CN103695342A - Bacillus having alga-lysing activity and application thereof - Google Patents
Bacillus having alga-lysing activity and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus having an alga-lysing activity and application of the bacillus in controlling cyanobacterial bloom. The bacillus sp. Lzh-5 which has an obvious alga-lysing activity is separated from Lake Taihu water body, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 8282; active alga-lysing ingredients, namely hexahydropyrrolo [1,2-alpha] pyrazine-1,4-dione and 3-isopropyl-hexahydropyrrolo [1,2-alpha] pyrazine-1,4-dione, are separated, purified and identified from a metabolic product of the bacillus, wherein median lethal doses LD50 of hexahydropyrrolo [1,2-alpha] pyrazine-1,4-dione and 3-isopropyl-hexahydropyrrolo [1,2-alpha] pyrazine-1,4-dione on microcystis aeruginosa 9110 are respectively 5.7mu g/mL and 19.4mu g/mL. The bacillus is applicable to research and production of novel biological algicides, and finally used for controlling cyanobacterial blooms of lakes.
Description
Technical field
The present invention relates to environmental microorganism field, particularly genus bacillus (Bacillus sp.) Lzh-5 and the application in blue-green alga bloom is controlled thereof of a strain secretion algicidal substances.
Background technology
The control techniques of current blue-green alga bloom can be summarized as physical method, chemical process and biological method.Physical method is sent out and be can be used as the subcontrol measure of algal bloom except algae etc. except algae and electromagnetic field except algae, flocculation as machinery, but shortcoming is that cost is very high, equipment is complicated, cures the symptoms, not the disease, and processing power is limited; Chemical process can directly be killed algae as chemical algicides such as weedicides, but the specificity of these chemical substances is poor, large to hydrobiont toxicity, and easily causes the residual and secondary pollution of objectionable impurities.
Based on physical method and chemical process, control the defects such as exist in wawter bloom expensive, highly difficult, high pollution, biological method, because of its Huan Bao ﹑ economic dispatch advantage, receives increasing concern.Molten algae bacterium (algae-lysing bacteria) for blue-green algae is the important component part of freshwater environment system, to reducing the biomass of blue-green algae, maintains the eubiosis and has vital role.
Therefore, those skilled in the art is devoted to screen the efficient molten algae active substance of efficient molten algae bacterium or the molten algae bacterial metabolism generation of separation and concentration, to develop microorganism algicide, in order to the control blue-green alga bloom problem of safety and efficiently.
Summary of the invention
In view of physical method in prior art with chemical process processing power is limited, the defect that easily causes secondary pollution, this technical problem to be solved is to find the efficient molten algae active substance of efficient molten algae bacterium and the molten algae bacterial metabolism generation of separation and concentration, in order to the control blue-green alga bloom problem of safety and efficiently.
For achieving the above object, the invention provides genus bacillus and the application of meta-bolites in blue-green alga bloom is controlled thereof that a strain has molten algae activity.
The invention discloses genus bacillus (Bacillus sp.) Lzh-5 that a strain has molten algae activity, Classification And Nomenclature is bacillus sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution's title is abbreviated as CGMCC, preserving number CGMCC No.8282, preservation date is on September 27th, 2013.Preservation mechanism address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101, phone: 86-10-64807355.
Further, the invention provides the application of above-mentioned genus bacillus Lzh-5 in controlling blue-green alga bloom.
Further, the application of the tunning that the invention provides above-mentioned genus bacillus Lzh-5 in controlling blue-green alga bloom.
Further, the invention discloses two kinds of effective constituents with molten algae activity in the tunning of above-mentioned genus bacillus Lzh-5, be respectively there is structural formula (I) and (II) shown in the compound of structure, the invention provides a kind of have structural formula (I) and (II) shown in the application in controlling blue-green alga bloom of the compound of structure.
Further, the invention provides a kind of molten phycomycete agent, it is characterized in that, in described molten phycomycete agent, comprise genus bacillus claimed in claim 1 (Bacillus sp.) Lzh-5.
Further, the invention provides a kind of molten algae medicament, it is characterized in that, the tunning that comprises genus bacillus claimed in claim 1 (Bacillus sp.) Lzh-5 in described molten algae medicament, described tunning is fermented liquid, concentrate of fermentation liquid, fermentation broth coarse extract or fermentation broth extract.
Further, the invention provides a kind of method of controlling blue-green alga bloom, comprise the steps:
1), fermentation genus bacillus Lzh-5 bacterial strain;
2), extractive fermentation liquid;
3), extraction liquid evaporate to dryness obtains crude extract;
4), crude extract water-soluble after for controlling blue-green alga bloom.
Preferably, in step 1), fermentation condition is, genus bacillus Lzh-5 is inoculated in the sterilizing beef-protein medium of pH7.0, and 28 ℃, 200rpm envrionment conditions bottom fermentation 48h, obtain described genus bacillus Lzh-5 fermented liquid.
Preferably, step 2) in, extraction agent is ethyl acetate, and in extraction system, the volume ratio of ethyl acetate and fermented liquid is 1:1, after mixing, puts into the vibrator 24h that vibrates, and isolated upper strata ethyl acetate solution is the extraction liquid of genus bacillus Lzh-5 fermented liquid.After extraction liquid evaporate to dryness, be fermentation broth coarse extract.Crude extract is through this area routine techniques means, and as the method for group chromatography, further purification can obtain fermentation broth extract.
Described genus bacillus Lzh-5 fermented liquid is respectively 4 of following algae strain days molten algae rates: microcystic aeruginosa PCC7806 is 88.5 ± 7.4%, microcystic aeruginosa 9110 is 81 ± 5.2%, the algae BN35 that quivers is 83.6 ± 4.5%, Microcystis viridis FACHB-979 is 78.2 ± 9.3%, chroococcoid FACHB-191 is 86.5 ± 7.9%, and chlamydomonas BS3 is 32.5 ± 11.3%; Within 6 days, molten algae rate is respectively: microcystic aeruginosa PCC7806 is 93.7 ± 3.4%, microcystic aeruginosa 9110 is 91.2 ± 6.3%, and the algae BN35 that quivers is 92.7 ± 1.5%, and Microcystis viridis FACHB-979 is 87.1 ± 7.5%, chroococcoid FACHB-191 is 91.4 ± 4.2%, and chlamydomonas BS3 is 23.6 ± 2.7%.
Described genus bacillus Lzh-5 fermented liquid, utilizes the meta-bolites of HPLC technology purifying genus bacillus Lzh-5, and concrete preparation method is as follows:
By described extraction liquid evaporate to dryness and after being dissolved in water, use 0.22 μ m aperture membrane filtration, filtrate is by the SupersilTM C18-EP semipreparative column of DIKMA company, and the HPLC that water and methyl alcohol are moving phase carries out preliminary purification, obtains molten algae composition;
By described molten algae composition, by DIKMA SupersilTM C18-EP analytical column, the HPLC that water and methyl alcohol are moving phase is further purified, and obtains two kinds of effective molten algae composition S-5A and S-5B.
In the meta-bolites of described genus bacillus Lzh-5, the chemical structure of effective molten algae composition is analyzed and is obtained by LC-MS ﹑ GC-MG and NMR.
When genus bacillus Lzh-5 is used for controlling blue-green alga bloom, the molecular ion peak of its effective molten algae composition S-5A is 155.0821m/z, and molecular weight analyte is 154.0742, and molecular formula is C
7h
10n
2o
2, proton nmr spectra result is
1h NMR (400MHz, D2O) δ 4.22 (s, 1H), 4.06 (dd, J=17.3,2.7Hz, 1H), 3.77 (d, J=17.3Hz, 1H), 3.44 (dd, J=8.7,4.8Hz, 2H), 2.41 – 2.12 (m, 1H), 2.09 – 1.71 (m, 3H); The molecular ion peak of effective molten algae composition S-5B is 197.1276m/z, and molecular weight analyte is 196.1212, and molecular formula is C
10h
16n
2o
2, proton nmr spectra result is
1h NMR (400MHz, D
2o) δ 4.20 (m, 1H), 4.07 (s, 1H), 3.47 (dd, J=16.3,8.9Hz, 1H), 2.38 (m, 1H), 2.26 (d, J=4.7Hz, 1H), 1.97 (s, 1H), 1.83 (s, 1H), 0.98 (d, J=7.2Hz, 1H), 0.76 (d, J=6.9Hz, 1H), carbon-13 nmr spectra result is
13c NMR (101MHz, D
2o) δ 172.23 (s), 167.12 (m), 60.34 (s), 58.72 (m), 45.20 (s), 28.89 (s), 28.10 (s), 21.55 (s), 17.95 (s), 15.06 (s).
Further, the meta-bolites hexahydropyrrolo that effective molten algae composition S-5A is described genus bacillus Lzh-5 is [1,2-a] pyrazine-1 also, 4-diketone, meta-bolites 3-sec.-propyl-hexahydropyrrolo that effective molten algae composition S-5B is described genus bacillus Lzh-5 is [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also.
Further, hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone the Lethal Dose 50 LD50 of microcystic aeruginosa 9110 is respectively to 5.7 μ g/mL and 19.4 μ g/mL.
The algicidal effect of genus bacillus Lzh-5 of the present invention has broad spectrum.Preparation method is simple for its fermented liquid, and preparation cycle is short.The fermented liquid of genus bacillus Lzh-5 has good algicidal effect to the microcystic aeruginosa 9110 in Taihu Lake and the algae BN35 that quivers etc., and microcystic aeruginosa is the main blue-green algae in the blue-green alga bloom of Taihu Lake.Genus bacillus Lzh-5 fermented liquid and acetic acid ethyl acetate extract all can be used for the control of blue-green alga bloom or other micro-algae, and algicidal effect is better.Wherein, genus bacillus Lzh-5 fermented liquid reaches 91.2 ± 6.3% to 6 of microcystic aeruginosa 9110 days molten algae rates.The meta-bolites hexahydropyrrolo of genus bacillus Lzh-5 also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-1,4-diketone has good lethal effect to the main blue-green algae microcystic aeruginosa 9110 in the blue-green alga bloom of Taihu Lake, can be used for research and development and the production of novel algicide, be finally applied to the control of lake blue algae wawter bloom.
Below with reference to accompanying drawing to genus bacillus Lzh-5 bacterial strain of the present invention and meta-bolites hexahydropyrrolo thereof also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-1, the effect of 4-diketone in blue-green alga bloom is controlled is described further, to understand fully object of the present invention, feature and effect.
Accompanying drawing explanation
Fig. 1 is the algicidal effect of molten phycomycete Lzh-5 to microcystic aeruginosa 9110;
Fig. 2 is that the fermented liquid acetic acid ethyl acetate extract of molten phycomycete Lzh-5 joins the algicidal effect on microcystic aeruginosa 9110 algae flat boards;
Fig. 3 be algicidal substances hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone join respectively the algicidal effect on microcystic aeruginosa 9110 algae flat boards;
Fig. 4 be algicidal substances hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone join respectively the algicidal effect to microcystic aeruginosa 9110 in algae liquid.
Embodiment
The screening of the molten algae bacterium of embodiment 1
The 10mL natural water samples that Mei Liangwan waters, Taihu Lake is gathered joins in the algae liquid of microcystic aeruginosa 9110 of 90mL logarithmic phase, get two days later yellow algae liquid and be coated with beef-protein medium agar plate by gradient dilution method, cultivate 24h for 28 ℃, get the flat board that bacterium colony density is moderate, according to the difference of colonial morphology, select different strains.
The bacterial strain screening is inoculated respectively in 10mL beef-protein medium, and 28 ℃, 200rpm cultivates 24h, and the cultured bacterium liquid of 10mL is added respectively in the algae liquid of 90mL logarithmic phase microcystic aeruginosa.In addition the beef-protein medium after 10mL sterilizing is added equally in 90mL algae liquid in contrast.The algae liquid of all experimental group and control group calculates its molten algae efficiency cultivate 48h in illumination box after, has chosen the strain bacterium that wherein molten algae efficiency is high and has further studied, and this bacterial strain code name is Lzh-5.
Algae liquid culture condition: microcystic aeruginosa 9110 use BG11 liquid nutrient mediums are cultivated, is positioned in illumination box, and 25 ℃, intensity of illumination 40 μ mol photons m
-2s
-1, light dark period is than being 12h:12h (illumination: dark).
Molten algae efficiency calculation method: molten algae efficiency (%)=(control group algae cell density-experimental group algae cell density)/control group algae cell density * 100.Wherein the algae cell density of microcystic aeruginosa is measured with blood counting chamber.
Fig. 1 is microcystic aeruginosa 9110 cell concns curve (molten phycomycete Lzh-5 (starting point concentration 1 * 10 over time when adding molten phycomycete Lzh-5
8cells/mL) (■) and blank (◆)).
The evaluation of embodiment 2 genus bacillus Lzh-5 bacterial strains
By methods such as morphologic observation, dyeing, biochemical reactions, 16srRNA gene sequencings, the strongest Lzh-5 of algicidal effect is identified, this bacterial strain is for belonging to G+ bacteria, cell is direct rod shape, 0.5～2.5 μ m * 1.2～10 μ m, often with paired or catenation, tool nose circle or square end.Cell dyeing great majority present Gram-positive when children cultivates age, with peritrichous, move.Gemma is oval, ovum circle, circular, can resist many poor environments.Have heat, pH and the various multifarious physiological properties of salt.Chemoheterotrophic bacteria, tool fermentation or respiratory metabolism type.Through 16srRNA gene sequencing and homology comparison, learn that in itself and GenBank, certain Bacillus strain has 99% homology, therefore be accredited as bacillus, called after genus bacillus Lzh-5.This bacterial classification has been preserved in national microbial strains preservation management committee's common micro-organisms center, preserving number CGMCC No.8282, and preservation date is on September 27th, 2013.Preservation mechanism address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, postcode: 100101, phone: 86-10-64807355.The accession number of the 16srRNA gene of this bacterial classification in GenBank is HQ896845.
The preparation method of embodiment 3 genus bacillus Lzh-5 fermented liquids and acetic acid ethyl acetate extract thereof
Genus bacillus Lzh-5 is inoculated in sterilizing beef-protein medium according to 1% inoculum size, and 200rpm shaking table obtains genus bacillus Lzh-5 containing fermented liquid after cultivating 48h at 28 ℃.Ethyl acetate is added in fermented liquid according to the ratio of 1:1, put into the vibrator 24h that vibrates, isolate upper solution, i.e. acetic acid ethyl acetate extract.By ethyl acetate evaporate to dryness, be dissolved in water, re-use after the filtering with microporous membrane in 0.22 μ m aperture for being further purified meta-bolites.
The algicidal effect of embodiment 4 genus bacillus Lzh-5 to different blue-green algaes and Eukaryotic Algae
By 6 parts of cultured (culture condition: beef-protein medium, 28 ℃, 200rpm cultivates 24h) 10mL genus bacillus Lzh-5 bacterium liquid adds respectively (90mL in the algae liquid of 6 strain blue-green algaes and eucaryon algae (table 1), all algae liquid is all cultivated logarithmic phase), the same aseptic beef-protein medium of 10mL that uses adds respectively (90mL in 6 kinds of algae liquid, all algae liquid is all cultivated logarithmic phase) (1:9) in contrast, in illumination box, cultivate the chlorophyll concentration of measuring respectively the algae liquid of all experimental group and control group after 4 days and 6 days, calculate its molten algae efficiency.Each sample is surveyed three Duplicate Samples, is expressed as the form of mean+SD.
The algae kind of using in experiment has 3 strains purchased from the aquatic institute in Wuhan algae kind storehouse, and other 3 strain screenings are from Taihu Lake water body.Experimental result shows, the fermented liquid of genus bacillus Lzh-5 has good algicidal effect (table 1) for most protokaryon algae kinds.This result shows that the molten algae ability of genus bacillus Lzh-5 has broad spectrum, is a kind of very potential molten algae bacterium.
The algicidal effect of table 1. genus bacillus Lzh-5 to 6 strain algae strains
Note: screen and obtain from the Mei Liangwan water body of Wuxi, Taihu Lake with the algae strain of asterisk (*) mark in table, all the other algae strains are all to buy from CHINESE FRESHWATER algae kind storehouse.
Genus bacillus Lzh-5 to the algicidal effect of the main blue-green algae microcystic aeruginosa 9110 in the blue-green alga bloom of Taihu Lake as shown in Figure 1.Fig. 1 shows, add after genus bacillus Lzh-5, prolongation in time of microcystic aeruginosa 9110 cell concns and declining, and prolongation in time of microcystic aeruginosa 9110 cell concns and rising in blank, nearly all microcystic aeruginosa 9110(> 91% in the time of the 6th day) death.Wherein adding the genus bacillus Lzh-5 starting point concentration of algae liquid is 1 * 10
8individual/mL.
The research of embodiment 5Lzh-5 bacterial strain algicidal mode
Genus bacillus Lzh-5 fermented liquid is made to acetic acid ethyl acetate extract according to the method described in step 3, with after centrifugal dryer evaporate to dryness, standby after dissolving with sterilized water.Finally that the same centrifugal drying of sterilizing beef-protein medium is concentrated in contrast.The contrast of the extraction liquid making according to the method described above and concentrated beef extract-peptone is added to respectively to the round scraps of paper (diameter 1cm) upper, is positioned on the BG11 solid plate that microcystic aeruginosa makes.Algae flat board put into illumination box cultivate within 2 days, observe afterwards circle scraps of paper algal control circle around formation to judge its algicidal effect.
The making method of microcystic aeruginosa agar plate: on every flat board, pour appropriate (about 20mL) BG11 nutrient agar (agar ratio 1.5%) into, standby after it solidifies.To after the centrifugal 20min of the cultured microcystic aeruginosa algae of 300mL liquid 4000g, discard supernatant, collect frustule precipitation and join (1% agar in the BG11 soft agar solid medium after 100mL sterilizing, be positioned in 53 ℃ of water-baths and prevent from solidifying), after shaking up, pour the BG11 solid plate upper strata (every flat board is approximately poured 20mL into) making into, after it solidifies, put into illumination box and cultivate also standby.
Experimental result as shown in Figure 2, shows that the fermented liquid extraction liquid of genus bacillus Lzh-5 has good algicidal effect, and this shows that this bacterial strain has the performance that the outer algicidal substances of secretion born of the same parents carries out molten algae.
The extraction purifying of molten algae effective constituent in embodiment 6 genus bacillus Lzh-5 tunnings
Utilize HPLC technology by the molten algae meta-bolites purifying of molten phycomycete Lzh-5, obtain two kinds of purifying substance S-5A and S-5B with molten algae function, concrete steps are as follows:
By the extraction liquid of genus bacillus Lzh-5 fermented liquid, by the SupersilTM C18-EP semipreparative column of DIKMA company, the HPLC technique means that water and methyl alcohol are moving phase is carried out preliminary purification, obtains molten algae effective constituent.Then by DIKMA SupersilTM C18-EP analytical column, the HPLC technique means that water and methyl alcohol are moving phase is further purified, and obtains two kinds of effective molten algae composition S-5A and S-5B, for Structural Identification.
The Identification of chemical structure of molten algae effective constituent in embodiment 7 genus bacillus Lzh-5 meta-bolitess
Utilize LC-MS ﹑ GC-MG and NMR to analyze the chemical structure that (nuclear magnetic resonance spectroscopy) has the meta-bolites of molten algae function.
Sample S-5A after purifying is carried out to LC-MS analysis, and the molecular ion peak that obtains sample S-5A is 155.0821m/z, and molecular weight analyte is 154.0742, and molecular formula is C
7h
10n
2o
2.
Sample S-5A after purifying is carried out to GC-MS analysis, with the structural similitude of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also of hexahydropyrrolo in GC-MS database, similarity index > 850.
Sample S-5A is carried out to hydrogen nuclear magnetic resonance spectrum analysis, obtains following result:
1H?NMR(400MHz,D
2O)δ4.22(s,1H),4.06(dd,J=17.3,2.7Hz,1H),3.77(d,J=17.3Hz,1H),3.44(dd,J=8.7,4.8Hz,2H),2.41–2.12(m,1H),2.09–1.71(m,3H)。
The LC-MS ﹑ GC-MS of sample S-5A and NMR result are analyzed, can determine that the meta-bolites S-5A with algicidal effect is also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of hexahydropyrrolo, its chemical structure is as shown below.
Sample S-5B after purifying is carried out to LC-MS analysis, and the molecular ion peak that obtains sample S-5B is 197.1276m/z, and molecular weight analyte is 196.1212, and molecular formula is C
10h
16n
2o
2.
Sample S-5B after purifying is carried out to GC-MS analysis, wherein 3-sec.-propyl-hexahydropyrrolo structural similitude of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also in S-5B and known references.
Sample S-5B is carried out to hydrogen nuclear magnetic resonance spectrum analysis, obtains following result:
1H?NMR(400MHz,D
2O)δ4.20(m,1H),4.07(s,1H),3.47(dd,J=16.3,8.9Hz,1H),2.38(m,1H),2.26(d,J=4.7Hz,1H),1.97(s,1H),1.83(s,1H),0.98(d,J=7.2Hz,1H),0.76(d,J=6.9Hz,1H)。
Sample S-5B is carried out to carbon-13 nmr spectra analysis, obtains following result:
13C?NMR(101MHz,D
2O)δ172.23(s),167.12(m),60.34(s),58.72(m),45.20(s),28.89(s),28.10(s),21.55(s),17.95(s),15.06(s)。
The LC-MS ﹑ GC-MS of sample S-5B and NMR result are analyzed, can determine that the meta-bolites S-5B with algicidal effect is also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of 3-sec.-propyl-hexahydropyrrolo, its chemical structure is as follows.
The molten algae meta-bolites of embodiment 8 genus bacillus Lzh-5 hexahydropyrrolo is [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and the 3-sec.-propyl-hexahydropyrrolo molten algae effect research of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also also
Use the method in the research of above-mentioned Lzh-5 bacterial strain algicidal mode, through experiment, find hexahydropyrrolo also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-1, on the BG11 solid plate that 4-diketone is made at microcystic aeruginosa, can form obvious algal control circle, blank can not form algal control circle (Fig. 3).
By hexahydropyrrolo also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] to add respectively blue-green algae be in microcystic aeruginosa 9110 to pyrazine-Isosorbide-5-Nitrae-diketone, makes hexahydropyrrolo also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also concentration of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone form respectively a series: 100 μ g/mL ﹑ 50 μ g/mL ﹑ 40 μ g/mL ﹑ 30 μ g/mL ﹑ 20 μ g/mL ﹑ 10 μ g/mL and 5 μ g/mL.To add hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also the microcystic aeruginosa 9110 of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone put into illumination box and cultivate and after 24 hours, measure molten algae rate.Experimental result shows, hexahydropyrrolo is [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also [1 also, 2-a] pyrazine-Isosorbide-5-Nitrae-diketone is respectively 5.7 μ g/mL and 19.4 μ g/mL (Fig. 4) to the Lethal Dose 50 LD50 of microcystic aeruginosa 9110 and synechococcus BN60.The meta-bolites hexahydropyrrolo that this result shows genus bacillus Lzh-5 also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone be the material of highly effective control blue-green algae.
In Fig. 4, add also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone and 3-sec.-propyl-hexahydropyrrolo microcystic aeruginosa 9110 survival rate change curves during [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also of different concns algicidal substances hexahydropyrrolo.
The algicidal effect of genus bacillus Lzh-5 of the present invention has broad spectrum.Preparation method is simple for its fermented liquid, and preparation cycle is short.The fermented liquid of genus bacillus Lzh-5 has good algicidal effect to the microcystic aeruginosa 9110 in Taihu Lake and the algae BN35 that quivers etc.Genus bacillus Lzh-5 fermented liquid and acetic acid ethyl acetate extract all can be used for the control of blue-green alga bloom or other micro-algae, and algicidal effect is better.Wherein, genus bacillus Lzh-5 fermented liquid reaches 91.2 ± 6.3% to 6 of microcystic aeruginosa 9110 days molten algae rates.The meta-bolites hexahydropyrrolo of genus bacillus Lzh-5 also [1,2-a] pyrazine-1,4-diketone and 3-sec.-propyl-hexahydropyrrolo also [1,2-a] pyrazine-1,4-diketone has good lethal effect to the main blue-green algae microcystic aeruginosa 9110 in the blue-green alga bloom of Taihu Lake, can be used for research and development and the production of novel algicide, be finally applied to the control of lake blue algae wawter bloom.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that those of ordinary skill in the art just can design according to the present invention make many modifications and variations without creative work.Therefore, all technician in the art, all should be in the determined protection domain by claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1. a strain has genus bacillus (Bacillus sp.) Lzh-5 of molten algae activity, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.8282, and preservation date is on September 27th, 2013.
2. the application of genus bacillus Lzh-5 as claimed in claim 1 in controlling blue-green alga bloom.
3. the application of the tunning of genus bacillus Lzh-5 as claimed in claim 1 in controlling blue-green alga bloom.
One kind have structural formula (I) or (II) shown in the application in controlling blue-green alga bloom of the compound of structure.
5. a molten phycomycete agent, is characterized in that, comprises genus bacillus claimed in claim 1 (Bacillus sp.) Lzh-5 in described molten phycomycete agent.
6. a molten algae medicament, it is characterized in that, the tunning that comprises genus bacillus claimed in claim 1 (Bacillus sp.) Lzh-5 in described molten algae medicament, described tunning is fermented liquid, concentrate of fermentation liquid, fermentation broth coarse extract or fermentation broth extract.
7. a method of controlling blue-green alga bloom, comprises the steps:
1), the genus bacillus Lzh-5 claimed in claim 1 that ferments;
2), extractive fermentation liquid;
3), extraction liquid evaporate to dryness obtains crude extract;
4), crude extract water-soluble after for controlling blue-green alga bloom.
8. method as claimed in claim 7, wherein in step 1), fermentation condition is, and genus bacillus Lzh-5 is inoculated in the sterilizing beef-protein medium of pH7.0, and 28 ℃, 200rpm envrionment conditions bottom fermentation 48h, obtain described genus bacillus Lzh-5 fermented liquid.
9. method as claimed in claim 7, wherein step 2) in, extraction agent is ethyl acetate.
10. method as claimed in claim 9, wherein step 2) in, the volume ratio of ethyl acetate and fermented liquid is 1:1.
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