bacterium TL, the Classification And Nomenclature of suggestion be fusiform Methionin genus bacillus (
lysinibacillus fusiformis).On May 11st, 2012, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, deposit number is CGMCC NO.6108.
below provide the present invention to utilize above-mentioned bacterial strains to process 5 embodiment of microcystic aeruginosa:
Algae liquid of the present invention is microcystic aeruginosa pure culture liquid, and algae kind is microcystic aeruginosa FACHB-905, and purchased from Wuhan hydrobiont institute of Chinese Academy of Sciences country's algae kind storehouse, therefore, the variation of chlorophyll-a concentration can characterize the removal effect of microcystic aeruginosa well.
Embodiment 1
What the present embodiment was processed is the fresh microcystic aeruginosa liquid of 100 mL, chlorophyll-a concentration 568.92 mg/m
3, pH value is 7.0.Specific embodiment step is as follows: first Methionin genus bacillus is cultured to logarithmic phase (28 h) under 30 ℃ of temperature, shaking speed 130 r/min conditions, then press bacterium algae than the throwing bacterium amount of 1:2,50 mL logarithmic phase bacterium liquid are inoculated in algae liquid, with sterilized water, make blank, the initial chlorophyll-a concentration of sampling and measuring is 379.28 mg/m
3, be placed in the circulate standing cultivation of illumination box of 12 h:12 h of 28 ℃, light intensity 2500 lux, light, every 24 h sampling and measuring chlorophyll-a concentrations.The algae liquid of processing through aforesaid method, during 96 h, chlorophyll-a concentration is 15.4 mg/m
3, clearance is up to 95.94%, and molten algae speed now still keeps higher level, is 2.977 mg/ (m
3h), removal effect is good.
Embodiment 2
The present embodiment difference from Example 1 is that the microcystic aeruginosa liquid chlorophyll-a concentration of processing is different, throws bacterium amount bacterium algae than different.What the present embodiment was processed is the fresh microcystic aeruginosa liquid of 100 mL, chlorophyll-a concentration 505.82 mg/m
3, pH value is 7.0.Specific embodiment step is as follows: first Methionin genus bacillus is cultured under 30 ℃ of temperature, shaking speed 130r/min condition to logarithmic phase (28 h), then press bacterium algae than the throwing bacterium amount of 1:5,20 mL logarithmic phase bacterium liquid are inoculated in algae liquid, with sterilized water, make blank, the initial chlorophyll-a concentration of sampling and measuring is 421.52 mg/m
3, be placed in the circulate standing cultivation of illumination box of 12 h:12 h of 28 ℃, light intensity 2500 lux, light, every 24 h sampling and measuring chlorophyll-a concentrations.The algae liquid of processing through aforesaid method, during 96 h, chlorophyll-a concentration is 89.73 mg/m
3, clearance is up to 78.71%, and molten algae speed now still keeps high level, is 5.90 mg/ (m
3h), removal effect is better.
Embodiment 3
The present embodiment is that the microcystic aeruginosa liquid chlorophyll-a concentration of processing is different, throws bacterium amount bacterium algae than different from embodiment 1,2 differences.What the present embodiment was processed is the fresh microcystic aeruginosa liquid of 100 mL, chlorophyll-a concentration 478.85 mg/m
3, pH value is 7.0.Specific embodiment step is as follows: first Methionin genus bacillus is cultured to logarithmic phase (28 h) under 30 ℃ of temperature, shaking speed 130 r/min conditions, then press bacterium algae than the throwing bacterium amount of 1:10,10 mL logarithmic phase bacterium liquid are inoculated in algae liquid, with sterilized water, make blank, the initial chlorophyll-a concentration of sampling and measuring is 435.32 mg/m
3, be placed in the circulate standing cultivation of illumination box of 12 h:12 h of 28 ℃, light intensity 2500 lux, light, every 24 h sampling and measuring chlorophyll-a concentrations.The algae liquid of processing through aforesaid method, during 96 h, chlorophyll-a concentration is 163.45 mg/m
3, clearance is 62.45%, molten algae speed now still keeps higher level, is 5.10 mg/ (m
3h), therefore visible, bacterium algae can suppress algal grown than the dosage of 1:10.
Embodiment 4
The present embodiment is that from embodiment 1,2,3 differences the microcystic aeruginosa liquid chlorophyll-a concentration of processing is different, and pH value is different, and the test photoperiod is different, and fixedly bacterium algae is than the throwing bacterium amount of 1:10.What the present embodiment was processed is 3 parts of fresh microcystic aeruginosa liquid, and every 1 part is 80 mL, and chlorophyll-a concentration is respectively 920.69 mg/m
3, 1066.46 mg/m
3with 1081.13 mg/m
3, pH value is all 7.5.Specific embodiment step is as follows: first Methionin genus bacillus is cultured to logarithmic phase (28 h) under 30 ℃ of temperature, shaking speed 130 r/min conditions, then press bacterium algae than the throwing bacterium amount of 1:10,8 mL logarithmic phase bacterium liquid are inoculated into respectively in above-mentioned 3 parts of algae liquid, with sterilized water, make blank, the initial chlorophyll-a concentration of sampling and measuring is 836.99 mg/m
3, 969.51 mg/m
3with 982.84 mg/m
3, be placed in 28 ℃, the standing cultivation of light intensity 2500 lux illumination box, wherein the photoperiod is respectively full dark, full exposure, the light 12 h:12 h that circulate, sampling and measuring chlorophyll-a concentration after 5 d.The algae liquid of processing through aforesaid method, after 5 d, chlorophyll a clearance is respectively 17.16%, 62.09% and 79.95%, and the visible ray 12 h:12 h that circulate are the suitableeest photoperiods of Methionin genus bacillus performance algae-lysing.
Embodiment 5
The present embodiment and above-mentioned 4 embodiment differences are that the microcystic aeruginosa liquid chlorophyll-a concentration of processing is different, bacterium liquid processing mode is different, and fixedly bacterium algae is than the throwing bacterium amount of 1:10, light 12 h:12h that circulate.What the present embodiment was processed is 4 parts of identical fresh microcystic aeruginosa liquid, and every 1 part is 60 mL, chlorophyll-a concentration 520.61 mg/m
3, pH value is 7.0.Specific embodiment step is as follows: first Methionin genus bacillus is cultured to logarithmic phase (28 h) under 30 ℃ of temperature, shaking speed 130 r/min conditions; Then according to following 4 kinds of modes, process logarithmic phase bacterium liquid: 1. bacterium source liquid (T1); 2. by bacterium liquid 1. high speed centrifugation (12000 r/min, 10 min) get supernatant liquor, through 0.22 μ m membrane filtration degerming, in dull and stereotyped checking supernatant liquor aseptic (T2); 3. the thalline after centrifugal in collecting 2., through sterilized water washing 3-4 time, prepares sterilized water bacteria suspension (T3); 4. by 1. high temperature (121 ℃) inactivation treatment 30 min(T4 of bacterium liquid); With bacterium algae, than the throwing bacterium of 1:10, measure the bacterium liquid that the above-mentioned 4 kinds of different modes of 6 mL were processed again, it is inoculated into respectively in algae liquid, with sterilized water, make blank, the initial chlorophyll-a concentration of sampling and measuring is 473.28 mg/m
3, be placed in the circulate standing cultivation of illumination box of 12 h:12 h of 28 ℃, light intensity 2500 lux, light, sampling and measuring chlorophyll-a concentration after 5 d.The algae liquid of processing through above-mentioned 4 kinds of methods, after 5 d, chlorophyll-a concentration is difference 123.92 mg/m
3, 280.60 mg/m
3, 1518.78 mg/m
3, 304.54 mg/m
3, and control group chlorophyll-a concentration is 1750.00 mg/m
3; When bacterium liquid processing mode is for 1. time, the clearance of chlorophyll a is 73.82%; When processing mode is for 2. time, the clearance of chlorophyll a is 40.71%; When processing mode is for 3. time, by known with control group contrast, Methionin genus bacillus thalline only shows certain algicidal effect in the early stage; When processing mode is for 4. time, the clearance of chlorophyll a is 35.65%.As can be seen here, Methionin genus bacillus contacts two kinds of modes of action of the direct molten algae of frustule by the outer indirect molten algae of non-proteolytic enzyme Algicidal substances of secretion born of the same parents and brings into play molten algae performance with thalline, show high efficiency.
Claims (4)
- Fusiform Methionin genus bacillus ( lysinibacillus fusiformis) degraded microcystic aeruginosa method, it is characterized in that carrying out according to following step:The single colony inoculation of fusiform Methionin genus bacillus is cultivated to 28 h to logarithmic phase in LB liquid nutrient medium under 30 ℃ of temperature, shaking speed 130 r/min conditions, according to the inoculum size of volume fraction 5%, be forwarded in fresh LB liquid nutrient medium again, under the same terms, cultivate 28 h to logarithmic phase, standby;Above-mentioned logarithmic phase Methionin genus bacillus is inoculated in fresh microcystic aeruginosa liquid than the volume ratio of 1:2-10 according to bacterium algae, in 28 ℃ of temperature, light intensity 2500 lux illumination box bacterium algae, cultivate altogether the microcystic aeruginosa of can degrading efficiently, the photoperiod arranges respectively light circulate 12 h:12 h, complete dark, full exposure;Wherein said fusiform Methionin genus bacillus is molten algae bacterium TL, and deposit number is CGMCC NO.6108.
- 2. the method for fusiform Methionin genus bacillus degraded microcystic aeruginosa according to claim 1, is characterized in that wherein said Methionin genus bacillus is untreated bacterium liquid, aseptic supernatant liquor, and thalline and high-temperature inactivation are processed bacterium liquid.
- 3. the method for fusiform Methionin genus bacillus degraded microcystic aeruginosa according to claim 1, is characterized in that wherein said fresh microcystic aeruginosa liquid Determination of Chlorophyll a starting point concentration is 478.85-1081.13mg/m 3, pH value is 7.0-7.5.
- 4. the method for fusiform Methionin genus bacillus according to claim 1 degraded microcystic aeruginosa, is characterized in that cultivating altogether in circulate 12 h:12 h illumination box bacterium algae of 28 ℃ of temperature, light intensity 2500 lux, light.
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| CN103952350B (en) * | 2014-04-24 | 2016-05-18 | 烟台地元生物科技有限公司 | Spindle lysine bacillus and microbial bacterial agent and their application |
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| CN112374616A (en) * | 2020-10-13 | 2021-02-19 | 神美科技有限公司 | Biodegradable slow-release denitrification carbon source and preparation method thereof |
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